Structural investigations of aroylhydrazones derived from nicotinic acid hydrazide in solid state and in solution

Article abstract of DOI:10.1016/j.saa.2013.01.028

Structural forms of aroylhydrazones derived from nicotinic acid hydrazide have been studied in the solid state by FT-IR spectroscopy and in solution by NMR, UV-Vis and ATR spectroscopy. The studied compounds were N′-benzylidene-3-pyridinecarbohydrazide (1), N′-(2,4- dihydroxyphenylmethylidene)-3-pyridinecarbohydrazide (2), N′-(5-chloro-2- hydroxyphenylmethylidene)-3-pyridinecarbohydrazide (3), and N′-(3,5- dichloro-2-hydroxymethoxyphenylmethylidene)-3-pyridinecarbohydrazide (4). The compound 1 adopted the most stable ketoamine form (form I, CONHNC) in the solid state as well as in various organic solvents. In mixtures of organic solvents with water the UV-Vis and ATR spectra implied intermolecular hydrogen bonding of 1 with water molecules. The presence of both tautomeric forms I and II (form II, COHNNC) was proposed for the solid substance and highly concentrated solutions of 2, whereas form I was detected as the predominant one in diluted solutions. For compounds 3 and 4 a coexistence of forms I and III (form III, CONHNHCCCO) was noticed in the solid state and in polar protic organic solvents. The conversion to form III was induced by increasing the water content in the solvent mixtures. This process was the most pronounced for compound 4. When exposed to daylight, an appearance of a new band was observed during time in the UV-Vis spectrum of 4 in organic solvent/water 1/1 mixtures, which implied that tautomeric interconversion was most likely followed by E/Z isomerisation.

Full text of DOI:10.1016/j.saa.2013.01.028

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 263–270
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Structural investigations of aroylhydrazones derived from nicotinic acid
hydrazide in solid state and in solution
a,
a
b
a,
⇑
⇑
-
´
ˇ
´
Nives Galic , Ivan Brodanac , Darko Kontrec , Snezana Miljanic
a Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia
Department of Organic Chemistry and Biochemistry, Ruder Boškovi´c Institute, Bijenicka 54, 10000 Zagreb, Croatia
b
-
ˇ
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
"
Substituents affected ketoamino–
enolimino equilibria of
aroylhydrazones.
"
Tautomerism was observed for
aroylhydrazone with two hydroxyl
groups.
"
"
Chloride substituents induced
formation of ketoamino tautomers.
When exposed to daylight E/Z
isomerisation in aqueous solutions
occurred.
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural forms of aroylhydrazones derived from nicotinic acid hydrazide have been studied in the solid
state by FT-IR spectroscopy and in solution by NMR, UV–Vis and ATR spectroscopy. The studied compounds
were N0-benzylidene-3-pyridinecarbohydrazide (1), N0-(2,4-dihydroxyphenylmethylidene)-3-pyridine-
carbohydrazide (2), N0-(5-chloro-2-hydroxyphenylmethylidene)-3-pyridinecarbohydrazide (3), and N0-
(3,5-dichloro-2-hydroxymethoxyphenylmethylidene)-3-pyridinecarbohydrazide (4). The compound 1
adopted the most stable ketoamine form (form I, ACOANHAN@CA) in the solid state as well as in various
organic solvents. In mixtures of organic solvents with water the UV–Vis and ATR spectra implied intermo-
lecular hydrogen bonding of 1 with water molecules. The presence of both tautomeric forms I and II (form II,
ACOH@NAN@CA) was proposed for the solid substance and highly concentrated solutions of 2, whereas
form I was detected as the predominant one in diluted solutions. For compounds 3 and 4 a coexistence of
forms I and III (form III, ACOANHANHAC@CACOA) was noticed in the solid state and in polar protic organic
solvents. The conversion to form III was induced by increasing the water content in the solvent mixtures.
This process was the most pronounced for compound 4. When exposed to daylight, an appearance of a
new band was observed during time in the UV–Vis spectrum of 4 in organic solvent/water 1/1 mixtures,
which implied that tautomeric interconversion was most likely followed by E/Z isomerisation.
Ó 2013 Elsevier B.V. All rights reserved.
Received 10 October 2012
Received in revised form 21 December 2012
Accepted 10 January 2013
Available online 31 January 2013
Keywords:
Aroylhydrazones
Tautomerism
Isomerisation
Spectroscopy
Introduction
compounds of this type possess antimicrobial, antituberculosis
and antitumor activities [1–3]. Recent studies have shown that nic-
The chemistry of aroylhydrazones has been intensively stud-
ied due to their analytical and biological applications. Many
otinic acid hydrazones could be considered as anti-inflammatory
and analgesic agents [4], and as a novel pharmacophore in the de-
sign of anticonvulsant drugs [5].
Aroylhydrazones can be involved in keto-enol tautomeric inter-
conversion (Scheme 1, forms I and II). Ketoamine tautomer (form I)
is usually the most stable form in solution and in the solid state
⇑
Corresponding authors. Tel.: +385 1 4606 191; fax: +385 1 4606 181 (N. Galic´),
tel.: +385 1 4606 202; fax: +385 1 4606 181 (S. Miljanic
´).
