Influence of regioisomerism on stability, formation kinetics and ascorbate oxidation preventive properties of Schiff bases derived from pyridinecarboxylic acids hydrazides and pyridoxal 5′-phosphate

Article abstract of DOI:10.1016/j.molliq.2017.07.106

The reaction between pyridoxal 5′-phosphate and hydrazides of 2-,3-, and 4-pyridinecarboxylic acids were studied in aqueous solution. The stability constants as well as the rate constants of formation of hydrazones derived from nicotinic and picolinic acids hydrazides were determined experimentally at pH values of 1.9; 6.6; 7.0; and 7.4. Isoniazid was found to form the least stable Schiff bases with PLP at every studied pH value. In the acidic medium, nicotinic acid derivative forms the most stable hydrazone. In the pH range of 6.6-7.4, the stability of PLP-NH and PLP-PH hydrazones vary in the opposite way. The rate of PLP-INH and PLP-PH Schiff bases formation decreases significantly in the pH range of 6.6-7.4, while the k1 value of PLP-NH remains nearly constant. The PLP-INH, PLP-NH, and PLP-PH were isolated as solids and characterized by means of NMR, IR-spectroscopy and fluorimetry. The PLPPH compound was found to fluoresce stronger than two other Schiff bases either in the solid phase and the solution. The PLP-INH hydrazone forms the 1:1 coordination compound with copper(II) ions, which stability is lg K ~ 5. Despite the stability of Cu(II)-PLP-INH complex lower than that of Cu(II)-EDTA, Cu(II)-CA complexes, sodium ethylenediaminetetraacetate and citric acid are less effective in preventing the copper-mediated oxidation of ascorbate.

Full text of DOI:10.1016/j.molliq.2017.07.106

Journal of Molecular Liquids 242 (2017) 1148–1155
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Influence of regioisomerism on stability, formation kinetics and
ascorbate oxidation preventive properties of Schiff bases derived from
☆
pyridinecarboxylic acids hydrazides and pyridoxal 5′-phosphate
b
Gamov G.A. a, , Kiselev A.N. , Aleksandriiskii V.V. a,b, Sharnin V.A. a
⁎
a
Research Institute of Thermodynamics and Kinetics of Chemical Processes, Ivanovo State University of Chemistry and Technology, 153000 Ivanovo, Sheremetevskii pr. 7, Russia
G.A. Krestov Institute of Solution Chemistry, Russian Academy of Sciences, 153045 Ivanovo, Akademicheskaya str. 1, Russia
b
a r t i c l e i n f o
Article history:
Received 12 May 2017
Received in revised form 3 July 2017
Accepted 25 July 2017
Available online xxxx
Keywords:
Pyridoxal 5′-phosphate
Pyridinecarboxylic acid hydrazide
Ascorbic acid
Stability
Hydrazone
Fluorescence
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
or its precursors' malabsorption from food and alcohol abuse [11], pyr-
idoxal 5′-phosphate interaction with drugs may lower significantly its
blood content.
An effective antitubercular drug isoniazid (isonicotinic acid hydra-
zide, INH) [12] is an example of agent decreasing PLP concentration in
blood [13]. INH binds either pyridoxal or pyridoxal 5′-phosphate into
stable Schiff base [14,15] thus acting as a toxin.
The mechanism of isoniazid bacteriostatic effect differs from its tox-
icity towards macroorganisms and relates to the free acyl radical forma-
tion under the action of KatG catalase-peroxidase enzyme of
mycobacteria [16]. This radical then reacts with NADH inactivating it.
Since the radical stability depends on the substituent localization in
the pyridine ring, the difference between biological activity of 2,4- and
3-isomers should be anticipated. Indeed, 4-isomer and 2-isomer
(picolinic acid hydrazide, PH) were found to provide the pronounced
antitubercular effect, while 3-isomer (nicotinic acid hydrazide, NH) is
inactive [17,18]. However, 6-aminonicotinic acid hydrazide possesses
antitubercular properties [18].
3-Hydroxy-2-methyl-5-[(phosphonoxy)methyl]-4-pyridinecarbo
xaldehyde (pyridoxal 5′-phosphate, PLP) is one of the most important
coenzymes in living organisms. PLP derivatives catalyze a variety of
metabolic processes including amino acids, lipids and carbohydrates
metabolism, and play the key role in the hormones, neurotransmitters
and heme biosynthesis [1,2]. The value of pyridoxal 5′-phosphate for
human beings is obvious from the fact that PLP low concentrations in
blood serum are associated with a number of pathologies such as sys-
temic inflammation [3], inflammatory bowel disease [4], rheumatoid ar-
thritis [5], epilepsy [6], periphery neuropathy [7], panic attacks [8],
depression [9], and anemia [10]. However, PLP level correction in the or-
ganism not always leads to the positive health response. Along with PLP
☆
Present contribution reports on thermodynamics and kinetics of the reactions
between pyridoxal 5′-phosphate and hydrazides of 2-, 3-, and 4-pyridinecarboxylic acids
in aqueous solution at pH values of 1.9, 6.6., 7.0, and 7.4. Hydrazones are isolated as
solids and studied by means of NMR, IR spectroscopy. The fluorescence of hydrazones
dissolved in neutral and alkali media is investigated. The hydrazones capability of
inhibiting the copper-mediated ascorbate oxidation is evaluated.
