Synthesis of Dicyanovinyl-Substituted 1-(2-Pyridyl)pyrazoles: Design of a Fluorescent Chemosensor for Selective Recognition of Cyanide

Article abstract of DOI:10.1021/acs.joc.7b02460

A fluorescence "turn-off" probe has been designed and successfully applied to detect cyanide (CN^-) based on a Michael-type nucleophilic addition reaction and intramolecular charge transfer (ICT) mechanism. For this research, a family of 3-aryl-

Full text of DOI:10.1021/acs.joc.7b02460

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Article
Synthesis of Dicyanovinyl-Substituted 1-(2-Pyridyl)pyrazoles: Design
of a Fluorescent Chemosensor for Selective Recognition of Cyanide
Jessica Orrego-Hernández, and Jaime Portilla
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Nov 2017
Downloaded from http://pubs.acs.org on November 24, 2017
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Synthesis of Dicyanovinyl-Substituted 1-(2-Pyridyl)pyrazoles: Design of a
Fluorescent Chemosensor for Selective Recognition of Cyanide
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Jessica Orrego-Hernández and Jaime Portilla*
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Bioorganic Compounds Research Group, Department of Chemistry, Universidad de los Andes,
Carrera 1 No. 18A-10, Bogotá 111711, Colombia
*Email: jportill@uniandes.edu.co
TOC graphic & Abstract
A fluorescence “turn-off” probe have been designed and successfully applied to detect cyanide
(CN-) based on a Michael-type nucleophilic addition reaction and intramolecular charge
transfer (ICT) mechanism. For this research, a family of 3-aryl-4-(2,2-dicyanovinyl)-1-(2-
pyridinyl)pyrazoles as donor-π-acceptor (D-π-A) systems have been synthesized in 58-66%
overall yield, by a three-step synthesis sequence starting from p-substituted acetophenones. The
product p-metoxyphenyl substituted showed good fluorescence emission and large Stokes shifts
in different solvents due to its greater ICT. Likewise, this probe evidenced high selectivity and
sensitivity, and fast recognition for CN- with a detection limit of 6.8 µM. HRMS analysis, 1H
NMR titration experiments and TD-DFT calculations were performed to confirm the
mechanism of detection and fluorescence properties of the chemodosimeter of CN-.
Additionally, fluorescent test paper was conveniently used to detect cyanide in aqueous
solution.
Keywords: Chemosensor, Dicyanovinyl, Fluorescence, 4-Formylpyrazole, Quantum yields
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INTRODUCTION
Recently, functionalized pyrazoles have attracted considerable attention due to their proven
utility as synthetic intermediates for the preparation of bioactive compounds,1 fluorescent
molecules,2 and coordination complexes.3 They have been used to synthesize a great number of
N-heterocycles with broad spectrum of biological activities,4 such as antifungal,4a
antitubercular,4b antiviral,4c antitumor,4d and antiparasitic4f activities. Notably, the pyrazole core
can act as an electron donor in a chromophoric system due to the presence of an electron-rich
nitrogen atom, which is favorable for application in dye chemistry.5 Tetrasubstituted pyrazoles
have been studied with UV-vis and fluorescence spectroscopy showing an intense blue
fluorescence and high quantum yields.6 Also, some pyrazoles are well studied as blue emitters
used as electroluminescent materials in organic light-emitting diodes (OLEDs).7 Due to the
broad applications in dye-sensitized solar cell (DSSCs)8 and second order nonlinear optical
devices arylsubstituted, pyrazoles are considered of great interest.9 In addition, these suitably
functionalized heterocycles are used as ligands to chelate metal cations, as well as receptors of
anions in the design of fluorescent chemosensors.10
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On the other hand, cyanide ion is present in water caused by contamination from industrial
waste, in cigarette smoke, and in substances present in some foods that releasing it.11 cyanide
in the human bloodstream causes cytotoxic hypoxia and cellular asphyxiation due to the
formation of a stable complex with cytochrome c oxidase that inhibits the function of this
enzyme. In addition, cyanide induces anaerobic metabolism, which leads the lactate
accumulation in the blood. The cytotoxic hypoxia and lactate accumulation affect the central
nervous system causing respiratory arrest and death.11-12 Due to the extreme toxicity of cyanide
in biological systems, the design of fluorescent probes based on small molecules,13 metal
complexes, 14 polymers,15 and nanoparticles16 is of great interest for the sensitive and selective
detection of cyanide.17 For the design of these chemosensors, fundamental photophysical
mechanisms such as photoinduced electron transfer (PET), intramolecular charge transfer
(ICT), metal–ligand charge transfer (MLCT), twisted intramolecular charge transfer (TICT),
and fluorescence resonance energy transfer (FRET) are commonly used.18
Consequently, the design of fluorescent probes based on intramolecular charge transfer (ICT)
to detect cyanide have been widely studied due to its donor-π-acceptor (D-π-A) properties,
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which can be modified favoring fluorescent and colorimetric changes.19 The dicyanovinyl group
is widely used as acceptor moiety in the design of conjugated D-π-A system. Additionally, this
acceptor group can react easily with cyanide breaking the conjugation of the D-π-A system who
leads the modification of the optical properties of those adducts.20
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Considering the electron rich and fluorescent properties of the pyrazole derivatives5-9 and our
continuing interest in the synthesis of novel molecules containing this heterocyclic moiety,1b,21
we proposed the synthesis of a family of 3-aryl-4-(2,2-dicyanovinyl)-1-(pyridin-2-yl)-1H-
pyrazoles 6a-c starting from acetophenones 1a-c (Scheme 1b). These compounds contain a
dicyanovinyl group, which acts as acceptor in the design of fluorescent chemosensors.5,20,22
Pyrazole-4-carbaldehydes 4a-c play a fundamental role in the achievement of these D-π-A
system 6a-c. The method usually employed in the synthesis of these aldehydes 4’ is based on
cyclization-formylation of N-arylhydrazones 3’23 under the conditions of the Vilsmeier–Haack
reaction (Scheme 1a).5,21e,24
Scheme 1. Strategy for the Synthesis of 3-Aryl-4-(2,2-dicyanovinyl)pyrazoles.
