Acid–Base Properties and Kinetics of Hydrolysis of Aroylhydrazones Derived from Nicotinic Acid Hydrazide

Article abstract of DOI:10.1007/s10953-016-0504-8

A series of aroylhydrazones were synthesized from nicotinic acid hydrazide and differently substituted benzaldehydes. The protonation constants of the 12 resulting hydrazones, as well as of the starting compounds, were determined in methanol/water 1/1 mix

Full text of DOI:10.1007/s10953-016-0504-8

J Solution Chem (2016) 45:1227–1245
DOI 10.1007/s10953-016-0504-8
Acid–Base Properties and Kinetics of Hydrolysis
of Aroylhydrazones Derived from Nicotinic Acid
Hydrazide
1
T. Benkovic D. Kontrec2 V. Tomisic A. Budimir3
´
N. Galic
ˇ ´1
•
•
•
•
´
1
Received: 16 May 2016 / Accepted: 19 July 2016 / Published online: 17 August 2016
Ó Springer Science+Business Media New York 2016
Abstract A series of aroylhydrazones were synthesized from nicotinic acid hydrazide and
differently substituted benzaldehydes. The protonation constants of the 12 resulting
hydrazones, as well as of the starting compounds, were determined in methanol/water 1/1
mixtures by spectrophotometric–potentiometric batch titrations. In order to determine the
effect of substituents on the stability of the C=N bond, the kinetics of hydrolysis of
hydrazones was studied spectrophotometrically in acidic and basic media. The HPLC
method was used for investigation of the hydrolysis of selected compounds, for which
equilibrium between hydrolysis and the condensation reaction was observed. The observed
rate constants, calculated from chromatographic and spectrophotometric data, are in good
agreement. Electrospray mass spectrometry was used for determination of reaction prod-
ucts. Oxidation and formation of quinones in basic media were observed for the 2,3- and
2,5-dihydroxy substituted benzaldehyde derivatives. On the other hand, the 2,4-dihydroxy
derivative showed higher stability compared to N0-salicylidene-3-pyridinecarbohydrazide.
Keywords Aromatic hydrazones Á HPLC Á Hydrolysis Á Mass spectrometry Á Protonation
constants Á UV spectrometry
Electronic supplementary material The online version of this article (doi:10.1007/s10953-016-0504-8)
contains supplementary material, which is available to authorized users.
&
&
1
A. Budimir
abudimir@pharma.hr
´
N. Galic
ngalic@chem.pmf.hr
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a,
10000 Zagreb, Croatia
2
ˇ
´
ˇ
Department of Organic Chemistry and Biochemistry, Ruder Boskovic Institute, Bijenicka 54,
¯
10000 Zagreb, Croatia
3
ˇ ´
Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica 1, 10000 Zagreb,
Croatia
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1 Introduction
Although the chemistry of aryl (Ar–NH–N=CH–R; R = alkyl or aryl) and aroyl (Ar–CO–
NH–N=CH–R; R = alkyl or aryl) hydrazones has been extensively studied for over a
century, they still attract considerable attention due to their versatile properties and
applications. Due to their unique structural properties, hydrazones have been used as
molecular switches and sensors for detecting metal cations, anions, and neutral molecules
[1–3]. Many compounds of this type are biologically active as anticancer, antibacterial,
antimicrobial, antidepressant, and anti-inflammatory agents [4–7]. Recent studies have
shown that nicotinic acid hydrazones can be considered as novel pharmacophores in the
design of new anticonvulsant drugs [8]. In addition, some 2-substituted phenyl derivatives
of nicotinic acid hydrazide have promising analgesic and anti-inflammatory activities [9].
The salicylaldehyde derivative of isonicotinoyl hydrazide was shown to be a selective iron
chelator with promising pharmacological properties [10]. However, pharmacokinetic
studies have pointed out its relatively short biological half-life due to hydrolysis of the
hydrazone bond, which is typical for this class of compounds [10–15].
For successful application of hydrazones as analytical reagents or biologically active
compounds, information about their physico-chemical properties, such as protonation
constants, kinetics of hydrolysis, etc., should be known. Due to their low solubility, and
possible hydrolysis in aqueous solutions, reliable values of protonation constants may be
difficult to obtain. Batch spectrophotometric–potentiometric titration has been proven to be
the most convenient method for such systems [16, 17]. Previously, we reported on the
structural properties of aroylhydrazones derived from nicotinic acid hydrazide in the solid
state and in solution [18–20]. The most stable form, stabilized by strong intramolecular or
solute–solvent hydrogen bonds, is the ketoamino tautomer with respect to the Ar–CO–NH–
N= part of the molecules and the enolimino tautomer regarding the salicylidene part of the
hydrazones. We have also recently reported on the kinetics of hydrolysis of aroylhydrazone
derivatives obtained by condensation reactions of nicotinic acid hydrazide with salicy-
laldehyde or o-vanilline [21]. The results suggested that in acidic solutions the rate-de-
termining step of hydrolysis is the attack of a water molecule on the protonated azomethine
group, whereas in basic medium it is the attack of water on the completely deprotonated
hydrazone. In continuation of our investigations on aroylhydrazones [18–23], in this paper
we present the protonation equilibria as well as kinetics of hydrolysis of 12 derivatives of
nicotinic acid hydrazide, which are shown in Fig. 1.