Schiff bases formed by pyridoxal/PLP and pyridinecarboxylic acids
hydrazides should be noted to have their own biological relevance. For
instance, pyridoxal isonicotinoyl hydrazone (PIH) prevents copper-me-
diated free radicals formation in presence of ascorbate thus providing
⁎
Corresponding author.
E-mail address: oxt705@isuct.ru (G.A. Gamov).
http://dx.doi.org/10.1016/j.molliq.2017.07.106
0167-7322/© 2017 Elsevier B.V. All rights reserved.

G.A. Gamov et al. / Journal of Molecular Liquids 242 (2017) 1148–1155
1149
the antioxidant activity [19]. From the other hand, PIH is capable of
Cu2+ binding and may show demetalling action useful for treatment
of Wilson disease, gene-determined disorder of copper metabolism
[19].
widths were set at 5 nm, scanning rate was 6000 nm min−1. The quartz
cell with absorbing layer thickness of 1 cm was used.
2.2. The determination of hydrazones stability constants
Taking all above into consideration, in the present contribution we
aim to figure out how\\CO\\NH\\NH2 group localization in the pyri-
dine ring influences the stability of Schiff bases derived from pyridoxal
5′-phosphate and pyridinecarboxylic acids hydrazides as well as rate
constants of Schiff bases formation in aqueous solutions at pH values
corresponding to the physiological ones. Hydrazones are also isolated
as solids and characterized by means of NMR, IR and fluorimetry. Be-
sides, Schiff bases ability of protecting the ascorbic acid (AA) from cop-
per-mediated oxidation is estimated.
The stability constants of Schiff bases PLP-NH, PLP-PH were deter-
mined using previously described method [22] using UV–Vis spectra
of sets of 8–10 solutions with PLP:NH, PLP:PH initial concentrations
ratio varying from 10:1 to 1:1. Before recording the spectra, all the mix-
tures were allowed to equilibrate during 36 h after preparing. The spec-
tral data were processed using FTMT software (see [29] and Refs. 16–18
within) which calculates the equilibrium constants via solving the fol-
lowing equations:
To the date, the reactions between pyridoxal 5′-phosphate and phar-
maceutical hydrazides (isoniazid, carbidopa, hydralazine) were de-
scribed in a few of papers [20–22].
Aλ ¼ ðε1½R1ꢀ þ ε2½R2ꢀ þ ε3½PꢀÞ ꢁ l;
ð1Þ
i
where Aλi is the absorbance value at wavelength λi; ε1, ε2, and ε3 are the
molar extinction coefficients of reagents and product; [R1], [R2], [P] are
the equilibrium concentrations of reagents and product; l is the thick-
ness of absorbing layer. Eq. (1) expresses both Beer–Lambert–Bouguer
law and independent light absorbance principle;
2. Experimental
2.1. Chemicals and apparatuses
Pyridoxal 5′-phosphate (abcr GmbH, Germany), isoniazid (Sigma-
Aldrich, USA), ascorbic acid (Hebei Welcome Pharmaceutical Co., Ltd.,
China), EDTA (Mosreaktiv, Russia), citric acid (Spektr-Khim, Russia),
CuCl2 (Reakhim, Russia) were used without additional purification.
The purity of reagents claimed by manufacturer was N99% (weight).
The hydrazides of 3- and 2-pyridinecarboxylic acids were synthesized
using ethyl esters of corresponding pyridinecarboxylic acids [23,24].
The impurities absence was controlled using 1H NMR spectroscopy
and GCMS (GCMS-QP2010 Ultra setup, Shimadzu, Germany). Helium
was used during GCMS procedure as a carrier gas at the flow-rate of
0.98 ml/min and pressure of 90 kPa. The total run time was 20 min. Hy-
drazides were injected as solutions in CH2Cl2 at Tinj = 180 °C. Column
oven temperature was 150 °C whilst ion source temperature was
200 °C. Purge flow (He) was 3.0 ml/min. Buffers with pH values of 6.6,
7.0, and 7.4 were prepared using Na2HPO4·12H2O and NaH2PO4·2H2O
(Spektr-Khim, Russia). The pH 1.9 value was set using preliminarily
standardized HCl. Acidity of buffers was controlled potentiometrically.
All the solutions were prepared using bidistilled water (κ = 3.6 μSm/
cm, pH = 6.6). The ionic strength value I = 0.25 close to that of eryth-
rocytes media [25] was set due to buffer mixture components (at pH
values of 6.6, 7.0, and 7.4) or using KNO3 (pH 1.9).