The novel D-π-A system 6a-c contain an electron rich pyrazole core bound to a dicyanovinyl
group (strong acceptor) in position 4 and p-substituted aryl group in position 3. The electronic
character in p-substituted aryl group was modified to evaluate the ICT effect in these structures.
In this work, the compound 6c (p-metoxyphenyl substituted) showed better fluorescence
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emission in different solvents, and large Stokes shifts due to the ICT is more favored. These
results were confirmed by TD-DFT calculations. In addition, the presence of 2-pyridil group on
pyrazole moiety of structural analogues of 6a-c has not yet been reported, which increases the
reactivity of electrophilic groups in this structure towards nucleophilic reagents.21e Therefore,
the detection of cyanide can carried out in short reaction time via a Michael-type nucleophilic
addition of cyanide to the dicyanovinyl group on 6c. Taking in to account this approach, the
compound 6c was studied as a “TURN-OFF” chemodosimeter of cyanide (CN-) showing a
selective detection with a good detection limit in acetonitrile, as well as visual detection of CN-
in paper test. It is important to note that the compound 6c is the first non-fused pyrazole used
as fluorescent chemodosimeter for detection of cyanide.
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RESULTS AND DISCUSSION
Synthesis. We started our work by synthesizing 2-(2-(1-arylethylidene)hydrazinyl)pyridines
3a-c23 via condensation reaction between acetophenones 1a-c and 2-hydrazinepyridyne (2).21e
The reaction provided the expected hydrazones 3a-c as colored solids in 76-92% yields, and
without any difficulty in the process. Structure of compounds 3a was solved by single-crystal
X-ray diffraction analysis25 (see Supporting Information, Figure S1). Subsequently, the
synthesis of 3-aryl-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehydes 4a-c was carried through the
cyclization-formylation of Vilsmeier–Haack of the corresponding hydrazone 3a-c.24c All
synthesis proceeded efficiently to give the pure 4-formylpyrazole 4a-c, in 80-85% yields, which
indicated the electronic demands of the substituents had little influence on the reactivity, as well
as in the synthesis of 3a-c (Scheme2). However, an impure solid was isolated in the process
after aqueous workup, and therefore the product had to be purified by flash chromatography,
unlike previous described analogous procedures.5,21e,24 Probably the presence of 2-pyridil group
on the product affect its solubility. The results of these two reaction steps are summarized below
(Scheme 3).
Scheme 2. Synthesis of 3-aryl-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehydes 4a-c.a
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a Reaction conditions: (i) 1 (6.00 mmol) and 2 (6.00 mmol) in EtOH (60.0 mL); (ii) DMF (0.6 mL,
~7.5 mmol), POCl3 (1.4 mL, 15.0 mmol) and 3 (2.5 mmol).
Continuing our study regarding the synthesis of the novel D-π-A systems 6a-c, we decided to
carry out the Knoevenagel reaction between those synthesized 4-formylpyrazoles 4a-c and
malononitrile (5) according to previous works.5,20,22 Gratifyingly, the reaction proceeded with
operational simplicity and high yields without requiring chromatographic purification. These
results allow us to conclude that indeed the presence of 2-pyridil group on 6a-c increases its
reactivity towards nucleophilic reagents. Also, the compound p-metoxyphenyl substituted 6c
showed better fluorescence emission (Scheme 3). All reactions offered the pure 3-aryl-4-(2,2-
dicyanovinyl)-1-(pyridin-2-yl)-1H-pyrazole 6a-c in 89-92% yield.
Scheme 3. Synthesis of 3-aryl-4-(2,2-dicyanovinyl)-1-(pyridin-2-yl)-1H-pyrazole 6a-c.a
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a Reaction conditions: 4 (0.50 mmol), 5 (0.50 mmol) and pyperidine (~10 mol %) in EtOH (5.0 mL). b
Photograph was taken using 10 μM solutions in acetonitrile. A hand-held UV lamp under long
wavelength (λ = 365 nm) was used.
The three D-π-A systems 6a-c were efficiently synthesized in three reaction steps starting from
the appropriate p-substituted acetophenone 1a-c and 2-hydrazinepyridyne (2). All synthesized
compounds (hydrazones 3a-c, formylpyrazoles 4a-c and products 6a-c) were adequately
characterized by 1H NMR, 13C NMR, analysis by HRMS and FTIR. The whole carbon skeleton
was assigned using 13C NMR spectra, combining with DEPT and two dimensional 1H, 13C shift
correlation HSQC and HMBC experiments (see Experimental Section and Supporting
Information for details).
Photophysical Properties of 6a-c. The UV-vis and fluorescence emission spectra of the
compounds 6a-c were done in dimethylsulfoxide (DMSO), dimethylformamide (DMF),
acetonitrile, ethanol (EtOH), and dichloromethane (DCM), ethyl acetate, and toluene (see
Supporting Information, Figures S2-S3 and Table S1). In the UV-vis spectra of 6a-c, the band
between 340-360 nm assigned to charge transfer (CT-bands)19 is bathochromically shifted by
increasing the donating strength of donor group in the order NO2 < H < MeO. The fluorescence
emission of compound 6c is quite significant versus 6a-b due to the presence of a strong
donating group like MeO that favors an higher intramolecular charge-transfer (ICT).19 The ICT
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in 6c is also responsible of higher Stokes shift and a bathochromic effect of fluorescence
spectrums with the increase of solvent polarity (Figure S3). The compound 6c showed the
highest relative quantum yields compared to 6b, meanwhile 6a have not fluorescence emission
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(Table S1). The relative quantum yields (φ ) of 6c in DMSO, DMF, EtOH, acetonitrile, DCM,
F
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ethyl acetate, and toluene are 0.038, 0.031, 0.014, 0.026, 0.009, 0.118, and 0.022 respectively.
