A Simple Isoniazid-Based N-Acylhydrazone Derivative as Potential Fluorogenic Probe for Zn2+ Ions

Article abstract of DOI:10.1007/s10895-020-02651-7

This study evaluated three isoniazid-based N-acylhydrazone derivatives (HL1, HL2, and HL3) varying their substituting groups (-H, -N(CH₃)₂, and -NO₂) as potential chemosensors for Zn²⁺ ions. To this end, the absorption and emission properties of these derivatives were investigated in the presence of Zn²⁺ ions. Results point to the derivative HL2 as the best chemosensor for Zn²⁺ ions because of its comparatively higher sensitivity. The color of this derivative changed from colorless to strong yellow with zinc addition, as indicated by the shift in UV-vis spectrum. Moreover, HL2 was the only derivative to emit fluorescence in the presence of Zn²⁺ ions, attributable to PET inhibition and bond isomerization promoted by coordination with this metal. LOD, LOQ, and binding constant values for HL2 + Zn²⁺ were 0.43?μmol.l^?1, 0.93?μmol.l^?1, and 5.04 × 10¹²?l.mol^?1, respectively. The fluorescence of HL2 with other metal ions (Fe³⁺, Mg²⁺, Na⁺, Cd²⁺, Cu²⁺, Co²⁺, Ni²⁺, Ca²⁺, and K⁺) was also investigated. Zn²⁺ yielded the best result without Cd²⁺ interferences. Job’s Plot showed that the stoichiometric ratio of the complex formed by HL2 and Zn²⁺ ions is 2:1 (ligand:metal). The strip test with adsorbed HL2 indicated fluorescence in the presence of zinc ions under 365?nm UV irradiation.

Full text of DOI:10.1007/s10895-020-02651-7

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Journal of Fluorescence
https://doi.org/10.1007/s10895-020-02651-7
ORIGINAL ARTICLE
A Simple Isoniazid-Based N-Acylhydrazone Derivative as Potential
Fluorogenic Probe for Zn2+ Ions
Daniela Corrêa Santos1,2 & Paulo José Sousa Maia1 & Marcos Antonio de Abreu Lopes Jr.1,3
Josué Sebastián Bello Forero3 & Andréa Luzia Ferreira de Souza1,3
&
Received: 14 July 2020 /Accepted: 9 November 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
This study evaluated three isoniazid-based N-acylhydrazone derivatives (HL1, HL2, and HL3) varying their substituting groups
(-H, -N(CH3)2, and -NO2) as potential chemosensors for Zn2+ ions. To this end, the absorption and emission properties of these
derivatives were investigated in the presence of Zn2+ ions. Results point to the derivative HL2 as the best chemosensor for Zn2+
ions because of its comparatively higher sensitivity. The color of this derivative changed from colorless to strong yellow with zinc
addition, as indicated by the shift in UV-vis spectrum. Moreover, HL2 was the only derivative to emit fluorescence in the
presence of Zn2+ ions, attributable to PET inhibition and bond isomerization promoted by coordination with this metal. LOD,
LOQ, and binding constant values for HL2 + Zn2+ were 0.43 μmol.l−1, 0.93 μmol.l−1, and 5.04 × 1012 l.mol−1, respectively. The
fluorescence of HL2 with other metal ions (Fe3+, Mg2+, Na+, Cd2+, Cu2+, Co2+, Ni2+, Ca2+, and K+) was also investigated. Zn2+
yielded the best result without Cd2+ interferences. Job’s Plot showed that the stoichiometric ratio of the complex formed by HL2
and Zn2+ ions is 2:1 (ligand:metal). The strip test with adsorbed HL2 indicated fluorescence in the presence of zinc ions under
365 nm UV irradiation.
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Keywords Zinc(II) Fluorescence N-acylhydrazone Isoniazid Chemosensor
Introduction
dysregulation of calcium homeostasis [3–5]. Furthermore,
zinc imbalance is directly related to diseases such as
Alzheimer, Parkinson, amyotrophic lateral sclerosis, prostate
cancer, diabetes, among others [6].
Zinc is the second most abundant transition metal in the hu-
man body; it is directly involved in many physiological and
pathological processes in living organisms [1]. Zn2+ ions are
key elements in structural components of numerous proteins
and act as catalysts in enzymes central to many cellular pro-
cesses [2]. Anomalous zinc levels can cause serious problems.
Zinc deficiency has been reported to cause chronic liver and
kidney diseases while its surplus can cause neurotoxicity and
For this reason, Zn2+ detection has attracted increasing in-
terest in the fields of chemistry and biology. Various methods
for detecting and quantifying zinc ions have been devised,
e.g., FAAS (Flame Atomic Absorption Spectrometry), elec-
trochemical methods, and ICP (Inductively Coupled Plasma
atomic emission spectrometry) [7]. However, these methods
require time-consuming pre-treatment of samples by experi-
enced professionals and costly equipment. In addition, detect-
ing zinc ions is not a trivial task as metals belonging to the
same group, which have similar chemical properties (e.g.,
cadmium), may interfere with its detection [8, 9].
* Andréa Luzia Ferreira de Souza
andrealuziasouza@yahoo.com.br
1
Universidade Federal do Rio de Janeiro, Campus Macaé Professor
Thus far, spectrofluorimetry has stood out as the most ef-
fective method for detecting Zn2+ ions in biological systems,
given that the d10 electronic configuration of this ion makes it
spectroscopically or magnetically silent [10]. In addition, the
highly sensitive, selective, and non-destructive nature of fluo-
rescence spectroscopy makes it a suitable technique for de-
tecting metal ions in small quantities.
Aloísio Teixeira, CEP, Macaé, RJ 27930-560, Brazil
2
Instituto de Macromoléculas, Universidade Federal do Rio de
Janeiro, Cidade Universitária, CEP, Rio de Janeiro, RJ 21941-598,
Brazil
3
Pós-Graduação em Química (PGQu), Instituto de Química,
Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de
Janeiro, RJ CEP21941-909, Brazil