CRiðtotalÞ ¼ ½Riꢀ þ ½Pꢀ;
ð2Þ
where CRi(total) is the total concentration of compound Ri. Eq. (2) ex-
presses material balance for Ri compound;
½Pꢀ
K ¼
;
ð3Þ
½R1ꢀ½R2ꢀ
where K is the equilibrium constant of reaction. Eq. (3) expresses acting
masses law.
In order to solve the Eqs. (1)–(3) jointly, one needs to minimize the
function:
z
X
ꢀ
ꢁ
2
F ¼
Aλ ð expÞ−Aλ ðcalcÞ
;
ð4Þ
i
i
q¼1
where Aλi(exp) and Aλi(calc) are the experimental and calculated absor-
bance values of solution number q; z is the total number of solutions.
The calculations were performed basing the experimental depen-
dencies of absorbance at 3–5 wavelengths on the reagents initial con-
centrations ratio (see examples of spectra on Figs. S1–S8). The molar
extinction coefficients of PLP, NH, PH required for calculations in FTMT
at every pH value and every wavelength were determined preliminarily
using calibration plots.
UV–Vis spectra of PLP, NH, and PH solutions of 5 · 10−5–2.0 ·
10−4 mol l−1 as well as mixtures of PLP + NH, PLP + PH were recorded
on double-beamed Shimadzu UV1800 spectrophotometer (USA) in the
wavelength range of 190–500 nm and absorbance range of 0–4. The
error of wavelength determination did not exceed 0.5 nm, the maximal
inaccuracy of absorbance measurements was of 0.006 units. The
temperature maintained at 298.2
0.1 K using external thermostat.
The quartz cells with absorbing layer thickness of 1 cm were used.
The elemental analysis of hydrazones was performed using FLASH
EA1112 setup (Termo Quest, Italy). Mass spectra (MALDI TOF) were
registered using Shimadzu Biotech Axima Confidence setup (Shimadzu,
USA). 1H, 13C and 31P NMR spectra of Schiff bases (D2O, pD ~ 12) were
registered using Avance III Bruker 500 NMR-spectrometer with 1H,
13C, 31P operating frequencies of 500.17, 125.77, and 202.47 MHz re-
spectively. The inaccuracy of chemical shift measurement in respect to
the external standard, HMDSO, was evaluated as 0.005 ppm. The sig-
nals in the spectra of studied compounds were assigned using literature
data [26] and NMR spectra predicting tools [27]. The IR spectra of Schiff
bases were recorded using Tensor 27 FTIR spectrometer (Bruker Optics,
Germany) with error of wavenumber determination of 1 cm−1. Spec-
tral bands were assigned using literature data [26,28].
2.3. The determination of rate constants of hydrazones formation
The spectrophotometrical kinetic experiments were performed at
the same concentration conditions and spectrophotometer setup as in
[22]. At the pH value of 1.9 the reaction proceeds too fast to reliably de-
termine its kinetic characteristics.
2.4. The synthesis of hydrazones
Synthesis was performed in accordance with Scheme 1. The prelim-
inarily heated to 90 °C solution of 0.309 g (1.25 mmol) of pyridoxal 5′-
phosphate in 25 ml of water was quickly added to the solution of
1.25 mmol of isoniazid or nicotinoyl hydrazide or picolinoyl hydrazide
in the 25 ml of water (at 90 °C). The reaction mixture was cooled during
1 h at room temperature. The precipitated crystalline products were
then filtrated, washed with small quantities of icy distilled water and ac-
etone and dried at 90 °C in the air until their weight became constant.
Fluorescence spectra were registered using RF6000 setup
(Shimadzu, USA) at the excitation wavelength λex = 300 nm in the
wavelength range of 310–550 nm. The excitation and emission slit

1150
G.A. Gamov et al. / Journal of Molecular Liquids 242 (2017) 1148–1155
Scheme 1. Formation of hydrazones derived from pyridoxal 5′-phosphate and hydrazides of 2, 3, and 4-pyridinecarboxylic acids.
2.4.1. {3-Hydroxy-4-[(E)-(isonicotinoylhydrazono)methyl]-2-methylpyridin-
5-yl}methylphosphate (Schiff base PLP-INH)
NH), 121.28 (C4 PLP), 61.95 (\\CH2\\PLP), 18.50 (\\CH3 PLP); 31P, δ,
ppm: s 3.76.