Due to the efficient emission fluorescence and large Stokes shift of 6c in acetonitrile (Figure
1), this solvent was selected in further studies of compound 6c as a “turn-off” fluorescent
chemodosimeter for anions due to the presence of the dicyanovinyl group as a Michael acceptor,
where the addition of nucleophile can interrupt the ICT in this compound leading the losing of
fluorescence emission.
Figure 1. Normalized UV−visible absorption (black line) and fluorescence (red line) spectrum
of 6c in acetonitrile solvent (10 µM). The sample was excited at 360 nm.
Fluorescence and UV-vis Response of 6c to Cyanide (CN-). Fluorescence spectra of 6c (50
µM) were taken with an excitation wavelength (λexc) of 360 nm in acetonitrile to observe the
response of fluorescence emission in presence of CN- and other anions (F-, Br-, I-, SCN-, NO3 ,
-
-
-
-
2-
-
-
NO2 , N3 , OAc-, ClO3 , SO4 , HSO4 , H2PO4 , PO43-, and S2-) dissolved in distillated water
(Scheme 4 and Figure 2). A substantial diminution of the fluorescence intensity up to 6-fold
was observed in presence of one equivalent of CN- without shifting of the emission band (λem
= 550 nm). In contrast, a small diminution of fluorescence intensity up to less than 1-fold was
observed when two equivalents of the other anions were used suggesting that the
chemodosimeter 6c is highly selective towards CN-. When the UV-vis spectra of 6c was taken
in presence of one equivalent of CN-, the disappearance of the CT band at 360 nm is almost
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completed due to the nucleophilic β-addition of CN- on the C=C bond of the dicyanovinyl group
of 6c. This addition leads an interruption of the D-π-A system, having a direct influence in the
diminution of fluorescence emission (Figure 3).
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Scheme 4. Fluorescence response of 6c in presence of different anions.a
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a
Photograph was taken using 10 μM solutions in acetonitrile-water (1%) and 1.0 equiv of the
respective anion. A hand-held UV lamp under long wavelength (λ = 365 nm) was used.
Figure 2. (a) Fluorescence spectra of 6c (50 μM) in acetonitrile-water (1%) in the presence of
1.0 equiv of CN- or 2.0 equiv of other anions λexc =360 nm. (b) Fluorescence intensity response
of 6c to CN- and other anions.The emission intensity was measured at λem = 550 nm.
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Figure 3. UV-visible absorption spectra of 6c (50 µM) in acetonitrile-water (1%) in the
presence of 1.0 equiv of CN- or 2.0 equiv of other anions.
On the other hand, UV-vis and fluorescence spectra of 6c upon addition of one equivalent of
CN- and two equivalents of other anions were measured with the aim to study in more detail
the selectivity of 3c towards CN-. In these competition experiments, it was found that the S2-
anion did not affect the reactivity of CN- and the other anions had a relatively low interference
-
-
3-
with exception of HSO4 , H2PO4 , and PO4 on the selectivity for CN- (see Supporting
Information, Figures S4-S5).
In order to understand the response of 6c towards CN- anion in presence of water, the UV-vis
absorption and fluorescence spectra of 6c were taken by increasing percentage of water in the
water-acetonitrile mixtures (see Supporting Information, Figures S6-S7). The UV-vis spectrum
of 6c did not show a significative change until the use of 60 % of water. After the use of 70 %
of water the bands at 290 and 360 nm were significantly reduced due to the low solubility of 6c
in water favoring the formation of non-soluble aggregates (Figure S6a). In addition, the
fluorescence emission at 550 nm in the spectrum of 6c showed a gradual diminution of
fluorescence intensity with the increment of percentage of water until 60 %. Interestingly, in
the range of 70-95 % of water the emission fluorescence spectra were blue shifted to 490 nm
with a high emission of fluorescence in comparison with the fluorescence emission of 6c in
pure acetonitrile (Figure S7a). The emission band at 490 nm of 6c by using 70-95% of water is
quite close the band emission of 6c in solid state (500 nm). This hypsochromic shift and
enhancement of fluorescence emission of 6c in water-acetonitrile solutions between 75-95%
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can be explained by aggregation induced emission (AIE) phenomena which is common in
dicyanovinyl derivatives.26
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Furthermore, the UV-vis absorption and fluorescence emission spectra of 6c with 2 equivalents
of CN- anion at different percentages of water-acetonitrile mixtures were taken (Figures S6b
and S7b), where the reactivity of CN- had not been affected up to of 10 % of water. Between 20
and 60 % of water, for the bands at 360 and 550 nm of compound 6c in the UV-vis and
fluorescence spectra respectively, it had not observed a significant decrease of those bands upon
addition of two equivalents of cyanide. The low selectivity of CN- is due to of solvation of
water that interferes in the reactivity of cyanide towards 6c. For acetonitrile-water solutions of
6c with 2 equivalents of cyanide between 70 and 95 % of water, the AIE phenomena was
principally observed in the fluorescence emission spectra. With these results, it was observed
that 10 % of water is the maximal amount of water in acetonitrile-water solutions of 6c for an
optimal detection of cyanide.
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Fluorescence and UV-vis spectra of 6c upon a regular increment of cyanide concentration
showed a gradual diminution of fluorescence intensity and absorption bands of 550 and 360 nm
respectively (Figures 4-5 and Table S1).
Figure 4. The absorption spectra of 6c (50 μM) in acetonitrile upon addition of increasing
concentrations of CN- (0-150 μM).
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Figure 5. Fluorescence intensity of 6c at 550 nm with different concentrations of CN-.