Fig. 1 Structures of aroylhydrazones derived from nicotinic acid hydrazide
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2 Experimental
2.1 Reagents
Aroylhydrazones 1–12 were prepared by mixing equimolar amounts of nicotinic acid
hydrazide (Fluka) with the corresponding aldehyde or ketone (Sigma). The reactions
were carried out in dry ethanol under argon atmosphere at 85 °C for 20 h. The solvent
was evaporated and the solids were suspended in CH2Cl2, filtered (G-3), rinsed with
CH2Cl2 (EtOH was used for 4, 5, 11 and 12), and dryed at 50 °C over 24 h. The
hydrazones were characterized by standard analytical procedures (NMR, IR, MS, and
HPLC).
2.2 Instruments
UV–Vis spectra were recorded by means of a Varian Cary 5 spectrophotometer equipped
with a thermostated cell compartment {temperature was maintained at (25 0.1) °C}. pH
values were measured with a Metrohm 827 pH LAB pH-meter using a combined glass—
Ag/AgCl electrode (Metrohm, 6.0255.100). Calibration of the electrode was done with
buffers prepared according to the literature [24].
Kinetic measurements were performed with a Varian Cary 50 spectrophotometer cou-
pled to an on-line data acquisition system. The cell compartment was thermostatted by the
use of a Varian refrigerated circulator bath. Temperature was kept at (25 0.1) °C.
Chromatographic measurements were carried out on an Agilent 1220 Series Infinity LC
(G4290C) equipped with built-in degasser, binary pump, autosampler, column oven, and
variable wavelength UV–Vis detector. The data were processed with OpenLAB CDS
ChemStation software.
Mass spectra were obtained on an Agilent 6420 Triple Quadrupole Mass Spectrometer.
Solutions were introduced directly via an Agilent 1200 HPLC system. The ESI MS spectra
were recorded in the range of m/z = 10 to 500 in both positive and negative ion modes.
The following conditions were used: capillary potential 4 kV, fragmentor voltage 135 V,
gas flow rate 0.6 dm-3Ámin-1, and gas temperature 300 °C.
2.3 Protonation Constants Measurements
Stock solutions of compounds 1–12 (c = 1 9 10-3 molÁdm-3) were prepared by dis-
solving the aroylhydrazones in methanol. Universal buffer mixtures (pH 2–12), consisting
of phosphoric, boric, and acetic acids and their salts, were used for pH adjustment.
Solutions for spectrometric determination of protonation constants were prepared as fol-
lows: 1 mL of buffer, 1 mL of sodium chloride (c = 1.0 molÁdm-3), and 3 mL of
deionized water were transferred to a 10 mL flask. Stock solution of a hydrazone (0.5 mL)
was added and the flask which was then filled with methanol. The working solution was
immediately transferred to a standard 1 cm quartz cuvette and absorption spectra were
recorded over the range 200–500 nm. Protonation constants were calculated by non-linear
least-squares analysis using the pHab2003 program from the Hyperquad program suite
[25]. Batch titrations were repeated at least three times. The protonation constants of
starting compounds (nicotinic acid hydrazide and aldehydes) were determined as well,
using the same procedure.
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2.4 Kinetics Measurements
The kinetics of hydrolysis was investigated by mixing equal volumes of a methanolic solution
of compounds 1–12 with a solution of hydrochloric acid or sodium hydroxide in a conventional
1 cm quartz cuvette. The concentration of hydrazones 1–12 after mixing was 5 9 10-5
molÁdm-3, whereas concentrations of hydrochloric acid ranged from 0.01to 0.2 molÁdm-3 and
that of sodium hydroxide ranged from 0.005 to 0.2 molÁdm-3. Measurements were performed
underpseudo-first-order conditions ([H?] or[OH-] C 100 9 [hydrazone]). The ionic strength
was kept constant (Ic = 1 molÁdm-3, NaCl). The absorbance changes in the course of
hydrolysis reactions were measured at selected wavelengths corresponding to maximum
absorbancedifferencebetweenthereactantsandtheproducts. Thekinetictraceswereevaluated
using the OriginPro 7.5 program, and the rate constants are reported as mean values of triplicate
measurements. In all experiments, except for compounds 3 and 5 in basic media, a single
exponential decay Eq. 1 of the measured absorbance versus time was successfully fitted to the
experimental data.
At ¼ DAexpðÀkobstÞ þ A1
ð1Þ
where DA is the total absorbance change, whereas At and A? are the absorbances at time
t and infinite time, respectively. The observed rate constant kobs is the calculated pseudo-
first-order rate constant. In the cases of compounds 3 and 5 in basic media, the spectral
changes (250–500 nm) were analyzed by multi-wavelength global fitting based on a two-
step reaction model incorporated in the Specfit 2.10 program [26].
Liquid chromatography was also used for investigation of the kinetics of the hydrolysis
of compounds 3, 4, 10, and 11. Gradient elution was performed {mobile phase A consisted
of phosphate buffer (0.05 molÁdm-3, pH 4) whereas mobile phase B consisted of HPLC
gradient grade methanol}. Injections (2 lL) were performed by using a software-operated
autosampler at regular time intervals (5 min). Separation was achieved on a Phenomenex
C18 Kinetex core shell column (50 9 3 mm, 2.6 lm). Temperature of the column was
maintained at (30 0.8) °C. The wavelength for simultaneous detection of hydrazones
and their hydrolysis products was selected from previously acquired UV–Vis spectra.