E-mail addresses: ngalic@chem.pmf.hr (N. Galic´), miljanic@chem.pmf.hr (S.
Miljanic´).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.01.028

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N. Galic et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 263–270
264
since it is stabilized by strong intermolecular or solute–solvent
hydrogen bonds [6–9]. However, upon complexation of metal cat-
ions, the tautomeric interconversion into the enolimine (form II)
commonly occurs [6–8,10]. If the hydroxyl group is situated in
ortho position regarding the C@N double bond, tautomeric equilib-
rium can also involve this part of the molecule (Scheme 1, form III).
Detailed investigations of Schiff bases derived from salicylaldehyde
and its derivatives have shown that the solvent polarity, as well as
the nature of the substituents in the salicylidene part of the mole-
cule affects the electron distribution in the system and the position
of the tautomeric equilibrium [11]. The polar solvents cause shift of
the interconversion to the ketoamine (form III), and the process is
accompanied by an appearance of a new band in absorption spec-
tra with kmax > 400 nm [11,12]. Electron donor groups in ortho and/
or para position (electron withdrawing substituents in meta posi-
tion) with respect to the C@N double bond also shift the equilib-
rium toward NH tautomer (form III) [11]. Limbach et al. have
shown that in Schiff bases derived from pyridoxal, protonation of
the pyridine nitrogen shift the tautomeric equilibrium to the
ketoamino form in the solid state and in the polar aprotic solvents
but only when there is aliphatic substituent on azomethine nitro-
gen. When the Schiff base nitrogen atom carries an aromatic sub-
stituent, protonation of ring nitrogen has little effect on the
equilibrium [13–15]. Apart from tautomeric interconversion, an
E–Z isomerisation of hydrazones due to the solvent effect, pH value
or UV irradiation can also occur (Scheme 1, form IV) [16–19]. Since
different tautomeric and isomeric forms can have diverse proper-
ties, the detailed study of such equilibria is relevant for successful
application of these compounds.
complexation of metal cations. As a consequence, these aroylhyd-
razones coordinate cations as doubly deprotonated, tridentate
O,N,O ligands both in the solution and in the solid state [10]. In
the present work, we extended the investigations of various equi-
libria (keto-enol tautomerism, E/Z interconversion, and hydrolysis)
to structurally related aroylhydrazones, derived from nicotinic acid
hydrazide and differently substituted benzaldehydes.
Experimental
Reagents
Compounds 1–4 were prepared by mixing equimolar amounts of
nicotinic acid hydrazide (Fluka) with aldehyde (benzaldehyde, 2,4-
dihydroxybenzaldehyde, 2-hydroxy-4-chlorobenzaldehyde and
3,5-dichloro-2-hydroxybenzaldehyde purchased from Sigma). The
reactions were carried out in dry ethanol under argon atmosphere
at 85 °C for 20 h. The solvent were evaporated from reaction mix-
tures and the solids were suspended in CH2Cl2 (EtOH was used for
2), filtered and dryed at 50 °C. Hydrazones were characterised by
standard methods. Deionized water and organic solvents of p.a. pur-
ity grade (Kemika) were used for spectroscopic measurements.
Apparatus
UV–Vis measurements were carried out using Varian Cary 3
spectrometer. The absorption spectra were recorded in the spectral
range from 200 nm to 500 nm. Conventional quartz cells (l = 1 cm)
were used throughout.
Previously we reported on the structural properties of N0-
salicylidene-3-pyridinecarbohydrazide and N0-(2-hydroxy-3-meth-
oxyphenylmethylidene)-3-pyridinecarbohydrazide [10,20,21]. In
solution and in the solid phase both compounds appear in form I
E [20]. E/Z interconversion as well as the formation of tautomeric
form III was not observed even in the systems containing water
[21]. The tautomeric interconversion into the form II occurred upon
1H and 13C NMR spectra were recorded on a Bruker Avance 600
spectrometer in deuterated DMSO. COSY spectra were recorded to
enable a complete assignment of signals. TMS was used as an inter-
nal standard.
FT-infrared spectra in transmission mode as well as in attenu-
ated total reflection (ATR) mode were acquired on a Bruker Equi-
nox 55 interferometer. Transmission spectra were obtained from
solid samples prepared as KBr pellets. For each pellet, 1 mg of
the solid sample and 100 mg of KBr were weighted, grounded in
an agate mortar and pressed. Spectra were recorded over the spec-
tral range between 4000 and 400 cmꢀ1 at 2 cmꢀ1 resolution. ATR
spectra were measured using the PIKE MIRacle ATR sampling
accessory with a diamond/ZnSe crystal plate. A pressure clamp
was used during the measurement of the solid samples, whereas
ATR spectra of the liquid samples were measured from a drop on
the crystal plate. The spectrum of the solvent or the solvents mix-
ture was used as a background while recording the ATR spectrum
of the respective hydrazone solution. The spectra were taken in the
single reflection configuration, over the spectral range 4000–
600 cmꢀ1 at 2 and 4 cmꢀ1 resolution for solids and liquids, respec-
tively. Thirty two scans were averaged to obtain a final spectrum.