Orange solid, Tdecomp = 276–277 °C. Rf = 0.92 (mobile phase NH3
25% aq., Polygram Sil G/UV254). Elemental, found (calcd.): C 45.91
(45.91), H 3.85 (4.13), N 15.65 (15.30), O 26.12 (26.21). Mz [Schiff
base + H] 367.17. λmax, nm (lg ε) at pH 7.4, H2O: 294 (4.23). IR:
3313s (ν(N\\H) hydrazide residue), 3087s, 3070, 3054s ν(C\\H) pyri-
dine rings, 2989s (νasym(\\CH3\\) and νasym(\\CH2\\) PLP residue),
1669s (ν(C_O) hydrazide residue), 1568s, 1490s, 1411s (ν(C_C) and
ν(C_N) pyridine rings), 1527m (ν(C_N), \\CH_N\\NH\\ group),
1298s (ν(C\\N) + δ(N\\H) hydrazide residue), 1223s (ν(≥C\\OH)
PLP residue), 1161m (ν(P_O) PLP residue). NMR (D2O, pD ~ 12) 1H,
δ, ppm: 8.47 s (1H,\\CH_), 8.28 d (2H, H2,6 INH, J3 = 5.22 Hz), 7.53
d (2H, H3,5 INH, J3 = 5.87 Hz), 7.43 s (1H, H6 PLP), 4.66 d (2H,\\CH2\\
PLP, J2,3 = 4.56 Hz), 2.16 s (3H, \\CH3 PLP); 13C, δ, ppm: 169.14
(NC_O), 158.92 (C3 PLP), 151.43 (\\CH_), 149.16 (C2,6 INH), 148.86
(C2 PLP), 144.48 (C4 INH), 134.01 (C6 PLP), 130.40 (C5 PLP), 122.19
(C3,5 INH), 120.46 (C4 PLP), 61.77 (\\CH2\\PLP), 18.36 (\\CH3 PLP);
31P, δ, ppm: s 3.76.
2.4.3. (3-Hydroxy-2-methyl-4-{(E)-[(pyridin-2-ylcarbonyl)hydrazono]
methyl}pyridin-5-yl)methylphosphate (Schiff base PLP-PH)
Yellowish orange solid, Tdecomp = 266–267 °C. Rf = 0.81 (mobile
phase NH3 25% aq., Polygram Sil G/UV254). Elemental, found (calcd.):
C 45.86 (45.91), H 4.20 (4.13), N 15.21 (15.30), O 26.18 (26.21). Mz
[Schiff base + H] 367.17. λmax, nm (lg ε) at pH 7.4, H2O: 299 (4.30).
IR: 3477s (ν(O\\H) PLP residue), 3292w (ν(N\\H) hydrazide residue),
3097w, 3066w (ν(C\\H) pyridine rings), 2994w, 2920
m
(νasym(\\CH3\\) and νasym(\\CH2\\) PLP residue), 1690vs (ν(C_O)
hydrazide residue), 1586s, 1466 m, 1407w (ν(C_C) and ν(C_N) pyr-
idine rings), 1305s (ν(C\\N) + δ(N\\H) hydrazide residue), 1236w
(ν(≥C\\OH) PLP residue), 1167 m (ν(P_O) PLP residue). NMR (D2O,
pD ~ 12) 1H, δ, ppm: 8.67s (1H, \\CH_), 8.47 dq (1H, H6 PH, J3
=
4.87 Hz, J4 = 1.52 Hz, J5 = 0.87 Hz), 7.87 dt (1H, H3 PH, J3 = 8.04 Hz,
J5 = 1.09 Hz), 7.84 td (1H, H4 PH, J3 = 7.61 Hz, J4 = 1.74 Hz), 7.55s
(1H, H6 PLP), 7.43 ddd (1H, H5 PH, J3 = 7.39 Hz, J3 = 5.00 Hz, J4
=
1.52 Hz), 4.80 d (2H,\\CH2\\PLP, J2,3 = 4.56 Hz), 2.24 s (3H,\\CH3
PLP); 13C, δ, ppm: 167.75 (NC_O), 160.71 (C3 PLP), 152.05 (\\CH_),
150.08 (C6 PH), 148.96 (C2 PLP), 140.49 (C2 PH), 138.36 (C4 PH),
132.35 (C6 PLP), 131.23 (C5 PLP), 126.58 (C3 PH), 124.67 (C5 PH),
123.29 (C4 PLP), 62.24 (\\CH2\\PLP), 18.76 (\\CH3 PLP); 31P, δ, ppm:
s 3.86.
2.4.2. (3-Hydroxy-2-methyl-4-{(E)-[(pyridin-3-ylcarbonyl)hydrazono]
methyl}pyridin-5-yl)methylphosphate (Schiff base PLP-NH)
Bright yellow solid, Tdecomp = 269–270. Rf = 0.91 (mobile phase
NH3 25% aq., Polygram Sil G/UV254). Elemental, found (calcd.): C 46.01
(45.91), 4.08 (4.13), 15.49 (15.30), 26.22 (26.21). Mz [Schiff base + H]
367.17. λmax, nm (lg ε) at pH 7.4, H2O: 296 (4.23). IR: 3599s (ν(O\\H)
PLP residue), 3343s (ν(N\\H) hydrazide residue), 3113s, 3052m
(ν(C\\H) pyridine rings), 2989s, 2911s (νasym(\\CH3\\) and
The atoms numbering is given (Scheme 2).