Hydrolyses of hydrazones (c = 5 9 10-5 molÁdm-3) were carried out in acidic and
alkaline methanol/water 1/1 (volume ratio) solutions. The concentration of acid used (HCl)
in working solutions was 0.01 molÁdm-3, whereas the concentration of base (NaOH) was
0.015 molÁdm-3
.
For MS measurements methanolic solutions of compounds 3, 4, 5, 7, 10, and corre-
sponding aldehydes were mixed with aqueous NaOH in order to get methanol/water 1/1
solutions. The final concentration of hydrazones (aldehydes) was 5 9 10-5 molÁdm-3
,
whereas that of NaOH was 0.1 molÁdm-3
.
3 Results and Discussion
3.1 Protonation Constants
Difficulties in obtaining reliable protonation constants of aroylhydrazones 1–12 were due
to their poor solubility (it was necessary to work at low concentrations) as well as slow
hydrolysis in mixed organic solvent/water solutions. For that reason, batch spectrophoto-
metric-potentiometric titrations were used in this work as the most convenient
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experimental method. The protonation constants were determined in methanol/water 1/1
mixtures. In order to investigate the solvent effect on protonation reactions, the corre-
sponding equilibrium constants for selected compounds were determined in MeOH/H2O
1/4 mixtures as well. All absorption spectra were recorded within 3 min after preparation
of the solutions. In that time no appreciable hydrolysis took place. As an example, the
dependence of the absorption spectrum of 2 on pH is shown in Fig. 2a and the corre-
sponding plot of the measured and calculated absorbances is given in Fig. 2b (the spec-
trophotometric titrations of all other hydrazones are shown in supporting information, SI,
Figs. S1–S11). The scheme of protonation pathway of aroylhydrazones is given in Fig. 3.
To provide more accurate assignation of pK values for aromatic hydrazones, comparison
with the protonation constants of corresponding starting compounds, nicotinic acid
hydrazide and aldehydes, was made. The latter constants are presented in Tables 1 and 2,
and those corresponding to compounds 1–12 in Tables 3 and 4. As can be seen in Table 2,
in the MeOH/H2O 1/4 system, the three pK values of nicotinic acid hydrazide, assigned to
the amide nitrogen (–NH–N), pyridine (ring) nitrogen (PyN) and amino group (–NH2),
were determined. However, in the system containing more organic solvent (MeOH/H2O
Fig. 2 a UV–Vis spectra of 2
(a)
1.2
0.9
0.6
0.3
0.0
and b dependence of absorbance
at 372 nm on pH in methanol/
water 1/1 mixtures:
c(2) = 5 9 10-5 molÁdm-3
,
l = 1 cm, Ic = 0.1 molÁdm-3
(NaCl), h = (25.0 0.1) °C;
filled square experimental,—
calculated
12.994
12.572
11.635
9.600
8.870
8.379
7.931
6.746
250
300
350
400
450
500
(b)
1.0
0.8
0.6
0.4
0.2
0.0
7
8
9
10
11
12
13
pH
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Fig. 3 Protonation equilibria of aroylhydrazones studied
Table 1 Protonation constants of starting compounds in methanol/water 1/1 mixtures, Ic = 0.1 molÁdm-3
(NaCl), h = (25.0 0.1) °C
log10 KiH
Compound
Assignment
Nicotinic acid hydrazide
11.98 0.01
1.40 0.02
8.56 0.01
10.39 0.01
6.88 0.01
10.62 0.01
7.43 0.01
10.84 0.01
8.91 0.01
8.12 0.02
8.30 0.01
7.02 0.01
7.52 0.01
5.86 0.01
5.28 0.01
10.70 0.01
–NH–N
–ring N or –NH2
2-Hydroxybenzaldehyde
–OH
2,3-Dihydroxybenzaldehyde
–OH (position 2)
–OH (position 3)
2,4-Dihydroxybenzaldehyde
2,5-Dihydroxybenzaldehyde
–OH (position 2)
–OH (position 4)
–OH (position 2)
–OH (position 5)
2-Hydroxy-3-methoxybenzaldehyde
2-Hydroxy-4-methoxybenzaldehyde
2-Hydroxy-3-chlorobenzaldehyde
2-Hydroxy-5-chlorobenzaldehyde
2-Hydroxy-3,5-dichlorobenzaldehide
2-Hydroxy-5-nitrobenzaldehyde
2-Hydroxyacetophenone
–OH
–OH
–OH
–OH
–OH
–OH
–OH
Uncertainties are expressed as standard deviations
1/1), only two protonation steps were observed (Table 1). The same trend was maintained
as well in protonation of hydrazones (Tables 3 and 4).