HO
OH
form II
N
N
N
HO
O
form I
N
N
H
Samples preparation
Stock solutions of compounds 1–4 (c = 1 ꢁ 10ꢀ3 mol dmꢀ3
)
N
were prepared in chloroform, dioxan, acetonitrile (MeCN), dimeth-
ylsulphoxide (DMSO), and methanol (MeOH). The working solu-
tions for UV–Vis measurements were prepared in a 10.0 mL flask
in pure organic solvents as well as in MeCN/water, DMSO/water
and MeOH/water mixtures by adding stock solution of 1–4 and
an appropriate amount of organic solvent or water. Concentration
O
O
form III
H
N
N
H
of the hydrazones in the working samples was 5 ꢁ 10ꢀ5 mol dmꢀ3
.
Working samples for measurement of ATR spectra were prepared
by dilution of an appropriate volume of the DMSO stock solutions of
compounds 1–4 (0.40 mol dmꢀ3) with DMSO and water, resulting in
the final hydrazone concentration of 0.20 mol dmꢀ3. Solutions of all
N
Scheme 1. Possible tautomeric forms of aroylhydrazones.

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N. Galic et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 263–270
studied compounds were prepared in pure DMSO, but in different
2
volume portions of solvents when DMSO was mixed with water.
While 1 was soluble in the solvent mixtures in the volume ratios,
V(DMSO)/V(H2O), of 9/1, 8/2, 7/3, 6/4 and 5/5, a poor solubility of 2
and 3 allowed preparation of solutions in the mixed solvents only
in ratios V(DMSO)/V(H2O) of 9/1 and 8/2. At the given concentration
compound 4 precipitated from the DMSO solution upon addition of
even a small amount of water.
1
3
4
O
8
13
N
N
6
12
11
N
H
9
7
5
2
10
1
Results and discussion
The structural analysis of N0-benzylidene-3-pyridinecarbohyd-
razide (1), N0-(2,4-dihydroxyphenylmethylidene)-3-pyridinecarbo-
hydrazide (2), N0-(5-chloro-2-hydroxyphenylmethylidene)-3-pyr
idinecarbohydrazide (3), and N0-(3,5-dichloro-2-hydroxymet
hoxyphenylmethylidene)-3-pyridinecarbohydrazide (4), shown in
Scheme 2, was performed by different spectroscopic techniques
both in the solid state and in the solution. The solid state structure
of compounds 1–4 was investigated by FT-IR spectroscopy,
whereas the NMR, UV–Vis and FT-IR spectroscopies were used
for the structural analysis of aroylhydrazones in solvents of differ-
ent polarities as well as in the mixtures of organic solvents and
water.
The compound 1, derived from benzaldehyde with no hydroxyl
group in ortho position to C@N double bond, was chosen as a mod-
el system since no tautomeric interconversion can occur in this
part of the molecule (form I to form III). The hydroxyl group situ-
ated in para position to C@N double bond in 2 can increase the ba-
sicity of the nitrogen atom, whereas chlorides in meta position
(compounds 3 and 4) can enhance the acidity of phenolic group
[11]. As a consequence, both substituents can shift the tautomeric
interconversion of aroylhydrazones to form III.
HO
7
OH
1
6
3
O
8
13
N
4
12
11
N
H
9
5
2
10
2
N
HO
7
1
6
3
O
8
13
N
4
12
11
N
H
Cl
9
5
10
3
N
Cl
2
FT-IR spectra of the solid samples
HO
7
1
6
3
O
8
Infrared spectra of the solid samples 1–4 were taken in attenu-
ated total reflection (ATR) mode as well as in transmission mode
(KBr pellets). The spectra obtained by two IR techniques were in
a very good agreement, and small spectral differences, if observed,
were attributed to the preparation procedures of the measured
samples (Figs. S1–S4). Given the resemblance between the spectra
obtained by two IR techniques, and the following study of the
DMSO/water solutions in the reflectance mode, a preliminary
assignment of vibrational bands is given in Table 1 only for ATR
spectra [9,16,21–25].
13
N
N
4
12
11
N
H
Cl
9
5
10
4
Scheme 2. Structures of compounds 1–4.
A strong band at 1649 cmꢀ1 in the spectrum of 1 was assigned
as the amide I band, originating mainly from the stretching vibra-
tion of the C@O group as well as from the CAN stretching and NH
bending to a lesser extent (Fig. 1a). A weak band at 1665 cmꢀ1 im-
plied that some carbonyl groups were not involved in interactions
with adjacent molecules [21]. In addition to vibrational modes of
the amide group, the NH deformation and the NAC@O stretching
contributed to the intense amide II band at 1551 cmꢀ1, whereas
the CAN stretching and the NH bending gave rise to the weak
amide III band at 1355 cmꢀ1. The deformation of the NH group pro-
duced a strong band observed at 1283 cmꢀ1. Stretching mode of
the C@N bond and stretching vibrations of the phenyl and pyridyl
a band at 1635 cmꢀ1 was assigned to the stretching vibration of the
C@O group present in the molecular structure of form I, disappear-
ance of the NAH stretching band from the high wavenumber re-
gion, appearance of the amide II band as a shoulder (1554 cmꢀ1
)
and broadening of the amide III band (1350 cmꢀ1) pointed to a
change of the amide group. A broad band peaking at 1608 and
1600 cmꢀ1, associated with the stretching modes of the aromatic
rings and C@N bond in hydrazones, indicated a change in the cen-
tral part of the molecule most likely due to a conversion of keto-
amine (form I) to enolimine (form II) [16]. Moreover, a broad
irregularly shaped band of moderate intensity at 1229 cmꢀ1, asso-
ciated with the bending of the enol OH group, supported presence
of form II, beside form I, in the solid state of compound 2.