2.5. The oxidation of ascorbate
ν
asym(\\CH2\\) PLP residue), 1684s (ν(C_O) hydrazide residue),
The study was performed analogously with described [19] with
some modifications. The 3.5 ml aliquot of the solution of ascorbic acid
(0.0001 mol l−1), or ascorbic acid (0.0001 mol l−1) + hydrazone
(0.0001 mol l−1) in the buffer with 6.6 pH was sampled into the stan-
dard 1 cm quartz cell. 2–3 drops of aqueous solution of CuCl2
(0.01 mol l−1) with preliminarily determined density were added to a
solution in the cell. Weighing the syringe containing CuCl2 before and
after addition allowed to determine the quantity of Cu2+ added. The ab-
sorbance of ascorbic acid solutions with/without adding of hydrazones/
Cu(II) ions was recorded during 300 s at wavelength of 265 nm. The
ascorbate molar coefficient of extinction at this wavelength is known
to be εAA = 14,500 [30] which is close to our own measurements.
1585s, 1487s, 1430s (ν(C_C) and ν(C_N) pyridine rings), 1520w
(ν(C_N),\\CH_N\\NH\\group), 1300s (ν(C\\N) + δ(N\\H) hydra-
zide residue), 1237m (ν(≥C\\OH) PLP residue), 1177s (ν(P_O) PLP
residue). NMR (D2O, pD ~ 12) 1H, δ, ppm: 8.82 dd (1H, H2 NH, J4
1.30 Hz, J5 = 0.87 Hz), 8.62 s (1H,\\CH_), 8.42 dd (1H, H6, NH, J3
4.78 Hz, J4 = 1.52 Hz), 8.12 dq (1H, H4 NH, J3 = 7.80 Hz, J4
1.73 Hz), 7.55s (1H, H6 PLP), 7.35 ddd (1H, H5 NH, J3 = 8.04 Hz, J3
=
=
=
=
5.00 Hz, J5 = 0.87 Hz), 4.76 d (2H,\\CH2\\PLP, J2,3 = 4.56 Hz), 2.25s
(3H,\\CH3 PLP); 13C, δ, ppm: 169.53 (NC_O), 159.44 (C3 PLP), 151.67
(\\CH_), 150.54 (C2 NH), 148.96 (C2 PLP), 148.05 (C6 NH), 136.55
(C4, NH), 133.69 (C6 PLP), 132.25 (C5 PLP), 130.68 (C3 NH), 124.13 (C5
Scheme 2. Schiff bases derived from pyridoxal 5′-phosphate and hydrazides of pyridinecarboxylic acids: a) isonicotinic; b) nicotinic; c) picolinic.

G.A. Gamov et al. / Journal of Molecular Liquids 242 (2017) 1148–1155
1151
Table 1
Stability constants, rate constants of formation and hydrolysis of Schiff bases derived from pyridoxal 5′-phosphate and hydrazides of pyridinecarboxylic acids at different pH.
Hydrazone
pH
1.94
6.6
7.0
7.4
PLP-INH [22]
lg K
4.51
–
–
5.33 0.12
–
–
5.02 0.13
–
–
4.61
4.72
4.21
28.39
k1, l mol−1 min−1
k2, min−1
lg K
49.44
30.31
1.23 · 10−3
5.8 · 10−4
1.77 · 10−3
5.16 0.25
21.74 0.86
PLP-NH
PLP-PH
4.64 0.28
5.05 0.12
22.89 0.46
2.0 · 10−4 6 · 10−5
5.04 0.11
26.55 1.33
2.4 · 10−4 7 · 10−5
k1, l mol−1 min−1
k2, min−1
lg K
25.20 0.40
5.8 · 10−4 4.1 · 10−4
5.14 0.18
1.5 · 10−4 1.0 · 10−4
4.64 0.13
k1, l mol−1 min−1
k2, min−1
57.36 0.76
14.80 1.74
4.2 · 10−4 1.8 · 10−4
3.4 · 10−4 1.4 · 10−4
In the all cases, we had associated the changes of absorbance with
decreasing of ascorbate concentration, which allows estimating it as
Figs. S1–S8) thus indicating the formation of π-π-p-π conjugated electron
system well-known for hydrazones [33,pp. 107–108].
ΔCAA = ΔA / εAA
.
One could observe the aldehyde proton signal in the 1H NMR spec-
trum of pyridoxal 5′-phosphate at 9.90 ppm. There are no signals in
this range in the spectra of synthesized compounds, however, the reso-
nance of\\CH_ group neighboring the imine bond appears at 8.47–
8.67 ppm. In the 13C NMR spectra, the signal of formyl group of PLP
(196.35 ppm.) moves upfield by 40 ppm confirming the \\CH_
formation.
The ascorbate oxidation in presence of EDTA and citric acid (CA) was
investigated in the same way.