The first protonation constant (K1H) of compounds 1–12 was assigned to protonation of
the amide group. The lowest value was determined for compound 1 since the negative
charge on the amide nitrogen can be stabilized most efficiently by an unsubstituted phenyl
ring. In all other hydrazones, the negative charge of the deprotonated hydroxyl group of the
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Table 2 Protonation constants of starting compounds in methanol/water 1/4 mixtures, Ic = 0.1 molÁdm-3
(NaCl), h = (25.0 0.1) °C
log10 KiH
Compound
Assignment
Nicotinic acid hydrazide
11.59
4.37
2.52
8.59
8.02
–NH–N
–ring N
–NH2
–OH
2-hydroxybenzaldehyde
2-hydroxy-3-methoxybenzaldehide
–OH
Table 3 Protonation constants
of compounds 1–12 in methanol/
water 1/1 mixtures, Ic = 0.1
molÁdm-3 (NaCl),
log10 K1H
log10 K2H
log10 K3H
Compound
1
10.50 0.02
12.77 0.03
[13
–
\2
h = (25.0 0.1) °C
2
8.88 0.01
10.29 0.03
9.52 0.03
11.08 0.02
8.71 0.03
8.52 0.01
7.76 0.02
8.53 0.02
7.11 0.01
6.40 0.01
8.11 0.02
\2
7.22 0.03
3
4
[13
8.36 0.04
5
12.85 0.02
12.92 0.02
[13
9.19 0.01
6
\2
\2
\2
\2
\2
\2
\2
7
8
12.11 0.03
12.18 0.04
11.67 0.03
11.17 0.02
[13
9
10
11
12
Uncertainties are expressed as
standard error of the mean
(N = 3 or 4)
Table 4 Protonation constants of compounds 2 and 6 in methanol/water 1/4 mixtures, Ic = 0.1 molÁdm-3
(NaCl), h = (25.0 0.1) °C
log10 K1H
log10 K2H
log10 K3H
Compound
2
6
12.10 0.04
11.97 0.02
8.74 0.01
2.46 0.04
2.48 0.05
8.56 0.004
Uncertainties are expressed as standard error of the mean (N = 3 or 4)
phenyl ring is already distributed over the whole system, and the corresponding protona-
tion constant of amide group has a significantly higher value, at least above 12. The values
of 11.67 and 11.17 for log10 K1H were determined for compounds 10 and 11 possessing –Cl
atoms and –NO2 group, respectively, i.e. strong electron-withdrawing substituents at the
salicylidene part of the molecule. The effect of substituents on the –NH–N protonation was
also noticed and reported for the isonicotinoyl and benzoyl hydrazones [27, 28] where an
extra ring nitrogen in the isonicotinoyl derivative or electron withdrawing substituent in
benzoylhydrazone lowered the pK value of the amide group.
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The second protonation constant (K2H) was assigned to protonation of the hydroxyl
group situated in the position ortho to the C = N double bond in compounds 2–12. The
bands centered around 330 nm in the UV spectra of hydrazones in neutral solutions are
associated with excitation of the aldehyde part of the molecule [19, 20]. Upon deproto-
nation of the phenolic oxygen this band decreased and a new broad one, assigned to the
deprotonated aldehyde, appeared approximately around or above 400 nm. The appearance
of a new band at higher wavelength due to deprotonation of hydroxyl group was also
observed in the spectrum of salicylaldehyde benzoylhydrazone [29]. The highest K2H value
(except for 5) was determined for compound 2. For substituted salicylaldehyde derivatives
this constant had lower values, which is in accordance with constants determined for
corresponding aldehydes under the same experimental conditions. The small difference
between K2H values of compounds 2 and 6 (8.88 vs. 8.71) could be a consequence of an
inductive effect of the –OCH3 group situated in the ortho position to the hydroxyl group.
The effect was more pronounced for meta derivative (compound 7, pK = 8.52), which was
consistent with decrease of pK value of the starting aldehydes (Table 1) as well as of
2-methoxyphenol (9.99) and 3-methoxyphenol (9.652) with respect to the pK of phenol
(9.99) in water [30].
Compared to the salicylidene derivative, in compounds with electron-withdrawing
substituents (8–11), a marked decrease of the K value of the hydroxyl group was
observed, and the effect was, as expected, most pronounced for 11 bearing a nitro group
in the para position relative to the hydroxyl group since this group in that position
possesses resonance along with an inductive withdrawing effect. The inductive effect of
chlorine was more pronounced when situated in the ortho (compound 8) rather than in
meta position (9) with respect to the –OH group, and was the most noticeable in
compound 10 with two chlorines on the phenyl ring. The results obtained are in
accordance to those of the corresponding starting compounds and to those reported for
phenols with the same substituents [31, 32]. As can be seen in the cases of compounds 2
and 6, the increase with organic solvent content caused an increase in pK values, which
agrees with the results obtained for protonation of hydroxyl groups in 4-(2-pyridy-
lazo)resorcinol in methanol–water mixtures [33]. The most significant difference between
the protonation constant values of the –OH group of the aldehydes and of hydrazine was
noticed for compound 12. The high value of 10.7 for 2-hydroxyacetophenon in com-
parison to 8.56 for 2-hydroxybenzaldehyde is a consequence of the electron-donating
effect of the methyl group, resulting in a stronger intramolecular hydrogen bond in the
ketone than in salicylaldehyde. Based on the crystal structure of 2 [21] and 12 [34], it is
reasonable to assume that the intramolecular H-bonds in hydrazones are preserved in
solution, but the effect of the methyl group is diminished.
The third protonation constant (K3H) of compounds 3–5 with two hydroxyl groups at the
phenyl ring was assigned to the protonation of 3-OH, 4-OH, and 5-OH groups, respec-
tively. The increased stability of the –OH group situated in the ortho position, which
resulted in a higher pK value, was due to the strong intramolecular hydrogen bond between
this group and the azomethine nitrogen, as shown in Fig. 2 [35]. For all other investigated
aromatic hydrazones the third protonation constant was assigned to protonation of the
pyridine nitrogen. Based on the experimental results (protonation of nicotinic acid
hydrazide, Table 1) we have concluded that such protonation in MeOH/H2O 1/1 systems
was taking place approximately at or below pH 2. However, due to the fast hydrolysis of
studied compounds below this pH value, the corresponding equilibrium constants were not
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determined. In MeOH/H2O 1/4 mixtures the pK corresponding to the pyridine nitrogen in
compounds 2 and 6 are higher, and amount to approximately 2.5.