The plot of fluorescence intensity vs concentration of CN- showed a linear range from 50-105
µM (Figure 6). The detection limit (LOD) was calculated as 6.8 µM (R2 = 0.9921). This LOD
is lower than the level goal (MCLG) permitted in drinking water according to USA EPA (0.2
ppm).27
Figure 6. (a) Fluorescence spectra of 6c (50 μM) in acetonitrile upon addition of increasing
concentration of CN- (0.1–105 μM, λexc = 360 nm). (b) Fluorescence intensity of 6c at 550 nm
with different concentrations of CN-.
1H NMR titration Experiments and HRMS-ESI Analysis. To demonstrate the reaction
1
process of 6c with cyanide and the nature of binding site, H NMR titration experiment was
carried out (Figure 7). During the titration, the concentration of 6c was kept constant, and the
[CN-]/[6c] ratio was increased. Accordingly, upon addition of cyanide to the solution of 6c, the
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Ha signal of the dicyanovinyl group located downfield is shifted to upfield. The protons of the
pyridyl group (H1, H2, H3, and H4) are upfield shifted due to the interruption of conjugation
and consequent formation of the stabilized carbanion. Additionally, the electrospray ionization
mass spectrum (HRMS-ESI) was obtained for the in situ intermediate 6c-CN formed from the
compound 6c and NaCN in acetonitrile, where the peak at m/z 353.1154 is assigned to the
negative charge specie [6c-CN]- (Figure 8).
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CH3
5
o
m
3'-4'
Ha
6'
5'
e)
d)
c)
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o
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ppm
ppm
1
Figure 7. H NMR spectra for titration experiment of 6c using a) 0.0, b) 0.4, c) 0.8, d) 1.2, and
e) 1.6 equiv of NaCN in [D6]DMSO at room temperature.
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Figure 8. HRMS (ESI) spectrum for the addition adduct of the reaction between 6c and CN-
in acetonitrile (left) and calculated molecular ion [M-] of the intermediate 1:1 6c-CN (right)
Computational calculation. To have a better understanding about the electronic structures of
compounds 6a-c, TD-DFT calculations were performed at the B3LYP/6-311(d,p) level (Figure
9. Also see Supporting Information, Figures S8-S12 and Table S2).28 Molecular orbital surfaces
of the HOMO and LUMO for the compounds 6a-c and 6c-CN are visualized in the Figure 9. In
the case of 6a, the HOMO is located on the 1-(2-pyridyl)-4-dicyanovinylpyrazole system and
the LUMO on the p-nitrophenyl group. For the contrary, the orbital HOMO in 6b is distributed
on all the molecule and the LUMO on the dicyanovinil group. For the compound 6c the HOMO
is mostly located on the p-metoxyphenyl group and the LUMO on the dicyanovinyl moiety.
The shape of the two frontier orbitals for 6c indicates a higher charge transfer (CT) from the
electron-donor to the electron-acceptor group in comparison with 6a-b. The calculated HOMO-
LUMO energy gaps for 6a-c are 3.04, 3.12, and 2.78 eV respectively (see Supporting
Information, Figure S9). From these results, the transference of electrons from HOMO to the
LUMO is relatively easier in 6c than in 6a-b. Thus, in comparison with 6b a bathochromic shift
of 6c is observed in the electronic absorption spectra (Table S2). These results are consistent
with the experimental results.
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LUMO
HOMO
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6a
6b
6c
6c-CN
Figure 9. Frontier molecular orbitals of 6a-c and the adduct 6c-CN in the ground state
For the adduct 6c-CN the distribution of the HOMO and LUMO is different in comparison with
6c (Figure 9). Due to the addition of the CN- on the dicyanovinyl group, the HOMO of 6c-CN
is confined in that fragment. Thus, the CT from the electron-donor to the electron-acceptor
group is interrupted in 6c-CN, causing a diminution of fluorescence emission. The higher
HOMO-LUMO energy gap of 6c-CN (3.25 eV) compared with 6c and the absence of the
absorption band around 360 nm in the electronic absorption spectra, confirms that, the CT
process through donor-π-acceptor system in this compound is disrupted.
Paper Test of 6c for Detection of Cyanide (CN-). The paper test of the compound 6c were
immersed in aqueous solutions (10 mM) of different anions (I-, Br-, F-, ClO3 , NO3 , NO2 ,
-
-
-
-
2-
-
-
-
HSO4 , SO4 , CN-, SCN-, CH3CO2 , H2PO4 , and N3 , where the fluorescence quench was
observed selectively in presence of CN- (Figure 10a). To evaluate the minimal amount of CN-
that the paper strips can detect, different concentrations of CN- from 0-10 mM were used. This
result showed that the minimal concentration of CN- detectable is 10 mM (Figure 10b). The
visual effect of the fluorescent test paper can be used for the selective detection of CN- in diverse
water samples.
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I- ClO3 NO3 NO2 HSO4 SO4 SCN- AcO- H2PO4 N3
(a)
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off
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6c 6c
5 mM
10 mM
0 mM 0.75 mM 1 mM
7.5 mM
2.5 mM
(b)
Figure 10. (a) Selective detection of CN- using the fluorescent test paper of 6c. (b) Detection
of CN- at different concentration using the fluorescent test paper of 6c. Photographs were taken
using a hand-held UV lamp under long wavelength (λexc = 365 nm).