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By means of fluorescence spectroscopy, metal ions can be
detected by their forming luminescent coordination com-
pounds [11]. This has considerably increased the importance
of N-acylhydrazone compounds in the field of coordination
chemistry because the nitrogen atom of the azomethine group
(C=N) and the carbonyl oxygen (C=O) can act as complexa-
tion sites for metal ions. These complexation sites improve
stability and enable the formation of a polydentate ligand [12].
This study aims at demonstrating the viability and effec-
tiveness of employing the fluorescence properties of
isoniazid-based N-acylhydrazones HL1, HL2, and HL3
(HL1–HL3) varying their substituting groups (-H, -
N(CH3)2, and -NO2) to detect zinc ions. To this end, the se-
lectivity and limit of detection (LOD) for Zn2+ ions in aceto-
nitrile (ACN) medium were investigated. This study also ex-
amined the behavior of the ligands in question in the presence
of Zn2+ and other metal ions by means of UV-vis and fluores-
cence techniques.
recrystallized in ethanol. The final products were character-
ized by melting point, quadrupole time-of-flight mass spec-
trometry (QTOF-EM), and 1H NMR (Fig. S1–S6).
N′-Benzylidene isonicotinohydrazide (HL1): white solid;
yield: 84%; mp: 189–190 °C; QTOF-EM found for
C
13H11N3O (M + Na+) m/z = 248.079433; 1H NMR
(400 MHz, DMSO-d6) δ: 12.02 (s, 1H, NH); 8.48 (s, 1H,
CH=N), 8.77 (d, 2H, J = 3.0 Hz, Ar-H), 7.82 (d, 2H,
J = 6.0 Hz, Ar-H), 7.73 (d, 2H, J = 3.0 Hz, Ar-H), 7.45–
7.16 (m, 3H, Ar-H).
N ′ - ( 4 - N , N - D i m e t h y l a m i n o b e n z y l i d e n e )
isonicotinohydrazide (HL2): yellow solid, yield: 81%. mp:
203–205 °C. QTOF-EM found for C14H16N4O (M + Na+)
m/z = 291.121632. 1H NMR (500 MHz, DMSO-d6) δ: 11.82
(s, 1H, NH), 8.78 (d, 2H, J = 5.0 Hz, Ar-H), 8.33 (s, 1H,
CH=N), 7.83 (d, 2H, J = 5.0 Hz, Ar-H), 7.58 (d, 2H,
J = 10.0 Hz, Ar-H), 6.78 (d, 2H, J = 10.0 Hz, Ar-H), 2.98
(s, 6H, N-CH3).
N′-(4-Nitrobenzylidene) isonicotinohydrazide (HL3): yel-
low solid; yield: 92%; mp: 265–267 °C. QTOF-EM found for
1
Experimental Section
C13H10N4O3 (M + Na+) m/z = 293.064511 (M + Na+), H
NMR (400 MHz, DMSO-d6) δ: 12.36 (s, 1H, NH), 8.82 (d,
2H, J = 6.0 Hz, Ar-H), 8.57 (s, 1H, CH=N), 8.33 (d, 2H,
J = 8.7 Hz, Ar-H), 8.04 (d, 2H, J = 8.7 Hz, Ar-H), 7.85 (d,
2H, J = 6.0 Hz, Ar-H).
The reagents isoniazid, benzaldehyde, 4-nitrobenzaldehyde,
and 4-(N,N-dimethylamino)benzaldehyde (Sigma-Aldrich®)
and glacial acid acetic (Vetec) were employed to synthesize
HL1–HL3. Chloride salts of all cations (Sigma-Aldrich®)
and HPLC-grade solvents (Tedia) were used without further
purification.
Measurement of Photophysical Properties of HL1–
HL3
The synthesis of N-acylhydrazone derivatives was carried
out in an Anton Paar Monowave 300. UV-vis absorption spec-
tra were recorded on a Shimadzu UV-2450 spectrophotometer
and steady-state fluorescence spectra were recorded on an
F9000 Edinburgh Instruments spectrofluorometer with a
450 W xenon arc lamp. TOF-EM analyses were performed
by means of a high-resolution mass spectrometer (Q-TOF
brand WATERS/MICROMASS model) with an 80–100 m/z
HL1–HL3 stock solutions were prepared in acetonitrile
(ACN). Aqueous solutions of the metal salts under investiga-
tion were prepared with 100 equiv. of respective ligand. UV-
vis spectra were obtained within the 200–500 nm range at
room temperature, with solutions of 0.8 μmol.l−1
,
0.6 μmol.l−1, and 1.6 μmol.l−1, for HL1–HL3, respectively.
Fluorescence assays were performed at room temperature,
using SUPRASIL quartz cuvettes with 10 mm pathlength
and transparent windows on all four sides. All the assays were
performed after mixing for 5 min to ensure uniformity.
Fluorescent spectra were recorded within the 400–680 nm
range. The fluorescence of compounds HL1–HL3
(0.8 μmol.l−1, 0.6 μmol.l−1 and 1.6 μmol.l−1, respectively)
was evaluated in the presence of 100 equiv. of Zn2+ ions
(λex = 290 nm, 356 nm, and 322 nm for HL1, HL2 and
HL3, respectively).
1
scanner. H NMR spectra were obtained on Bruker FT-
400 MHz and FT-500 MHz spectrometers (USA) with
DMSO-d6 as solvent at the Farmanguinhos-FIOCRUZ (RJ)
laboratory. Chemical shifts are reported in delta (δ) units rel-
ative to the singlet (0 ppm) of tetramethylsilane (TMS).
Melting points of the derivatives were determined on a
Fisatom melting-point apparatus.
Microwave Assisted-Synthesis of Isoniazid-Based N-
Acylhydrazones Derivatives (HL1–HL3)
As to titration, HL2 concentration was kept constant
(0.6 μmol.l−1), while Zn2+ concentration varied from 0 to
160 equiv. (λex = 356 nm, 400–680 nm). Limit of detection
(LOD) and limit of quantification (LOQ) were estimated with
the standard deviation of blank measurement. Selectivity as-
says were conducted at constant HL2 concentration
(0.6 μmol.l−1), 100 equiv. of the metal ions under investiga-
tion (Na+, Cd2+, Zn2+, Fe3+, Mg2+, Ca2+, and Cu2+), and
In a 30 mL vial, a mixture of isoniazid (3.0 mmol), aldehyde
(3.0 mmol), and glacial acetic acid (~5 drops) in ethanol
(15 mL) was stirred (1000 rpm) under microwave irradiation
for 45 min at 140 °C [13]. After the reaction mixture was left
to cool down to room temperature, the solid was filtered off
and washed in cold ethanol. Then, the crude product was