3.2 Hydrolysis
As a continuation of our previous work [21], in this study we investigated the kinetics of
hydrolysis of aroylhydrazones 1–12 in methanol/water 1/1 mixtures in both acidic and
basic conditions under the pseudo-first-order conditions. The time resolved UV absorption
spectra of all compounds in acidic solutions exhibited well-defined isosbestic
point(s) throughout the course of the reaction (Fig. 4 and Figs. S12–S22), indicating that
there was no accumulation of intermediate products between the aroylhydrazone and the
aldehyde resulting from a hydrolysis reaction. A single exponential decay of the absor-
bance at the selected wavelengths versus time (Fig. 4 and Figs. S12–S22) was successfully
fitted to the experimental data, implying first-order dependence with respect to the
aroylhydrazones.
The measurements showed that under the investigated acidic conditions
{c(HCl) = 0.01–0.20 molÁdm-3}, in accordance with our previous results [21], the
observed rate constant increased linearly with increase in H? concentration:
kobs ¼ k0 þ k00½HþŠ
ð2Þ
as shown in Fig. 5 for the compounds 4 and 9, and in Figs. S40–S49 for the other studied
compounds. These findings point to the first-order dependence with respect to [H?]. The
effect was more pronounced for compounds 4 and 7 bearing electron-donating groups in
their para positions relative to the C=N double bond.
The hydrolysis mechanism of hydrazone in acidic media involves the protonation of the
C=N nitrogen followed by nucleophilic attack of water and subsequent cleavage of the
0.7
1.0
0.6
0.5
0.8
0.4
0.6
0
10
20
t/min
30
40
0.4
0.2
0.0
250
300
350
400
Fig. 4 Time resolved UV spectra of compound 2 in methanol/water 1/1 mixture. The first spectrum was
recorded 2 min after preparation of solution, while other spectra were collected every 2 min over an hour:
c(2) = 5 9 10-5 molÁdm-3, c(H?) = 0.10 molÁdm-3, Ic = 1 molÁdm-3 (NaCl), l = 1 cm, h = (25.0
0.1) °C. The inset represents the time dependence of absorbance at 331 nm
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(a)
(b)
20
16
12
8
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4
0
0.00
0.05
0.10
0.15
0.20
0.00
0.05
0.10
0.15
0.20
[H+] / mol dm
[H+] / mol dm
Fig. 5 Dependence of kobs for a 4 and b 9 on H? concentration in methanol/water 1/1 mixtures: c(2 or
9) = 5 9 10-5 molÁdm-3, Ic = 1.0 molÁdm-3 (NaCl), h = (25.0 0.1) °C
(i)
(fast)
(slow)
(fast)
(fast)
(ii)
(iii)
(iv)
Fig. 6 Proposed mechanism of acid hydrolysis of hydrazones studied [21]
C–N bond (Fig. 6) [21]. The kinetic results obtained herein are consistent with this
mechanism.
According to the mechanism shown in Fig. 6, the dependence of the observed rate
constant on H? concentration can be expressed as:
k2K1½HþŠ
kobs ¼ kÀ2
þ
ð3Þ
1 þ K1½HþŠ
By taking into account the protonation measurements, the value of K1 is rather low, and
the product K1[H?] is much lower than 1, so Eq. 3 becomes:
kobs ꢀ kÀ2 þ k2K1½HþŠ
ð4Þ
which is in agreement with the observed linear dependence of kobs on [H?]. By comparison
of Eqs. 2 and 4 it is obvious that k00 = k2K1 whereas k0 = k-2. The values of the product
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Table 5 Values of the product
Compound
(k–2 r)/10-3 s-1
(k2K1 r)/10-3 dm3Ámol-1Ás-1
k2K1 and constant k-2 (Fig. 6) for
the acidic hydrolysis of aroylhy-
drazones 1–12 in methanol/water
1/1 mixtures: Ic = 1 molÁdm-3
(NaCl), h = (25.0 0.1) °C
1
&0
20.2 0.2
21.1 0.3
11.3 0.2
91.1 0.7
22.5 0.2
20.6 0.3
60.6 0.2
5.8 0.1
2
&0
3
0.26 0.03
&0
4
5
&0
6
&0
7
&0
8
0.84 0.01
0.46 0.02
0.94 0.04
2.1 0.1
&0
9
11.4 0.1
7.8 0.3
10
11
12
12.2 0.5
38.1 0.2
Uncertainties are expressed as
standard deviations
k2K1 and rate constant k-2 determined by a linear fit of the experimental data are listed in
Table 5.
A linear relationship between kobs and concentration of H? ions with negligible inter-
cept in the case of compounds 1, 2, 4–7, and 12 indicates the ‘‘irreversibility’’ of the
studied reaction under the experimental conditions. On the contrary, the existence of a
significant intercept in the case of compounds 3 and 8–11 suggests equilibrium between
species II and III (step (ii) in the proposed reaction mechanism, Fig. 6).