rings contributed to a band of moderate intensity at 1602 cmꢀ1
.
Other bands in the range from 1600 to 1300 cmꢀ1 were mostly
attributed to the stretching of the aromatic moieties in the molec-
ular structure of 1.
Interestingly, in the ATR spectra of the solids 3 and 4 two well
separated bands were obtained in the spectral range of the carbonyl
group stretching (Fig. 1c and d). Intense bands at 1669 cmꢀ1 and
1657 cmꢀ1 were assigned to the C@O stretching of the amide group
in compounds 3 and 4, respectively, whereas those observed at the
higher wavenumbers, 1688 cmꢀ1 (3) and 1695 cmꢀ1 (4), implied an
Unlike the well defined vibrational bands in the spectrum of 1,
clearly indicating the presence of form I, a pattern of the overlap-
ping bands between 1650 and 1500 cmꢀ1 in the ATR spectrum of
2 implied existence of various molecular forms (Fig. 1b). Although

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N. Galic et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 263–270
266
Table 1
Preliminary assignment of the vibrational bands in the ATR spectra of the solid samples 1–4 and their solutions in dimethylsulphoxide in the spectral range 1700–1100 cmꢀ1
.
Wavenumber (cmꢀ1
Solid
)
Assignment
DMSO solution
1
2
3
4
1
2
3
4
1688 m
1669 s
1695 m
1657 s
m
m
m
m
m
m
m
C@O (form III)
1665 w
1649 s
1679 s
1673 s
1677 s
1680 s
C@O +
C@O +
m
m
CAN + d NH (amide I)
CAN + d NH (amide I)
1635 s
1630 m
C@N conjugated (form II)
1608 m
1600 w
1577 m
1554 sh
1508 m
1610 w
1599 w
1590 w
1542 m
C@N;
C@N;
m
m
CC/CN (pyridyl);
CC/CN (pyridyl);
m
m
CC (phenyl)
CC (phenyl)
1602 w
1588 m
1551 s
1596 w
1590 w
1544 m
1603 w
1589 m
1562 m
1608 s
1603 w
1591 m
1558 m
1601 sh
1590 m
1558 m
1591 m
1561 w
1513 m
CC/CN (pyridyl);
m CC (phenyl)
d NH +
m
NAC@O (amide II)
m
m
m
m
m
m
m
m
m
m
CC (phenyl)
CC (phenyl)
CC (phenyl)
CC (phenyl, pyridyl)
CC (phenyl)
CC (phenyl, pyridyl)
CC (pyridyl)
CC (phenyl, pyridyl)
CAN + d NH (amide III)
CC (pyridyl)
1492 w
1473 w
1492 w
1481 w
1476 w
1463 w
1480 s
1484 w
1481 m
1473 w
1464 w
1449 s
1452 m
1434 w
1418 w
1377 w
1349 m
1447 w
1421 w
1427 w
1416 w
1432 w
1419 w
1369 w
1336 s
1431 m
1420 m
1379 w
1344 m
1450 w
1416 w
1417 w
1422 m
1374 w
1349 m
1355 w
1330 w
1350 w
1324 w
1295 m
1366 m
1285 s
1356 w
1326 w
1287 s
1298 m
1284 m
1277 m
1286 s
1294 s
d NH; d OH
d NH
d OH
d OH (form II)
dip CH
m CAN
dip CH
dip CH
m CAO
1283 s
1280 vs
1271 s
1265 sh
1223 m
1271 sh
1274 m
1229 m
1222 w
1203 w
1176 w
1154 w
1131 m
1214 w
1187 m
1218 m
1197 w
1179 m
1164 m
1229 w
1195 w
1223 m
1184 m
1200 w
1169 w
1178 w
1167 w
1153 w
1127 w
1185 w
1150 m
1118 m
1149 m
1154 w
1117 w
1161 w
1136 w
Abbreviations: sh, shoulder; w, weak; m, medium; s, strong; vs, very strong;
m
, stretching; d, deformation; ip, in plane.
Table 2
1H NMR spectra of 1–4 in dimethylsulphoxide.