3. Results and discussion
3.1. Thermodynamics and kinetics of hydrazones formation
Hydrazones are able to exist as a mixture of E,Z-isomers due to the
\\CH_N\\group presence in the molecule [33,pp. 48–52]. However,
there is the only set of signals in the 1H, 13C NMR spectra of PLP-INH,
PLP-NH, PLP-PH hydrazones. Our quantum chemical calculations of
neutral and zwitter-ionic species of non-ionized Schiff bases (not
shown) have revealed that the E-isomers are more stable (Gz-GE is ca.
42 kJ mol−1 for zwitter-ions and 8 kJ mol−1 for molecular species).
Therefore, hydrazones probably precipitate as E-isomer.
The results of spectrophotometric determination of the equilibrium
constant as well as rate constants of direct (k1) and inverse (k2) reaction
of Schiff base formation at different pH are summarized (Table 1).
The k2 values (Table 1) were calculated using well-known equation:
Kr ¼ k1=k2
ð5Þ
The errors of lg Kr and k1 values (Table 1) are the half-width of the
confidence interval with a confidence probability of 0.95 and sample
size of three experiments. The inaccuracies of k2 values include the de-
termination errors of all values used for k2 calculations.
Taking into account the peculiarities of distribution of electronic
density in pyridine, one could anticipate the similarity between Schiff
bases derived from hydrazides of 2- and 4-pyridinecarboxylic acids.
However, Table 1 data show no significant differences between
hydrazones stability except for 7.4 pH, where PLP-INH Schiff base has
the smallest value of lg K. Interestingly, PLP-NH hydrazone is the most
stable at pH 1.9.
The stability of each hydrazone changes by ~0.5 log units in the pH
range of 6.6–7.4 (Table 1). These variations are caused by the change
of equilibrium concentrations of different dissociated species of pyri-
doxal 5′-phospate with different binding ability towards hydrazides
(see e.g. [21].).
No frequencies characteristic for\\NH2 groups of INH, NH, PH and
CHO-group of PLP could be found in the IR spectra of Schiff bases (see
Section 2.4), which confirms hydrazones formation.
Being heated in the argon atmosphere, hydrazones decompose in
the temperature range of 267–277 °C (see Section 2.4). IR-analysis of
the evolving gases shows the predominant presence of aqueous vapor,
CO2 and NH3.
The synthesized Schiff bases fluoresce in the solution (Fig. 1).
Fig. 1 data show that the alkali media promote the fluorescent prop-
erties of Schiff bases (strongest emission at λem = 360 nm). Neutral
buffer solutions allows only weak fluorescence of PLP-NH and PLP-PH
at λem ~ 480 nm. In the alkali media the emission spectra of PLP-INH
and PLP-PH are similar (the latter fluoresce more intensively). The
The hydrazide of nicotinic acid seems to be the most hazardous com-
pound for a healthy tissue, where pH of medium is close to 7.4, since its
hydrazone is the most stable and forms readily enough at this pH value
(Table 1). The picolinic acid hydrazide could be optimal PLP-binding
agent in some tumors with acidic interstitial fluid [31,32].
The rate of PLP-INH and PLP-PH hydrazones formation decreases
significantly in the pH range of 6.6–7.4, while k1 value of PLP-NH re-
mains nearly constant.
3.2. Hydrazones synthesis and fluorescence
The studied Schiff bases form under the relatively mild conditions
and require no non-aqueous solvents and high temperatures during
the synthesis. When the preliminarily heated solutions of PLP and
INH, NH, PH with equal concentrations are mixed, the crystalline insol-
uble product begins to form quickly and, then, could be filtered easily.
Schiff bases formation could be proved by the comparison of UV–Vis,
NMR (1H, 13C) and IR spectra of reagents and product.
For example, all the reaction mixtures start to absorb intensively (ε =
17,000–22,500) in the range of 294–303 nm (depending on pH value, see
Fig. 1. Fluorescence of hydrazones derived from PLP and hydrazides of 4-, 3- and 2-
pyridinecarboxylic acids in the neutral and alkali media (solvent water).

1152
G.A. Gamov et al. / Journal of Molecular Liquids 242 (2017) 1148–1155
PLP-NH compound emits weaker at 360 nm, but shows the fluorescence
in the long-wave range (weak peak at 505 nm).
It is worth noting that the only PLP-PH hydrazide lights with bright
green in the crystalline form when being irradiated by mercury UV-
lamp (λex = 365 nm).
3.3. Copper-mediated oxidation of ascorbate in the presence of hydrazones
Ascorbic acid, an efficient reductant, oxidizes to the dehydroascorbic
acid because of two consecutive one-electron processes [30]. Ascorbate
oxidation accelerates in the presence of transition metal ions such as
Cu(II) [19] and Fe(III) [34]. The ascorbate was found [30] to be quite sen-
sitive to even traces of metals-catalyzers, and, therefore, it could serve
as an indicator of solution contamination by metals. Pyridoxal isoniazid
hydrazone has been earlier observed to slow the ascorbate oxidation in
the presence of copper(II) ions due to forming the complexes with Cu2+
[19].