Since the protonation of the C=N nitrogen is the key factor in the hydrolysis of
aroylhydrazones in an acidic environment, a possible explanation of the results obtained
for compounds 8–11 is that electron-withdrawing –Cl and –NO2 groups decrease the
electron density on the C=N nitrogen and at the same time decrease its affinity for pro-
tonation, preventing hydrolysis of the hydrazone bond. This is consistent with a previous
study on hydrazone basicity [36] as well as with the stability study of salicylaldehyde
isonicotinoyl hydrazone analogs [10].
The electron-donating groups (–OH in the para-position of compound 4 and –OCH3 in
the para-position of compound 7 relative to the C=N double bond) were shown to
accelerate the hydrolysis in acidic media. These groups donate electrons to the azomethine
nitrogen via conjugation of C=N and aromatic ring, so the nitrogen becomes more basic,
and thus can be more easily protonated.
Additionally, the UV spectra of reaction mixtures recorded after the hydrolysis reaction
indicated that, in the case of compounds 1, 2, 4–7, and 12, the reaction proceeded to completion,
i.e. k–4 in step (iv) (Fig. 6) of the proposed reaction mechanism was negligible. Indeed, the UV
spectrum of the solution after reaction completion matched the sum of the spectra of the cor-
responding aldehyde and nicotinic acid hydrazide, the final products of hydrolysis. On the other
hand, this situation was not observed for compounds 3 and 8–11, suggesting the existence of an
equilibrium between the final hydrolysis products and intermediate IV (Fig. 6). Therefore, it can
be concluded that in these cases under acidic condition the hydrolysis reaction did not proceed to
completion. To confirm this we performed additional chromatographic experiments. The
kinetics of hydrolysis of compounds 3, 4, 10, and 11 in acidic medium was followed by means of
HPLC. The results are in agreement with those obtained by UV spectrometry. As can be seen in
Figs. 7 and S23, the hydrolysis of compound 4 was complete, whereas that of other compounds
was partial, anda after certaintime, the peak correspondingtothe hydrazonedid not decrease any
further. The retention times of reaction products, aldehydes and nicotinic acid hydrazide, were
123

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J Solution Chem (2016) 45:1227–1245
determined by separate experiments. The kobs values calculated from chromatographic data are
in good agreement with those determined spectrophotometrically. For compound 4, the kobs
calculated from UV data is (9.54 0.04) 9 10-4 s-1 and the one calculated from HPLC data is
(9.41 0.02) 9 10-4 s-1. For compound 10, the kobs calculated from UV data is
(4.37 0.01) 9 10-4 s-1 and the one calculated from HPLC data is (4.4 0.2) 9 10-4 s-1
For compound 11, the values are and
(11.422 0.003) 9 10-4 s-1
(10.027 0.003) 9 10-3 s-1, respectively.
.
In the [OH-] range investigated {c(NaOH) = 0.005–0.20 molÁdm-3}, negligible
spectral changes of compounds 1, 4, and 12 occurred indicating the lack of considerable
hydrolysis under these conditions (Figs. S24, S26, S34). On the other hand, the time
resolved absorption spectra of compounds 2 and 6–11 showed well-defined isosbestic
point(s), and a single exponential decay of the measured absorbance at the selected
wavelengths versus time were successfully fitted to the experimental data (Figs. S25, S28–
S33). The variations of the corresponding kobs with [OH-] are given in Fig. 8 and
Figs. S48–S53 in the supplementary information and obey the following relationship:
k6K5½OHÀŠ
kobs
¼
ð5Þ
1 þ K5½OHÀŠ
according to the mechanism proposed in Fig. 9.
(a)
(b)
100
80
60
40
20
0
aldehyde hydrazone
hydrazide
a)
93 min
72 min
51 min
30 min
9 min
0
1
2
3
4
5
0
20
40
60
80
100
t / min
t / min
(c)
(d)
hydrazone
hydrazide
aldehyde
80
70
60
50
40
123 min
98 min
73 min
48 min
23 min
3 min
4
0
1
2
3
0
20
40
60
80
100
120
t / min
t / min
Fig. 7 Selected chromatograms of a 4 and b 10 and time dependence of hydrazone peak area of c 4 and
d 10 in methanol/water 1/1 mixtures: c(4 or 10) = 5 9 10-5 molÁdm-3, c(H?) = 0.10 molÁdm-3; the
signals were monitored at 231 and at 259 nm, respectively
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J Solution Chem (2016) 45:1227–1245
1239
1.8
1.5
1.2
0.9
0.6
0.3
0.0
0.00
0.04
0.08
0.12
0.16
0.20
Fig. 8 Dependence of kobs for
2
on the OH- concentration in methanol/water 1/1 mixture:
c(2) = 5 9 10-5 molÁdm-3, Ic = 1.0 molÁdm-3 (NaCl), h = (25.0 0.1) °C; filled square experimen-
tal;—calculated
-O
-O
O
C
O
C
K5
H
N
N
C
H
+
OH-
N
N
C
H
+
H2O
(v)
(fast)
N
N
-O
-O
O
O
k6
H
C
C
N
N
C
H
C
N
N
H
+
H2O
(vi)
(slow)
rds
N
N
OH
-O
-O
O
C
H
O
k7
k-7
H
H
C
(vii)
N
N
H
C
(fast)
(fast)
C
N
N
H
N
O-
N
N
OH
-O
-O
O
H
O
H
k8
H
C
N
N
H
C
(viii)
C
N
NH2
+
C
O
N
O-
Fig. 9 Proposed mechanism of basic hydrolysis of hydrazones studied [21]
At low OH- concentrations, K5[OH-] is much lower than 1 and the reaction rate
becomes kobs = k6K5[OH-], whereas at higher hydroxide concentrations, where K5[OH-]
is much higher than 1, the reaction rate becomes kobs = k6. The rate-determining step in
alkaline solutions is believed to be attack of a water molecule on deprotonated hydrazone.