CONCLUSIONS
To sum up, we have synthesized a family of 3-aryl-4-(2,2-dicyanovinyl)-1-(pyridin-2-yl)-1H-
pyrazoles 6a-c in 58-66% overall yield. The products were obtained in three reaction step
(Condensation, Vilsmeier–Haack, Knoevenagel) starting from acetophenones 1, in high
isolated yields at each step. All synthesized compounds were adequately characterized by
spectroscopic and HRMS analysis, and the structure of the hydrazone 3a was confirmed by
single-crystal X-ray diffraction analysis. This synthetic methodology could be used to yield
other interesting compounds with photophysical and photochemical properties starting from the
valuable 4-formyl-1-(2-pyridyl)pyrazoles 4. Remarkably due to a better intramolecular charge
transfer (donor-π-acceptor system) in 6c, this compound showed a bathochromic shift in the
UV-vs absorption spectra, higher fluorescence emission in different solvents, and high Stokes
shifts in comparison with its analogs 6a-b. Therefore, the compound 6c was studied as a
“TURN-OFF” chemodosimeter of CN-, the results showed a selective detection towards CN- in
comparison with other anions set with a detection limit of 6.8 µM. HRMS analysis and
spectroscopic studies like absorption, fluorescence, and NMR spectra revealed the formation of
1
adduct 6c-CN via Michael addition of CN- on the C=C bond of the probe, even H NMR
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titration experiments were successfully performed. TD-DFT calculations showed the higher
charge transfer in the compound 6c compared with 6a-b, likewise, an interruption of this ICT
in the intermediate 6c-CN. Essay of fluorescent test paper showed visual selective detection of
CN- anion in aqueous solution (10 mM) that can be applied in the analysis of contaminated
water. As a final point, this protocol to design a fluorescent probe with structure analogous to
6c has not been previously reported. Thus, we expect to extend its use regarding the design of
novel fluorimetric and ratiometric chemosensors for anions or even metal ion detection, based
in others Michael acceptors like pyrazoles with benzothiazolium salt moiety or in N-(2-
pyridyl)pyrazole moiety as N,N-type ligand, respectively.
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EXPERIMENTAL SECTION
General Information. All reagents were purchased from commercial sources and used without
further purification, unless otherwise noted. All starting materials were weighed and handled in
air at room temperature. The reactions were monitored by TLC and visualized using an UV
lamp (254 or 365 nm) and/or with p-anisaldehyde and H2SO4 in EtOH. Flash chromatography
was performed on silica gel (230-400 mesh). NMR spectra were recorded at 400 MHz (1H) and
100 MHz (13C) at 298 K using tetramethylsilane (0 ppm) as the internal reference. NMR
spectroscopic data were recorded in CDCl3 or [D6]DMSO using the residual non-deuteriated
1
signal as the internal standards for H NMR and the deuteriated solvent signal as the internal
13
standard for C NMR spectroscopy. DEPT spectra were used to assign the carbon signals.
Chemical shifts () are given in ppm and coupling constants (J) are given in Hz. The following
abbreviations are used for multiplicities: s = singlet, d = doublet, t = triplet, and m = multiplet.
Melting points were determined using a capillary melting point apparatus and are uncorrected.
IR spectra were recorded on a FT-IR spectrophotometer using KBr discs. Spectra are reported
in frequency of absorption in cm-1, and only selected resonances are reported. High-resolution
mass spectra (HRMS) were recorded using a Q-TOF spectrometer via electrospray ionization
(ESI). Crystallographic data were recorded on a diffractometer using graphite-monochromated
Mo K radiation ( = 0.71073 Å). Structures were solved using an iterative algorithm29a and
subsequently completed by a difference Fourier map and refined using the program
SHELXL2014.29b The electronic absorption and fluorescence emission spectra were recorded
in quartz cuvettes having a path length of 1 cm. UV-vis and fluorescence measurements were
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performed at room temperature (20 ºC). For fluorescence measurements, both the excitation
and emission slit widths were 5 nm. Hydrazinylpyridine 2 was prepared using a known
procedure.21e
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Synthesis and Characterization
General Procedure for the Synthesis of 2-(2-(1-Arylethylidene)hydrazinyl)pyridines 3a-
c.23 To a solution of freshly synthesized 2-hydrazinepyridyne (2)21e (656 mg, 6.0 mmol) in 40
mL of ethanol was added the appropriate acetophenone 1a-c (6.0 mmol) in 20 mL of ethanol at
room temperature. The reaction mixture was stirred at reflux during 2 h. Then the reaction was
allowed to cool to room temperature and the solvent was evaporated under reduced pressure.
The solid residue was purified by recrystallizetion from ethanol/water to afford the desired
hydrazone derivatives 3a-c as colored solids in good yields.
2-(2-(1-(4-Nitrophenylethylidene)hydrazinyl)pyridine 3a. Following the general procedure, for
the reaction with 4-nitroacetophenone (1a, 992 mg, 6.0 mmol), the hydrazone 3a was obtained
as orange crystals suitable for X-ray diffraction analysis (1416 mg, 92%). Mp 159-160 °C.
FTIR: ν = 3421, 3202, 3004, 1584, 1513, 1439, 1323, 1274 cm-1. 1H NMR (CDCl3, 400 MHz):
 = 2.29 (s, 3H), 6.86 (t, J = 6.0 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.67 (t, J = 7.2 Hz, 1H), 7.92
(d, J = 9.0 Hz, 2H), 8.16 (d, J = 4.1 Hz, 1H), 8.21 (d, J = 9.0 Hz, 2H), 8.46 (s, 1H) ppm. 13C{1H}
NMR (CDCl3, 100 MHz):  = 2.0 (CH3), 108.0 (CH), 116.9 (CH), 123.7 (CH), 126.0 (CH),
138.5 (CH), 140.3 (C), 144.6 (C), 147.3 (CH), 156.3 (C) ppm. HRMS (ESI+): calcd. for
+
C13H13N4O2 257.1039 [M + H]+; found 257.1040.