(a)
0.3
Proton
d (ppm)
0.2
0.1
1
2
3
4
H (C1) (O)
H (C2)
H (C3) (O)
H (C4)
H (C5)
H (C7)
H (C10)
H (C11)
H (C12)
H (C13)
H (N)
7.762 (dd)
7.4731 (m)
7.4731 (m)
7.4731 (m)
7.762 (dd)
8.484 (s)
12.064 (s)
6.405 (dd)
9.987 (s)
6.376 (d)
7.354 (d)
8.531 (s)
9.094 (s)
8.769 (d)
7.567 (dd)
8.276 (d)
11.358 (s)
12.316 (s)
6.967 (dd)
7.321 (dd)
–
7.689 (d)
8.644 (s)
9.111 (s)
8.782 (d)
7.578 (dd)
8.291 (d)
11.185 (s)
12.648 (s)
–
7.679 (d)
–
7.608 (d)
8.582 (s)
9.118 (s)
8.806 (d)
7.598 (t)
8.302 (d)
12.362 (s)
0.0
0.3
(b)
0.2
0.1
9.104 (s)
8.779 (d)
7.576 (dd)
8.281 (d)
12.021 (s)
0.0
0.2
A
(c)
(d)
of the aroylhydrazone molecules having a hydroxyl group at the
ortho position with respect to the C@N bond, it was very likely that
form III was formed. Owing to the electron withdrawing ability,
chloride substituents on the phenyl moiety facilitated transfer of
the hydrogen atom from the hydroxyl group to the nitrogen atom
of the C@N group. Nevertheless, strong bands at 1271 cmꢀ1 (3)
and 1277 cmꢀ1 (4), assigned to the OH deformation modes, indi-
cated the presence of the form I.
0.1
0.0
0.1
0.0
1H and 13C NMR spectra
NMR spectral data of compounds 1–4 in DMSO are given in Ta-
bles 2 and 3. A complete assignment of signals corresponding to
aromatic protons was enabled by 2D (COSY) 1H NMR spectra (data
not shown). In the 1H NMR spectra of 2–4 strong deshielding of hy-
droxyl protons (d > 12 ppm) due to intramolecular hydrogen bonds
was observed. The similar downfield shifts of the AOH signals were
recorded for analogues aroylhydrazones derived from nicotinic
3500 3000 1800 1600 1400 1200 1000 800
Wavenumber / cm-1
600
Fig. 1. ATR spectra of the solid substances 1 (a), 2 (b), 3 (c) and 4 (d).
additional carbonyl group in the molecular structure. Given the
possibility of tautomeric conversion in the phenylmethylidene part

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N. Galic et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 263–270
Table 3
form I E [8,20,21]. The signals of azomethine protons in aroylhyd-
razones in a Z configuration would be at lower field, approximately
around 8.32 ppm, as in the salicylaldehyde benzoyl hydrazone, due
to increased electron density of the HC@N double bond [16].
Although the intramolecular OH—N@C bond was noticed, that
was not the case with the tautomeric interconversion leading to
form III, since no splitting of methine proton signal was observed
in the spectra of 2–4.
ThepresenceofformI forcompounds 1–4 inDMSO was confirmed
by 13C NMR spectra. The signals observed at ꢂ160 ppm are typical for
C@Omoietyofamidegroup(formI), whilethesignalsofthesamecar-
bon atom (C8) in the tautomeric form III should be at ꢂ100 ppm
[9,17]. In addition, the signals at ꢂ180 ppm typical for C1 atom in
form III were missing in the spectra of aroylhydrazones 2–4.
13C NMR spectra of 1–4 in dimethylsulphoxide.
C atom
d (ppm)
1
2
3
4
1
2
3
4
5
6
7
8
9
10
11
12
13
129.317
127.666
130.714
127.666
129.317
134.614
148.949
162.195
129.668
152.741
149.066
124.043
135.896
161.553
103.163
161.40
108.284
131.767
110.937
149.01
160.005
129.304
152.739
150.104
124.048
135.804
156.531
118.702
131.402
123.521
127.959
121.107
146.741
162.049
129.075
152.926
149.135
124.053
135.927
152.731
123.443
130.819
121.997
128.832
121.133
147.907
162.084
128.528
153.132
149.178
124.066
135.987
UV–Vis spectra
acid hydrazide and salicylaldehyde or o-vanillin [20,21]. The sig-
nals of NH protons in compounds 1–4 were observed at (11.2–
12.4) ppm, indicating hydrogen bond formation between solvent
molecules and amide proton. The signals at (8.5–8.6) ppm, as-
signed to azomethine protons, pointed out that all investigated
aroylhydrazones in DMSO adopted the most stable conformation,
The absorption electronic spectra of 1–4 were acquired in sol-
vents of different polarities as well as in the mixtures of organic
solvents with water (Table 4). The spectra were recorded immedi-
ately after the preparation of solutions and during the time, up to
several days. The bands at ꢂ220 nm, attributed to the
p ?
p⁄ tran-
sition of the amide C@O group in investigated hydrazones, was also
Table 4
UV–Vis spectra of 1–4 in different solvents.