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Scheme 1 Synthetic route to
obtain N-acylhydrazones
derivatives HL1–HL3
ligand concentration equal to 0.6 μmol.l−1. The solutions were
prepared with ACN at 25 °C, λex = 356 nm, and within the
400–680 nm range.
and estimated from linear relationships between maximum
emission intensity at 579 nm and Zn2+ concentration.
Job’s Plot was performed in a front-face fluorometer with
concentrations of HL2 and Zn2+ ions varying from 0 to
20 μmol.l−1 in ACN, at 25 °C, λex = 356 nm, and 379–
700 nm scan.
Aggregation
Front-face fluorometry was also used for the aggregation as-
say, in which HL2 concentration varied from 5.0–
500 μmol.l−1 with concentration of Zn2+ ions at 100 equiv.
of HL2, scan within the 380–680 nm range, and λex at
360 nm.
Calculations of Binding Constants and LOD
The binding constant for HL2 was determined by means of
fluorescence titration and by plotting a graph of Fmax vs. [M]
μmol.l−1. Limit of detection (LOD) and limit of quantification
(LOQ) of HL2 for Zn2+ ions in ACN were measured with the
help of blank standard method (LOD = 3σ/m; LOQ = 10σ/m)
Strip Test
Strip tests were conducted in two steps. First, 3 × 1 cm filter
paper strips were immersed in an HL2-ACN solution (1 ×
10−5 mol.l−1). After drying, the strips were immersed in aque-
ous zinc chloride solution at different concentrations (from
1 × 10−3 to 1 × 10−2 mol.l−1). Fluorescence responses of the
strips were analyzed under 365 nm UV-irradiation.
Results and Discussion
The isoniazid-based N-acylhydrazone derivatives HL1–HL3
were synthesized by the Schiff Base condensation reaction as
described in our previous study (outlined in Scheme 1) [13].
Microwave-assisted reactions of isoniazid with respective al-
dehyde (benzaldehyde, 4-(N,N-dimethylamino)benzaldehyde,
and 4-nitrobenzaldehyde dissolved in ethanol), for 45 min, at
140 °C yielded white (HL1) and yellow (HL2 and HL3)
1
Fig. 1 UV–vis absorption spectra for HL1–HL3 (0.8 μmol.l−1
0.6 μmol.l−1 and 1.6 μmol.l−1, respectively) in ACN at 25 °C
,
solids. Then, these solids were recrystallized in ethanol. H
NMR results and quadrupole time-of-flight mass spectrometry