2-(2-(1-(Phenylethylidene)hydrazinyl)pyridine 3b.23 Following the general procedure, for the
reaction with acetophenone (1b, 720 mg, 6.0 mmol), the hydrazone 3b was obtained as yellow
crystals (1016 mg, 80%). Mp 139-140 °C (Lit. 138-139 °C).23 FTIR: ν = 3446, 3204, 3003,
2832, 1595, 1580, 1440, 1244 cm-1. 1H NMR (CDCl3, 400 MHz):  = 2.77 (s, 3H), 6.78 (t, J =
6.0 Hz, 1H), 7.31-7.44 (m, 4H), 7.62 (t, J = 7.3 Hz, 1H), 7.80 (d, J = 7.6 Hz, 2H), 8.14 (d, J =
4.7 Hz, 1H), 8.30 (s, 1H) ppm. 13C{1H} NMR (CDCl3, 100 MHz):  = 12.3 (CH3), 107.8 (CH),
115.8 (CH), 125.7 (CH), 128.31 (CH), 138.2 (CH), 138.8 (C), 143.2 (C), 147.2 (CH), 157.1 (C)
ppm. HRMS (ESI+) calcd. for C13H14N3 212.1188 [M + H]+; found 212.1191.
+
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2-(2-(1-(4-Methoxyphenylethylidene)hydrazinyl)pyridine 3c.23 Following the general
procedure, for the reaction with 4-methoxyacetophenone (1c, 900 mg, 6.0 mmol), the
hydrazone 3c was obtained as yellow crystals (1100 mg, 76%). Mp 110-111 °C (Lit. 100-
103 °C).23 FTIR: ν = 3422, 3178, 3011, 2941, 1500, 1439, 1288, 1138 cm-1. 1H NMR (CDCl3,
400 MHz):  = 2.24 (s, 3H), 3.84 (s, 3H), 6.76 (t, J = 6.0 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 7.39
(d, J = 8.5 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 8.06 (s, 1H), 8.12 (d, J =
4.4 Hz, 1H) ppm. 13C{1H} NMR (CDCl3, 100 MHz):  = 12.2 (CH3), 55.3 (CH3), 107.6 (CH),
113.7 (CH), 115.6 (CH), 127.0 (CH), 131.5 (C), 138.2 (CH), 143.1 (C), 147.3 (CH), 157.2 (C),
159.9 (CH) ppm. HRMS (ESI+) calcd. for C14H16N3O+: 242.1293 [M + H]+; found 242.1288.
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General Procedure for the Synthesis of 3-Aryl-1-(pyridin-2-yl)-1H-pyrazole-4-
carbaldehydes 4a-c. To a flask containing dry N,N-dimethylformamide (0.58 mL, 7.5 mmol)
cooled to 0ºC was added phosphoryl chloride (1.4 mL, 15.0 mmol) for about 1 h maintaining
the temperature. The corresponding hydrazone 3a-c (2.5 mmol) was added, and the mixture
was stirred at 80 °C for 3 h. After the completion of the reaction, the mixture was cooled to
room temperature, poured into ice-chilled water, and neutralized with 10% sodium bicarbonate
solution. The deposited solid was filtered off, washed with cold water and dried under high
vacuum to afford the crude product in good yield. The solid residue was purified by flash
chromatography on silica gel (eluent: CH2Cl2) to afford the pure product 4a-c.
3-(4-Nitrophenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde 4a. Following the general
procedure, for the reaction with 2-(2-(1-(4-nitrophenylethylidene)hydrazinyl)pyridine (3a, 640
mg, 2.5 mmol), the 4-formylpyrazole 4a was obtained as a white solid (590 mg, 80%). Mp 209-
210 °C. FTIR: ν = 3447, 3153, 3104, 2851, 1687, 1602, 1560, 1456, 1342 cm-1. 1H NMR
(CDCl3, 400 MHz):  = 7.36 (t, J = 6.9 Hz, 1H), 7.93 (t, J = 6.9 Hz, 1H), 8.13 (d, J = 7.6 Hz,
1H), 8.25 (d, J = 8.6 Hz, 2H), 8.34 (d, J = 8.6 Hz, 2H), 8.50 (d, J = 4.4 Hz, 1H) ppm. 13C{1H}
NMR (CDCl3, 100 MHz):  = 113.0 (CH), 122.9 (C), 123.2 (CH), 123.7 (CH), 129.7 (CH),
135.0 (CH), 137.7 (C), 139.2 (CH), 148.2 (C), 148.5 (CH), 150.1 (C), 151.3 (C), 183.5 (CH)
+
ppm. HRMS (ESI+): calcd. for C15H11N4O3 295.0831 [M + H]+; found 295.0827.
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3-(Phenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde 4b.24d Following the general
procedure, for the reaction with 2-(2-(1-(phenylethylidene)hydrazinyl)pyridine (3b, 528 mg,
2.5 mmol), the 4-formylpyrazole 4b was obtained as a yellow solid (530 mg, 85%). Mp 103-
105 °C. FTIR: ν = 3131, 3054, 2848, 2735, 1689, 1596, 1459, 1359, 1192 cm-1. 1H NMR
(CDCl3, 400 MHz):  = 7.29 (t, J = 5.9 Hz, 1H), 7.45-7.53 (m, 3H), 7.86-7.90 (m, 3H), 8.12 (d,
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J = 8.4 Hz, 1H), 8.49 (d, J = 4.1 Hz, 1H) ppm. C{1H} NMR (CDCl3, 100 MHz):  = 113.1
(CH), 122.5 (C), 122.8 (CH), 128.6 (CH), 128.9 (CH), 129.4 (CH), 131.3 (C), 132.4 (CH),
139.0 (CH), 148.4 (CH), 150.4 (C), 154.7 (C), 184.9 (CH) ppm. HRMS (ESI+): calcd. for
C15H12N3O+ 250.0980 [M + H]+; found 250.0974.