Solvent
k (nm) (10ꢀ4 /L molꢀ1 cmꢀ1
e )
1
2
3
4
*
Chloroform
342 (0.96)
299 sh (1.42)
290 (1.74)
244 (1.35)
343 (0.92)
304 sh (1.85)
294 (2.22)
242 (1.47)
292 (1.99)
293 (2.14)
*
Dioxan
337 (1.18)
298 (1.66)
289 (1.98)
343 (1.03)
304 (1.88)
294 (2.20)
242 (1.49)
*
Acetonitrile (MeCN)
336 (1.09)
297 (1.79)
286 (2.08)
239 sh (1.49)
220 (1.92)
b
338 (1.01)
301 (2.22)
291 (2.53)
242 sh (1.61)
293 (2.15)
217 (1.53)
DMSO
MeOH
455 (0.09)
338 (1.19)
307 (1.97)
296 (2.08)
400 (0.12)a,c
339 (1.02)
303 (1.97)
292 (2.23)
221 (2.27)
336 (2.46)
304 (1.34)
294 (1.08)
400 (0.05)a
334 (2.40)
303 (1.47)
246 (1.25)
216 (1.93)
339 (1.48)
301 (1.51)
291 (1.69)
400 (0.03)a,c
339 (1.21)
299 (1.60)
289 (1.87)
219 (2.04)
302 (2.18)
300 (2.33)
218 (1.61)
MeCN/H2O 1/1
400 (0.05)a
331 (2.58)
302 (1.69)
244 (1.40)
216 (1.99)
400 (0.03)a,c
335 (1.12)
298 (1.75)
286 (2.02)
239 sh (1.46)
220 (1.96)
400 (0.12)a,c
337 (1.00)
302 (2.12)
292 (2.39)
222 (2.14)
296 (2.36)
217 (1.51)
b
DMSO/H2O 1/1
MeOH/H2O 1/1
400 (0.01)a,c
339 (1.28)
298 (1.71)
290 (1.94)
407 (0.22)a,c
337 (0.99)
304 (1.91)
294 (2.10)
333 (2.49)
302 (1.53)
290 sh (1.10)
300 (2.44)
400 (0.05)a
332 (2.58)
302 (1.69)
243 (1.37)
215 (1.95)
400 (0.02)a,c
337 (1.23)
298 sh (1.84)
289 (2.13)
218 (2.09)
400 (0.33)a,c
337 (0.91)
302 (1.86)
293 (2.06)
222 (2.09)
298 (2.61)
218 (1.63)
*
Not soluble.
a
The bands corresponding to the absorption of the form III.
b
c
The bands corresponding to the absorption of the form III appeared during the time.
The E/Z photoizomerization occurred.

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1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.5
0.4
0.3
0.2
0.1
0.0
(a)
A405
A
A
DMSO
0.8
0.6
0.4
0.2
0.0
DMSO/H2O 9/1
DMSO/H2O 8/2
DMSO/H2O 7/3
DMSO/H2O 6/4
DMSO/H2O 5/5
336 nm
0
10 20 30 40 50 60
t / min
384 nm
t = 2 min
t = 24 h
t = 48 h
t = 72 h
t = 96 h
406 nm
300
350
400
450
500
250
300
350
400
450
500
Wavelength / nm
Wavelength / nm
Fig. 3. UV–Vis spectra of 4 in DMSO/H2O 1/1 mixture during the time when
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(b)
solution was exposed to light. Inset: The absorbance at 405 nm during the time
A
when solution was kept in the dark. c(4) = 5 ꢁ 10ꢀ5 mol dmꢀ3
.
1
2
3
4
5
6
DMSO
DMSO/H2O 9/1
DMSO/H2O 8/2
DMSO/H2O 7/3
DMSO/H2O 6/4
DMSO/H2O 5/5
bond formation between the ANH protons and solvent molecules
[21]. No change in the absorption spectra of 1 in above mentioned
solvents was observed during the time. Small increase in absor-
bance at 336 nm with increase of water content in DMSO/H2O mix-
tures in UV spectra of 2 also indicated the formation of hydrogen
bonds. The appearance of a new band centred around 400 nm, as-
signed to form III, was recorded in 1/1 organic solvent/water mix-
tures. Slight increase in absorbance, (ꢂ0.03 after 24 h and ꢂ0.09
after 120 h) at 400 nm was noticed in MeCN/water mixture,
whereas the increase in absorbance in DMSO/water system at
same wavelength was even lower, ꢂ0.02 after 120 h. The shift of
tautomeric equilibrium to form III, by adding water to the system,
was very pronounced for compound 4 (Fig. 2b), and in much less
extent for 3. In the absorption spectra of 4 a new, broad band in
the spectral region (380–500) nm appeared. It should be pointed
out that for 4 (and less for 3) the prominent change in the absorp-
tion spectra were observed in solvents containing water during the
time, but only if the solutions were exposed to light. As far as the
solutions were kept in the dark, no time dependence of absorbance
was noticed. As an example, the UV–Vis spectra of 4 in the DMSO/
H2O 1/1 system are shown in Fig. 3. The absorbance at 294 nm de-
creased, the band with kmax = 336 nm disappeared as well as the
one with kmax = 406 nm, whereas the new band (kmax = 384 nm)
appeared. These spectral changes, accompanied by the occurrence
of isosbestic points at 274 nm and 361 nm, can be assigned to the
E/Z photoisomerisation of aroylhydrazone as suggested on
Scheme 3. The E/Z photoisomerisation was already reported for
ketoenamines including hydrazones [27]. Although the formation
of form III was noticed in spectra of 2, the light induced additional
changes in the absorption spectra were not. Presumably that can
be a consequence of the presence of two hydroxyl groups in 2
which probably stabilize E form forming additional hydrogen
bonds in the system [11].