Authors [19] described a number of the reactions passing in the so-
lution of ascorbate and Cu(II) ions:
Fig. 3. Time-dependent variation of ascorbate concentration in the presence of PLP-INH
hydrazone at 6.6 and 7.4 pH values. C0(AA) = 0.0001 mol l−1, C0(PLP-INH) =
CuðIIÞ þ ascorbate→CuðIIÞ ꢂ ascorbate→CuðIÞ þ ascorbyl
ð6Þ;
ð7Þ;
0.0001 mol l−1
.
–
CuðIÞ þ O2→CuðIIÞ þ O2
absorbance measurement determines the variation of ascorbate con-
centration seen on the plot and the irregular character of observed
changes.
All the hydrazones possess similar protecting activity towards ascor-
bic acid at 6.6 pH in the absence of Cu2+ ions (Fig. 4). When CuCl2 is
added to the system, PLP-PH hydrazone is the least agent preventing
ascorbate oxidation.
–
CuðIÞ þ O2 þ 2Hþ→CuðIIÞ þ H2O2
ð8Þ;
ð9Þ;
−
2O2 þ 2Hþ→O2 þ H2O2
Authors [19] had shown the pyridoxal isonicotinoyl hydrazone to
form complex with Cu(II) ions thus competing for metal with ascorbate.
This complexation probably prevents the reduction of Cu(II) to Cu(I),
which could react with dissolved oxygen generating such strong oxi-
dants as O–2 and H2O2. The PLP-INH hydrazone is also capable of forming
the coordination compound with Cu2+ (see an example on Fig. 5).
Copper ion seems to disturb π-π-p-π conjugated electron system of
hydrazone since it interacts with\\NH: atom. Therefore, the absorbance
at 295 nm becomes less intensive while bathochromic shift is observed
(new peak at 410 nm).
CuðIÞ þ H2O2→CuðIIÞ þ OH– þ ꢁOH:
ð10Þ:
The Cu(II) capability of accelerating the ascorbate oxidation produc-
ing both hydrogen peroxide and hydroxyl radical was confirmed in the
recent detailed study [35].
Fig. 2 shows the dependencies of ascorbate (AA) concentration on
time in the presence/absence of PLP-INH hydrazone and Cu(II) ions.
The Schiff base formed by pyridoxal 5′-phosphate could be assumed
to protect ascorbic acid as effectively as pyridoxal isoniazid hydrazine
[19] does.
The Fig. 5 data allow estimating the complexation equilibrium con-
stant using FTMT software analogously with stability constants of Schiff
bases (lg K = 4.8 0.5).
Replacing the buffer with pH value of 6.6 by one with pH 7.4 leads to
insignificant differences (Fig. 3). The experimental inaccuracy of
Fig. 4. Time-dependent decreasing of ascorbate concentration in the presence of PLP-INH,
Fig. 2. Time-dependent decreasing of ascorbate concentration in the presence/absence of
PLP-NH, PLP-PH hydrazones and presence/absence of Cu(II) ions. C0(AA)
0.0001 mol l−1, C0(PLP-INH, PLP-NH, PLP-PH) = 0.0001 mol l−1, C0(Cu) = 0.00005–
0.00007 mol l−1
=
PLP-INH hydrazone and Cu(II) ions. C0(AA)
= , C0(PLP-INH) =
0.0001 mol l−1
0.0001 mol l−1, C0(Cu) = 0.00005–0.00007 mol l−1
.
.

G.A. Gamov et al. / Journal of Molecular Liquids 242 (2017) 1148–1155
1153
ascorbate. Though authors [19] had found it is unlikely that ligands
bind the forming free radicals, Schiff bases should be noted to be oxi-
dized by H2O2 as it follows from weeklong NMR control of PLP-PH
+ H2O2 mixture at pH 12 we have performed. The number of signals
in the low-field range of spectra decreased from 6 to 5. It could be a con-
sequence of the process:
R1\\CO\\NH\\N_CH\\R2→R1\\CO\\NH\\NH\\CO\\R2;
ð11Þ
where \\CH_ group transforms into NC_O and its 1H NMR signal
disappears.
The oxidation of analogous compound, 2-pyridylcarboxaldehyde
isonicotinoylhydrazone, by the Scheme (11) was found to be catalyzed
by Fe(III) ions [39]. Thereby, the PLP-INH, PLP-NH, PLP-PH hydrazones
may also react readily with H2O2 evolving in the presence of Cu(II). In
our opinion, these suggestions require some further investigations.
4. Conclusions
Fig. 5. Complex formation between Cu(II) and PLP-INH hydrazone in aqueous solution at
6.6 pH. The values in plot description stand for initial molar concentrations.
The reaction between pyridoxal 5′-phosphate and hydrazides of 2-,
3-, and 4-pyridinecarboxylic acids were studied in aqueous solution.