3-(4-Methoxyphenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde 4c. Following the general
procedure, for the reaction with 2-(2-(1-(4-methoxyphenylethylidene)hydrazinyl)-pyridine (3c,
640 mg, 2.5 mmol), the 4-formylpyrazole 4c was obtained as a white solid (580 mg, 83%). Mp
102-103 °C. FTIR: ν = 3146, 2967, 2836, 2776, 1675, 1598, 1517, 1474, 1252 cm-1. 1H NMR
(CDCl3, 400 MHz):  = 3.87 (CH3), 7.02 (d, J = 8.6 Hz, 2H), 7.28 (t, J = 6.9 Hz, 1H), 7.85-7.88
(m, 3H), 8.10 (d, J = 7.6 Hz, 1H), 8.46 (d, J = 4.4 Hz, 1H) ppm. 13C{1H} NMR (CDCl3, 100
MHz):  = 55.3 (CH3), 113.1 (CH), 114.1 (CH), 122.3 (C), 122.6 (CH), 123.9 (C), 130.2 (CH),
132.8 (CH), 139.0 (CH), 148.3 (CH), 150.4 (C), 154.3 (C), 160.6 (C), 184.8 (CH) ppm. HRMS
(ESI+): calcd. for C16H14N3O2 280.1086 [M + H]+; found 280.1091.
+
General Procedure for the Synthesis of 3-aryl-4-(2,2-dicyanovinyl)-1-(pyridin-2-yl)-1H-
pyrazoles 6a-c. To a flask containing an equimolar mixture of 1-(pyridin-2-yl)-1H-pyrazole-4-
carbaldehyde 4a-c and malonitrile (5, 33 mg, 0.50 mmol) in 5 mL of ethanol was added
pyperidine (~10 mol %) as catalyst. The mixture was stirred at reflux for 2 h. Then this was
allowed to cool to room temperature and the deposited solid was filtered off, washed with cold
ethanol and dried under high vacuum to afford products 6a-c in high yields.
4-(2,2-Dicyanovinyl)-3-(4-nirophenyl)-1-(pyridin-2-yl)-1H-pyrazole 6a. Following the general
procedure, for the reaction with 3-(4-nitrophenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-
carbaldehyde (4a, 147 mg, 0.5 mmol), the 4-dicyanovinylpyrazole 6a was obtained as a white
solid (154 mg, 90%). Mp 253-255 °C. FTIR: ν = 3447, 3150, 2231, 1587, 1526, 1463, 1353,
1221 cm-1. 1H NMR ([D6] DMSO, 400 MHz):  = 7.53 (m, 1H), 8.00 (d, J = 8.2 Hz, 2H), 8.05-
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8.13 (m, 2H), 8.32 (s, 1H), 8.37 (d, J = 8.2 Hz, 2H), 8.60 (d, J = 4.4 Hz, 1H) ppm. C{1H}
NMR ([D6] DMSO, 100 MHz):  = 80.2 (C), 113.0 (CH), 122.9 (C), 123.2 (CH), 123.7 (CH),
129.7 (CH), 135.0 (CH), 137.7 (C), 139.0 (CH), 113.8 (C), 113.9 (C), 115.0 (C), 124.0 (CH),
124.1 (CH), 129.2 (CH), 130.3 (CH), 136.3 (C), 140.2 (CH), 147.9 (C), 148.9 (C), 149.1 (CH),
151.7 (CH), 153.0 (CH) ppm. HRMS (ESI+): calcd. for C18H11N6O2 343.0943 [M + H]+; found
+
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343.0940.
4-(2,2-Dicyanovinyl)-3-(phenyl)-1-(pyridin-2-yl)-1H-pyrazole 6b. Following the general
procedure, for the reaction with 3-(phenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde (4b,
125 mg, 0.5 mmol), the 4-dicyanovinylpyrazole 6b was obtained as a white solid (132 mg,
89%). Mp 227-228 °C. FTIR: ν = 3447, 3153, 3031, 2228, 1583, 1525, 1463, 1222 cm-1. 1H
NMR (CDCl3, 400 MHz):  = 7.34 (t, J = 7.1 Hz, 1H), 7.54-7.58 (m, 5H), 7.78 (s, 1H), 7.90 (t,
J = 8.0 Hz, 1H), 8.(d, J = 8.2 Hz, 1H), 8.52 (d, J = 4.8 Hz, 1H) ppm. 13C{1H} NMR (CDCl3,
100 MHz):  = 79.9 (C), 113.2 (CH), 113.4 (C), 114.0 (C), 114.9 (C), 123.3 (CH), 129.1 (CH),
129.2 (CH), 129.5 (CH), 129.9 (CH), 130.2 (C), 139.0 (CH), 148.7 (CH), 150.0 (C), 150.9
(CH), 156.5 (C) ppm. HRMS (ESI+): calcd. for C18H12N5 298.1093 [M + H]+; found 298.1092.
+
4-(2,2-Dicyanovinyl)-3-(4-methoxyophenyl)-1-(pyridin-2-yl)-1H-pyrazole 6c. Following the
general procedure, for the reaction with 3-(4-methoxyphenyl)-1-(pyridin-2-yl)-1H-pyrazole-4-
carbaldehyde (4c, 140 mg, 0.5 mmol), the 4-dicyanovinylpyrazole 6c was obtained as a yellow
solid (151 mg, 92%). Mp 217-219 °C. FTIR: ν = 3447, 3156, 2226, 1597, 1510, 1463, 1263,
1224 cm-1. 1H NMR (CDCl3, 400 MHz):  = 3.89 (CH3), 7.06 (d, J = 8.7 Hz, 2H), 7.32 (t, J =
7.1 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.75 (s, 1H), 7.87 (t, J = 6.7 Hz, 1H), 8.08 (d, J = 8.2 Hz,
1H), 8.50 (d, J = 4.0 Hz, 1H) ppm. 13C{1H} NMR (CDCl3, 100 MHz):  = 55.5 (CH3), 79. (C),
113.2 (CH), 113.4 (C), 114.1 (C), 114.7 (CH), 114.8 (C), 122.5 (C), 123.2 (CH), 129.5 (CH),
130.4 (CH), 139.0 (CH), 148.7 (CH), 150.0 (C), 151.2 (CH), 156.3 (C), 161.0 (C) ppm. HRMS
(ESI+): calcd. for C19H14N5O+ 328.1198 [M + H]+; found 328.1199.