1
2
3
6
3
5
4
2
6
1
300
350
400
450
500
Wavelength / nm
Fig. 2. UV–Vis spectra of 1 (a) and 4 (b) in DMSO and DMSO/H2O mixtures:
V(DMSO)/V(H2O) 9/1, 8/2, 7/3, 6/4 and 5/5 acquired immediately after preparation
of solutions. c(1) = c(4) = 5 ꢁ 10ꢀ5 mol dmꢀ3. Arrows show the change in absor-
bance with the increase in water content in DMSO/water mixtures.
noticed in the UV spectra of nicotinic acid hydrazide. The bands in
the spectral region (286–307) nm can be assigned to the azome-
thine C@N bonds and aromatic rings [26], while the bands above
330 nm to the part of the aroylhydrazones originated from alde-
hydes [10,21]. That was confirmed by recording the absorption
spectra of the starting aldehydes under the same experimental
conditions.
On the basis of the comparison of NMR data for 1–4 in DMSO,
and absorption data in various solvents (Table 4), it was concluded
that in solution all investigated aroylhydrazones predominantly
adopted the most stable configuration (form I E). As expected, in
the UV–Vis spectra of compound 1 in solvents of different polari-
ties, no absorption above 400 nm was observed since there is no
possibility of tautomeric interconversion to form III. Also, no
hydrolysis in solvents containing water was recorded. In the
absorption spectra of 2–4 in polar, protic solvents and in the mix-
tures of organic solvent with water, the band centred around
400 nm appeared, indicating the presence of form III. The effect
was most pronounced in the electronic absorption spectra of 4 as
there were two chloride atoms in meta positions, influencing the
tautomeric equilibrium. As for 1, the hydrolysis was not observed
for 2–4.
ATR spectra
A tentative assignment of the ATR spectra of compounds 1–4 in
DMSO (0.20 mol dmꢀ3) is given in Table 1 [9,16,21ꢀ25]. In the
spectra of all solutions the intense bands around 1680 cmꢀ1
and 1290 cmꢀ1 were dominant. The former band was assigned to
the C@O stretching of the amide group (amide I) and the latter
to the mixed NH and OH bending vibrations. Both bands indicated
the presence of form I of the studied hydrazones (Fig. 4). However,
by comparing the ATR spectrum of 2 with the spectra of other
studied aroylhidrazones, broader and weaker amide II
(1561 cmꢀ1) and amide III (1356 cmꢀ1) bands were observed,
accompanied by the appearance of a strong band at 1630 cmꢀ1
The influence of water content in the mixed organic solvent–
water system on keto-enol tautomeric interconversion and possi-
ble E/Z isomerisation of 1–4 was studied more detailed in the case
of DMSO and acetonitrile. The volume ratio (V(organic solvent)/
V(water)) was changed from 9/1 to 5/5. The addition of water into
the system caused increase in absorbance at 303 nm (DMSO) and
at 297 nm (MeCN) in the case of 1 for approximately 0.12 units
(Fig. 2a). These spectral changes can be assigned to the hydrogen

´
269
N. Galic et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 263–270
Cl
HO
O
E OH form
−
N
Cl
N
H
Cl
O
N
Cl
Cl
O
NH
O
h
ν
O
HN
H
N
N
H
Cl
N
N
E NH form
−
Z NH form
−
Scheme 3. Possible mechanism of E/Z isomerisation of 4.
0.010
0.008
0.006
0.010
0.005
0.000
0.005
DMSO
(a)
DMSO/H2O 9/1
DMSO/H2O 8/2
DMSO/H2O 7/3
DMSO/H2O 6/4
DMSO/H2O 5/5
A
0.004
(b)
(c)
(d)
0.002
0.000
1800
1700
1600
1500
1400
1300
1200
1100
A
0.000
0.010
Wavenumber / cm-1
Fig. 5. ATR spectra of 1 in DMSO and DMSO/water mixtures: V(DMSO)/V(H2O) 9/1,
8/2, 7/3, 6/4 and 5/5. c(1) = 0.20 mol dmꢀ3
.
0.005
0.000
spectrum of 2 in the solid state, but was not observed either in
the UV–Vis spectrum or in the NMR spectra of the respective
DMSO solution. Discrepancy between the electronic spectrum
and the vibrational spectrum was attributed to the different con-
centrations, 5 ꢁ 10–5 mol dmꢀ3 and 0.20 mol dmꢀ3, used in UV–
Vis and ATR measurements, respectively. It was suggested that in
the diluted solution solute–solute intermolecular interactions,
which otherwise stabilize enolimine molecules, weakened, en-
abling interconversion of the molecules to the most stable keto-
amine form I. In addition, a detection of only form I by NMR
spectroscopy, contrary to the observation of forms I and II by IR
spectroscopy, was associated with different time scales of these
two spectroscopic methods. Ketoamine–enolimine equilibrium
was most likely fast on the NMR time scale, and slow on the IR time
scale [16]. Although forms I and III were present in the solid state,
new bands indicative of form III were not observed in the spectra of
DMSO solution of hydrazones 3 and 4 having chloride substituents
on the benzene ring (Fig. 4c and d).