The stability constants as well as the rate constants of formation of
hydrazones derived from nicotinic and picolinic acids hydrazides were
determined experimentally at pH values of 1.9; 6.6; 7.0; and 7.4. Isoni-
azid was found to form the least stable Schiff bases with PLP at every
studied pH value. In the acidic medium, nicotinic acid derivative forms
the most stable hydrazone. In the pH range of 6.6–7.4, the stability of
PLP-NH and PLP-PH hydrazones vary in the opposite way. The rate of
PLP-INH and PLP-PH Schiff bases formation decreases significantly in
the pH range of 6.6–7.4, while the k1 value of PLP-NH remains nearly
constant.
The PLP-INH, PLP-NH, and PLP-PH were isolated as solids and char-
acterized by means of NMR, IR-spectroscopy and fluorimetry. The PLP-
PH compound was found to fluoresce stronger than two other Schiff
bases either in the solid phase and the solution (λem = 360 nm). The lu-
minescence spectrum of PLP-INH hydrazone is similar with that of 2-
isomer, however, it emits much weaker. The peculiarity of nicotinic
acid hydrazide derivative is the additional weak emission in the long-
wave range (505 nm).
If it is the thermodynamic stability of complex with metal which de-
termines the protecting action of hydrazones towards ascorbate in the
mixture with Cu(II), it would be reasonable to assume the ligands bind-
ing the copper ions more effectively to exhibit more oxidation
preventing action. However, there is no direct link between lg K and
protecting effect (Fig. 6).
The inhibition of copper-mediated ascorbate oxidation by the strong
chelator EDTA is slightly less pronounced than that by PLP-INH
hydrazone. This observation in in correspondence with previous find-
ings for pyridoxal isonicotinoyl hydrazone [19]. Citric acid forming at
pH N 5.5 the copper(II) complexes of Cu2(CA)42− composition (lg K =
5.5–6.0 [36,37]) protects the ascorbic acid even less. (Fig. 6). Ascorbate
oxidation preventive action of citric acid along with a number of other
organic acids is studied in details in [38].
Free radical scavenging and competing for oxidizer are other proba-
ble explanations of oxidation preventive action of Schiff bases towards
The PLP-INH, PLP-NH, and PLP-PH hydrazones ability of preventing
the copper-mediated ascorbate oxidation was studied. All the Schiff
bases are efficient antioxidants, however the derivative of 2-isomer is
the least protective agent. The protective activity of hydrazones does
not change significantly in the physiological range of pH values (6.6–
7.4).
The PLP-INH hydrazone forms the 1:1 coordination compound with
copper(II) ions, which stability is lg K ~ 5. Despite the stability of Cu(II)-
PLP-INH complex lower than that of Cu(II)-EDTA, Cu(II)-CA complexes,
sodium ethylenediaminetetraacetate and citric acid are less effective in
preventing the copper-mediated oxidation of ascorbate.
The hydrazones derived from hydrazides of 2-, 3-, and 4-
pyridinecarbopylic acids and PLP could be considered as analogues of
known biologically active compound, pyridoxal isonicotinoyl
hydrazone. The latter is known as low-toxic, membranotropic [40],
demetalling towards copper [19,41] (but not calcium/magnesium
[42]), antioxidant [43]. They are easy to synthesize, sufficiently stable,
capable of fluorescing (especially, PLP-PH), bind the copper ions effi-
ciently, which make it possible to use them in medicine, e.g., Wilson dis-
ease treatment. PLP-PH Schiff base could be also used in vivo as
luminescent sensor.
The ability of PLP-INH, PLP-NH, and PLP-PH hydrazones to protect
the ascorbic acid from oxidation is also valuable, since ascorbic acid,
but not its oxidized form, dehydroascorbic acid, reduces the malignant
potential of human melanoma [44]. Thus, the hydrazones could be
Fig. 6. Time-dependent decreasing of ascorbate concentration in the presence of PLP-INH/
EDTA/citric acid (CA) and presence/absence of Cu(II) ions. C0(AA) = 0.0001 mol l−1
,
C0(PLP-INH, EDTA, CA) = 0.0001 mol l−1, C0(Cu) = 0.00005–0.00007 mol l−1
.

1154
G.A. Gamov et al. / Journal of Molecular Liquids 242 (2017) 1148–1155
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Conflict of interests
Authors declare none.
Acknowledgements
The study was carried out with financial support of Russian Founda-
tion of Basic Research (project 16-33-60017) and under grant of Council
on Grants of the President of the Russian Federation (project
14.Z56.16.5118-MK).
The equipment of Joint Research Center “Upper Volga Regional Cen-
ter of Physical and Chemical Research” was used to perform NMR and IR
study. Prof. G.V. Girichev (ISUCT) is greatly acknowledged for his help
with quantum chemical calculation performance.
We also would like to thank prof. S.V. Makarov (ISUCT) for kind per-
mission to use spectrofluorimeter and A. Levantovskii (Magicplot Sys-
tems, LLC) who developed MagicPlot software used for spectral data
processing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.molliq.2017.07.106.
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