The parameters obtained by this study are collected in Table 6, and are consistent with our
previous studies in dioxane/water solvent [21].
In the case of compounds 3 and 5, the time resolved UV spectra indicate a multiple step
process (Figs. 10 and S27). Absorbance versus time traces obtained from the time resolved
spectra, indicated in the inset of Fig. 10, show biphasic character, and can be very well
fitted by considering two consecutive processes, both of which are pH independent
(Fig. 11). The exact nature of the two processes is not clear. The first process probably
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J Solution Chem (2016) 45:1227–1245
Table 6 Values of the parame-
ters K5 and k6 (Fig. 9) for the
basic hydrolysis of compounds 2
and 6–11 in methanol/water 1/1
mixtures: Ic = 1 molÁdm-3
Compound
(k6 r)/10-5 s-1
(K5 r)/dm3Ámol-1Ás-1
2
19 0.2
18 0.5
12 0.2
32 0.2
14 0.1
26 0.2
26 0.1
65
64 15
37
6
6
(NaCl), h = (25.0 0.1) °C
7
3
8
166 21
211 30
331 68
347 44
9
10
11
Uncertainties are expressed as
standard deviations
1.2
0.9
0.6
1.2
0.9
0.6
0.3
0.0
0
200
400
600
800
1000
t/min
250
300
350
400
450
500
Fig. 10 Time resolved UV–Vis spectra of compound 3 in methanol/water 1/1 mixture: c(3) = 5 9 10-5
molÁdm-3, c(OH-) = 0.07 molÁdm-3, Ic = 1 molÁdm-3 (NaCl), l = 1 cm, h = (25.0 0.1) °C; the inset
represents time dependence of absorbance at 330 nm
relates to hydrolysis of fully deprotonated hydrazones to the deprotonated nicotinic acid
hydrazide and 2,3- or 2,5-dihydroxybenzaldehyde (Fig. 9), while the second step possibly
relates to the oxidation of dihydroxybenzaldehyde.
To provide more detailed insight into the mechanism of hydrolysis of compounds 3 and
5 in basic media, the stabilities of nicotinic acid hydrazide, 2,3- and 2,5-dihydroxyben-
zaldehyde were investigated under the same experimental conditions. Nicotinic acid
hydrazide was proven to be stable on the basis of negligible spectral changes during the
monitored time (Fig. S35), whereas changes in the absorption spectra of 2,3- and 2,5-
dihydroxybenzaldehyde (Figs. S36 and S37) are evident. The bands at 427 nm (2,3-di-
hydroxybenzaldehyde) and 453 nm (2,5-dihydroxybenzaldehyde) decreased, while the
band at 304 nm increased, showing that those two aldehydes underwent some additional
reactions, most likely oxidation to their respective semiquinone radicals and quinones. In
fact, autooxidation of various ortho- and para-dihydroxy compounds to the quinones in
alkaline media has been previously observed [37, 38]. Unfortunately, we were not able to
obtain kinetic data which could be reliably processed, so a future study is needed to resolve
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J Solution Chem (2016) 45:1227–1245
1241
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Fig. 11 Dependence of kobs on
the OH- concentration for
compound 3 in methanol/water
1/1 mixture:
kobs1
c(3) = 5 9 10-5 molÁdm-3
Ic = 1.0 molÁdm-3 (NaCl),
h = (25.0 0.1) °C
,
kobs2
0.03
0.06
0.09
0.12
0.15
0.18
0.21
[OH-]/mol dm-3
this issue quantitatively. The same change (the appearance of another peak) was also
noticed in chromatograms of pure 2,3- and 2,5-dihidroxybenzaldehydes in basic media
acquired over time. In order to shed some more light on possible products of these reac-
tions, mass spectrometry was used (next section).
3.3 Mass Spectrometry
The full scan MS spectra of selected compounds (3, 4, 5, 7, 10, and the corresponding
aldehydes) in basic medium were acquired in both positive (ESI?) and negative (ESI–) ion
modes. The compounds were selected based on the following characteristics: 7 and 10
hydrolyzes completely in basic media according to the proposed scheme (Fig. 9), com-
pound 4 is stable under the experimental condition used, and compounds 3 and 5 are those
for which the formation of quinones was suspected. The MS spectra of the corresponding
aldehydes were acquired to confirm the proposed hydrolysis products based on the UV
data. As can be seen in Fig. 12a, almost the only signal in the ESI– MS spectrum of 7,
recorded immediately after the preparation of solution, was one at m/z = 270 assigned to
the [M–H]- ion. However, in the spectrum of 7 recorded after one day, the signal of the
deprotonated hydrazone molecule disappeared, and several signals at m/z = (151, 136,
123, and 108) arose (Fig. 12b). The signals were assigned to deprotonated aldehyde
{Mr(A) = 152} and some fragment ions: [A–CH3]-, [A–CO]-, and [A–CO–CH3]-. The
ESI– MS spectrum of 2-hydroxy-4-methoxybenzaldehyde revealed the same pattern, with
no change during time (Fig. S54). Based on these results, it can be concluded that the
products of hydrolysis in basic medium are the starting compounds, nicotinic acid
hydrazide and 2-hydroxy-4-methoxy-benzaldehyde, whereas fragment ions of aldehyde
were formed during the electrospray process.