Procedure for obtaining Sodium 1,1,2-tricyano-2-(3-(4-methoxyphenyl)-1-(pyridin-2-yl)-
1H-pyrazol-4-yl)ethan-1-ide 6c-CN. To a NMR tube containing 4-(2,2-dicyanovinyl)-3-(4-
methoxyophenyl)-1-(pyridin-2-yl)-1H-pyrazole (6a, 10 mg, 0.031 mmol) in 0.7 mL of [D6]
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DMSO was added NaCN (0.050 mmol, 1.6 equiv) to afford the adduct 6c-CN in 100%
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conversion. H NMR ([D6] DMSO, 400 MHz):  = 3.82 (CH3), 4.58 (s, 1H), 7.04 (d, J = 8.8
Hz, 2H), 7.37 (t, J = 6.3 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.97 (d, J = 8.0, 1H), 8.01 (t, J = 6.7
Hz, 1H), 8.50 (d, J = 4.3 Hz, 1H), 8.57 (s, 1H) ppm. 13C{1H} NMR ([D6] DMSO, 100 MHz):
 = 16.9 (C-Na+), 28.1 (CH), 55.3 (CH3), 111.6 (CH), 114.1 (CH), 118.4 (C), 121.8 (C, CN),
122.1 (CH), 124.5 (C), 126.9 (CH), 128.3 (C, 2CN), 129.2 (CH), 139.8 (CH), 148.5 (CH), 150.3
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-
(C), 150.5 (C), 159.6 (C), 164.8 (C, NaCN) ppm. HRMS (ESI+): calcd. for C20H13N6O2
353.1156 [M - Na]-; found 353.1154.
Chemosensor Design
UV-vis absorption and fluorescence studies. The solvochromic studies of the compounds 6a-c
were carried out with 0.01 mM stock solutions in dimethylsulfoxide, dimethylformamide,
ethanol, acetonitrile, dichloromethane, ethyl acetate, and toluene. The 0.08 mM stock solutions
of the chemodosimeter 6c were prepared in water-acetonitrile (99:1 v/v). The salts used in stock
solutions of anions were NaI, KBr, NaF, NaClO3, KNO3, NaNO2, NaHSO4, Na2SO4, NaCN,
NaSCN, NaCH3CO2.3H2O, NaH2PO4, Na3PO4, NaN3, and NaS2. Inorganic salts were dissolved
in distilled water to afford 1 mM aqueous solution. Aliquots of stock solution of 6c was diluted
to 5 mL to make the final concentration of 50 μM. In the selectivity and competition
experiments of 6c towards CN- and other anions, the fluorescence emission spectra were
recorded at λexc = 360 nm from 50 μM of the chemodosimeter in an acetonitrile solution upon
addition of one equivalent of various metal ions. The fluorescence intensities were measured at
λem = 550 nm. Fluorescence response in photographs was excitation at 365 nm using a UV lamp.
Determination of the relative quantum yields. The relative quantum yields were obtained by
using anthracene (φF = 0.28 in ethanol at 340 nm) as reference and calculated according to the
following equation.30
푠푡
퐹
1 − 10−퐴 휂푥2
푥
휑푓,푥 = 휑푓,푠푡
∙
∙
∙
휂푠2푡
푥
퐹
푠푡
1 − 10−퐴
where x and st indicate the sample and standard solution, respectively, φ is the quantum yield,
F is the integrated area of the emission, A is the absorbance at the excitation wavelength, and η
is the index of refraction of the solvents.
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Determination of the detection limit. The limit of detection (LOD) of 6c for CN- was obtained
by 3Sb/k, where Sb is the standard deviation of the blank measurements (by 10 times), and k is
the slope from the plot fluorescence intensity I versus [CN-].30e,31
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DFT calculations Theoretical calculations were obtained using DFT performed using Gaussian
09.28 The DFT calculations employed the B3LYP hybrid functional and the 6-311G+(d,p) basis
set. All geometries were optimized in the ground state without solvent effects. Time-dependent
(TD-DFT) calculations were performed on optimized geometries. The visual platform used in
this work was Avogadro 1.2.0 to analyze the output files performed in the calculations.32
Preparation of test paper
Filter paper was immersed in an acetonitrile solution (1 mM) of compound 6c, and dried in an
oven at 30 ºC for 4 h. The papers were tested in aqueous solutions (10 mM) of different anions
(NaI, KBr, NaF, NaClO3, KNO3, NaNO2, NaHSO4, Na2SO4, NaCN, NaSCN,
NaCH3CO2.3H2O, NaH2PO4, and NaN3 and irradiated with a UV lamp at 365 nm. Additionally,
the test papers were studied in aqueous solutions of NaCN varying the concentration from 0-10
mM.
AUTOR INFORMATION
Corresponding Author
E-mail: jportill@uniandes.edu.co
ORCID
Jaime Portilla: 0000-0002-8206-7481 786
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
The authors are grateful for the financial support from Universidad de los Andes and the
Colombian Institute for Science and Research (COLCIENCIAS, project code: 120465843502),
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and the High Performance Computing (HPC) services of Vicerrectoria de Investigaciones of
Universidad de los Andes. We also acknowledge Edwin Guevara (Universidad de los Andes)
for acquiring the mass spectra and Dr. Eduardo C. Escudero-Adan (X-ray Diffraction Unit of
Institute of Chemical Research of Catalonia (ICIQ) in Tarragona-Spain) for his help with the
analysis of the X-ray crystallographic data.
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ASSOCIATED CONTENT
Supporting Information
CIF for compound 3a and copies of H and 13C{1H} NMR spectra for all compounds,
1
spectroscopic properties and computational calculation of 6a-c and of 6c-CN. The Supporting
Information is available free of charge on the ACS Publications website at DOI:
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