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Fig. 2 Qualitative colorimetric effect of HL1–HL3 with Zn2+ ions in ACN at 25 °C
(QTOF-EM) spectra of these recrystallized solids are shown
in the supporting information section below (Fig. S1–S6).
presented here. The ligands HL1 and HL3 did not change
color when in contact with Zn2+ ions in ACN (Fig. 2), either.
The behavior of N-acylhydrazone HL2 in the presence of
Cd2+, Cu2+, Fe3+ ions was also investigated and compared to
that in the presence of Zn2+ ions in ACN. In the assays with
metal ions, their presence frequently caused HL2 to exhibit
non-significant changes (Fig. S7). However, in the case of
Cd2+, Cu2+, Fe3+, and Zn2+ ions, a distinctive absorption shift
was observed, as shown in Fig. 3. UV–vis absorption spectra
of acylhydrazone HL2 in the presence of different metal ions
is shown in Fig. S8.
Colorimetric Sensing of HL1-HL3
The absorption spectra of free HL1–HL3 in dilute ACN so-
lutions (abs. ~ 0.2) is characterized by two main absorption
bands in the 200–500 nm region as shown in Fig. 1. The bands
at higher energy are ascribed to π → π* transitions (εHL1
=
3.29 × 105, εHL2 = 4.99 × 104, and εHL3 = 7.6 ×
104 L mol−1 cm−1) in the aromatic system of the compounds
while the low intensity bands at lower energy are attributed to
π → π* transitions (εHL1 = 1.07 × 105, εHL2 = 1.94 × 105, and
As to HL2 in the presence of Zn2+ ions, absorbance bands
at 259 nm and 356 nm shifted to 240 nm and 364 nm, respec-
tively. These changes show that adding Zn2+ enlarged the
conjugated system of the ligand, implying that the nitrogen
atom of the C=N group and the oxygen atom of the C=O
group of the ligand have successfully coordinated to the cen-
tral Zn2+ ion [14–16]. For HL2 in the presence of Cu2+ ions,
ε
HL3 = 6.2 × 104 L mol−1 cm−1) of C=N and C=O groups [14].
The addition of a solution of Zn2+ ions to HL2 leads to its
color changing from colorless to strong yellow (Fig. 2). Since
HL1 and HL3 showed no changes in absorption spectra in the
presence of Zn2+, only the absorption spectrum of HL2 will be
Fig. 3 UV–vis absorption spectra of acylhydrazone HL2 in the presence
of different metal ions (1 equiv.) in ACN at 25 °C
Fig. 4 UV-vis titration spectra for HL2 (0.6 μmol.l−1) from 0 to 160
equiv. of Zn2+ ions