0.005
0.000
1800
1700
1600
1500
1400
1300
1200
1100
Wavenumber / cm-1
Fig. 4. ATR spectra of 1 (a), 2 (b), 3 (c) and 4 (d) in DMSO. c = 0.20 mol dmꢀ3
.
(Fig. 4b). The new band was assigned to the stretching of the C@N
bond in the molecular structure of the enolimine tautomer, conju-
gated with the nearby pyridine ring and the hydrazone C@N bond
[9,25]. The existence of form II was also suggested by the FT-IR

´
N. Galic et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 263–270
270
Given that water can induce interconversion of tatutomeric
form II, involving the carbonyl group on the pyridine ring, was no-
ticed in the solid substance and at high concentration in solution.
On the other hand, chlorine atoms in meta positions on the phenyl
ring enhanced acidity of the hydroxyl group in ortho position,
resulting in formation of ketoamine form III. In diluted solutions
form III was favoured in polar organic solvents and in mixtures
with water. In aqueous environment it was stable in the dark,
but was subjected to an additional equilibrium when exposed to
daylight. There were strong indications that E/Z photoisomerisa-
tion occurred.
and/or isomeric forms of the hydrazone molecules [16,19], which
was observed for the compounds 2–4 in organic solvent/water
mixtures by UV–Vis absorption spectroscopy, the ATR spectra of
aroylhydrazones (0.20 mol dmꢀ3) in DMSO/water mixtures were
recorded. Thereby, DMSO/water solutions of 1 were prepared in
the volume ratios V(DMSO)/V(H2O) 9/1, 8/2, 7/3, 6/4 and 5/5, while
compounds 2 and 3 were soluble only in the mixed solvents of
V(DMSO)/V(H2O) 9/1 and 8/2. Moreover, the addition of water
caused precipitation of the compound 4 from the DMSO solution
even at concentration of 0.02 mol dmꢀ3, preventing a structural
study of 4 in DMSO/water mixtures by infrared spectroscopy.
In the ATR spectrum of the DMSO solution of 1 the prominent
bands at 1679 cmꢀ1 and 1285 cmꢀ1, assigned to the carbonyl group
stretching and the amino group bending, respectively, changed the
most significantly when water was added in the system. By
increasing the water content in the solvent mixture, both bands
decreased in intensity, got broader and shifted (Fig. 5). With regard
to the spectrum of 1 in pure DMSO, a downward shift of 21 cmꢀ1
for the C@O stretching band and an upward shift of 15 cmꢀ1 for
the NH bending band were observed in the spectrum of the mix-
ture containing equal volumes of DMSO and water. The obtained
spectral changes indicated an involvement of 1 in interactions with
water through the amide group [21,28]. The broadening of the
band assigned to the stretching of the C@N bond (1603 cmꢀ1) also
pointed to predominant hydrogen bonding of 1 with water mole-
cules, which was consistent with the findings based on the UV–
Vis spectra. The intensities of the amide II (1562 cmꢀ1) and the
amide III (1366 cmꢀ1) bands were not affected by the water con-
tent increase, confirming the stability of the ketoamine form I of
1 in the DMSO/water mixtures.
The ATR spectra of 2 and 3 in DMSO and in solutions containing
water did not differ significantly. For both 2 and 3 the same trend
as for 1 was observed concerning the bands originated from the
C@O stretching as well as the NH and OH bending (spectra not
shown). Along with decrease in intensity and broadening, the
amide I band at 1673 cmꢀ1 (2) and 1677 cmꢀ1 (3) was shifted to-
wards lower wavenumbers for 7 cmꢀ1 (2) and 5 cmꢀ1 (3), whereas
the band corresponding to the polar groups deformation at
1287 cmꢀ1 (2) and 1286 cmꢀ1 (3), appeared at wavenumbers high-
er for 3 cmꢀ1 in the spectra of compounds in DMSO/water mixture,
V(DMSO)/V(H2O) 8/2. Regardless of water content in the studied
mixtures, significant changes either in position or in intensity of
the bands assigned to the C@N stretching modes in forms I and II
for 2 and in form I for 3, were not observed. It was assumed that
at high aroylhydrazone concentration the water content up to
20% did not affect tautomeric equilibria in the studied systems,
but rather induced hydrogen bonding with water molecules, stabi-
lizing in that way the present forms in the solutions.
Acknowledgements
This work was supported by the Ministry of Science, Education
and Sports of the Republic of Croatia (Projects 119-1191342-2959,
119-1191342-2960, 119-1191342-1083 and 098-0982904-2910).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.01.028.
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