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J Fluoresc
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