The ESI–MS data of compound 10 are similar (Fig. S55). The signal at m/z = 308 ([M–
H]- ion), present in MS spectrum of 10 immediately after the solution preparation, dis-
appeared from the MS spectrum acquired after 24 h, while those corresponding to
deprotonated aldehyde [A–H]-, as well as for fragment ion [A–CO]- at m/z = 189 and
161, appeared. There were no changes in the MS spectrum of 3,5-dichloro-2-hydroxy-
benzaldehyde during time.
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J Solution Chem (2016) 45:1227–1245
(a)
100
80
60
40
20
0
270
292
300
100
200
400
m/z
(b)
100
80
60
40
20
0
151
292
aldehyde
fragment ions
136
108
123
100
200
300
400
m/z
Fig. 12 ESI=MS spectrum of compound 7 (Mr = 271) in basic methanol/water 1/1 solution immediately
after the preparation of solution (a) and after 24 h (b): c(7) = 5 9 10-4 molÁdm-3
,
c(NaOH) = 0.1 molÁdm-3
In MS spectra of hydrazones 7 and 10 acquired in the positive ion mode (t = 2 min),
the most intensive signals are those of [M ? Na]? ions. These signals were not observed in
the ESI? MS spectra of compounds 7 and 10 recorded after 24 h.
The MS spectrum of 4 acquired immediately after preparation of the solution, and after
24 h, were almost the same, with an intense signal corresponding to the [M–H]- ion at m/
z = 256. These findings are in agreement with those obtained by spectrometric measure-
ments, revealing the stability of 4 in basic media. Also, in the MS spectra of 2,4-dihy-
droxybenzaldehyde collected over time, no changes were observed, and the most intensive
signal was at m/z = 137, corresponded to the [M–H]- ion.
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J Solution Chem (2016) 45:1227–1245
1243
On the contrary, the time dependence MS spectra of the other two hydrazones with two
hydroxyl groups on the benzene ring, compounds 3 and 5, are completely different
(Fig. S56). In the ESI– MS spectra recorded immediately after the preparation of solution,
the base signals at m/z = 256 are assigned to the deprotonated hydrazone molecules. These
signals are missing in the spectra acquired after 24 h, and also, the signals corresponding to
aldehydes are not observed. However, a new one, at m/z = 225 assigned to the [M–H–31]-
ion, appeared. The loss of 31 units can be assigned to the loss of a CH2OH group. To get more
detailed insight into the structure of the ion, MS/MS experiments were conducted (Fig. S57),
and the proposed fragmentation mechanism, involving the formation of radical anion, is
shown in Fig. S58. The formation of radical ions was also proposed previously in MS/MS
analysis of phenolic compounds, including caffeic acid, with two hydroxyl groups on the
benzene ring [39]. In the MS spectra of 2,3- and 2,5-dihydroxybenzaldehydes acquired
immediately after the preparation of solutions, the signals of [M–H]- ions at m/z = 137 are
present, but they absent in the spectra collected after 24 h, suggesting the oxidation of
dihydroxy benzaldehydes to quinones. All the above findings are in complete agreement
with those obtained by the UV and HPLC measurements.
4 Conclusions
In this work the protonation constants of 12 differently substituted aromatic hydrazones
derived from nicotinic acid hydrazide, and the corresponding starting compounds, were
determined in methanol/water 1/1 mixtures by spectrophotometric-potentiometric batch
titrations. The electron-withdrawing substituents (–Cl and –NO2 groups) situated at the 3
and/or 5 position on the salicylidene part of the hydrazone molecule lowered the K1H value
assigned to protonation of the amide nitrogen, and also the K2H value which refers to
protonation of the hydroxyl group at the ortho position relative to the C=N double bond.
The effect is most pronounced for the derivative with –NO2 group situated at the para
position regarding the hydroxyl group, as both resonance and inductive electron-with-
drawing properties of this group contribute to the overall distribution of negative charge in
the hydrazone molecule.
The hydrolytic stability of hydrazones was studied in both acidic and basic media by
means of spectrophotometric, HPLC, and MS measurements. The kinetic data obtained
from different techniques are in excellent agreement. The hydrazones were hydrolyzed to
their starting compounds, nicotinic acid hydrazide and differently substituted benzalde-
hydes. As in the case of acid–base equilibria, the substituents had significant effects on the
kinetics of the hydrolytic processes. The electron-withdrawing substituents decreased
hydrolysis in acidic media, whereas the electron-donating groups (–OH, –OCH3) had the
opposite effect. The hydroxyl group at position 4 at the salicylidene part of the molecule
and methyl group at the azomethine carbon atom, prevented hydrolysis of the hydrazones
in basic media. In addition, the hydroxyl groups at position 3 and 5 led to the autooxidation
of the hydrolytic products, and conversion of aldehydes to their respective quinones.
The results described above can help in targeted design of the hydrazone-based or
analogous compounds aimed for biological and/or analytical purposes.
Acknowledgments This work was supported by the Croatian Science Foundation (project IP-2014-09-
4841).
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