Synthesis and characterization of a series of isoniazid hydrazones. Spectroscopic and theoretical study

Article abstract of DOI:10.1016/j.molstruc.2016.12.018

A family of hydrazones of isoniazid and a group of hydroxybenzalaldehydes (vanillin, 5-bromovanillin, 5-chlorosalicylaldehyde and 5-bromosalicylaldehyde) were obtained and fully characterized. The results, including theoretical data, are comparatively ana

Full text of DOI:10.1016/j.molstruc.2016.12.018

Accepted Manuscript
Synthesis and characterization of a series of isoniazid hydrazones. Spectroscopic and
theoretical study
Verónica Ferraresi-Curotto, Gustavo A. Echeverría, Oscar E. Piro, Reinaldo Pis-Diez,
Ana C. González-Baró
PII:
S0022-2860(16)31308-4
10.1016/j.molstruc.2016.12.018
MOLSTR 23216
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 21 June 2016
Revised Date: 17 November 2016
Accepted Date: 5 December 2016
Please cite this article as: V. Ferraresi-Curotto, G.A. Echeverría, O.E. Piro, R. Pis-Diez, A.C. González-
Baró, Synthesis and characterization of a series of isoniazid hydrazones. Spectroscopic and theoretical
study, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2016.12.018.
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ACCEPTED MANUSCRIPT
Synthesis and characterization of a series of isoniazid hydrazones. Spectroscopic and theoretical
study
1
2
2
1
Verónica Ferraresi-Curotto , Gustavo A. Echeverría , Oscar E. Piro , Reinaldo Pis-Diez , Ana C.
González-Baró
1
*
1
CEQUINOR (CONICET, UNLP), CC 962, B1900AVV, La Plata, Argentina.
IFLP (CONICET, UNLP), CC 67, B1900AVV, La Plata, Argentina.
2
*Corresponding author: agb@quimica.unlp.edu.ar
1

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Abstract
A family of hydrazones of isoniazid and a group of hydroxybenzalaldehydes (vanillin, 5-
bromovanillin, 5-chlorosalicylaldehyde and 5-bromosalicylaldehyde) were obtained and fully
characterized. The results, including theoretical data, are comparatively analyzed along with the
already reported hydrazone of o-vanillin. The crystal structures of three compounds were
determined. The hydrazones obtained from halogenated aldehydes are isomorphic and chiral to
+
-
-
each other. Structures are further stabilized by (pyr)NH ···Cl and OwH···Cl bonds. The vanillin
hydrazone shows a conformer that differs from the previously reported. Neighboring molecules
are linked to each other through OH···N(pyr) bonds, giving rise to a nearly planar polymeric
structure. The conformational space was searched and geometries were optimized both in the gas
phase and including solvent effects by DFT. Results are extended to describe the 5-bromovanillin
hydrazone. FTIR, NMR and electronic spectra were measured and assigned with the help of
computational calculations.
Keywords: Crystal structures; spectroscopy; DFT calculations; Isoniazid; Schiff bases.
1
. Introduction
The hydrazone functional group has been extensively studied [1 and references therein].
Hydrazones play important roles in various fields, from organic synthesis and medicinal
chemistry to supramolecular chemistry, and have many applications. The straightforward
synthesis, the possibility of including different substituents, and the stability towards hydrolysis
of hydrazones can be cited as reasons for their attractiveness.
The chemical versatility of hydrazones can be mainly attributed to the functional diversity of the
azomethine group, particularly the C=N–N moiety. The structure of a hydrazone shows (i)
nucleophilic imine and amino-type (more reactive) nitrogens, (ii) an imine carbon that has both
electrophilic and nucleophilic character, (iii) configurational isomerism stemming from the
intrinsic nature of the C=N double bond, and (iv) in most cases an acidic N–H proton. These
structural motifs give determines the physical and chemical properties of the hydrazone, playing
a crucial role in its applications.
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Hydrazones are usually obtained by condensation of one hydrazine or hydrazide with a ketone or
aldehyde. The hydrazide here employed is Isoniazid, the hydrazide of isonicotinic acid (INH, see
Figure 1). It has been recognized as an effective antituberculous agent and it is still employed in
the treatment and prevention of this disease. However, actual formulations containing INH show
several undesired side effects. On the other hand, it has been determined that hydrazones
obtained from the condensation of isoniazid and some hydroxy-aldehydes are less toxic while
preserving activity. This fact can be attributed to the inactivation of the NH group of INH upon
2
hydrazone formation [2 and references therein]. Moreover, hydrazones from INH are interesting
chemical systems because they possess an additional nucleophilic cite, the pyridine nitrogen
atom.
Figure 1. Structure of Isoniazid.
As part of our study on Schiff base formation with aromatic hydroxy-aldehydes [2, 3], a complete
study of a family of hydrazones of INH was carried out with a group of aldehydes depicted in
Figure 2, including vanillin (4-hydroxy-3-methoxybenzaldehyde, Va) and its derivative 3-bromo-
4
-hydroxy-5-methoxybenzaldehyde (5-bromovanillin, BrVa). Two derivatives of salicylaldehyde
are also included, namely 5-chloro-2-hydroxybenzaldehyde (5-chlorosalicylaldehyde, ClSal) and
5
-bromo-2-hydroxybenzaldehyde (5-bromosalicylaldehyde, BrSal).
Figure 2.Structure of the hydroxyl-aldehydes. (a) Vanillin, (b) Br-vanillin, (c) 5-
Chlorosalicylaldehyde, (d) 5-Bromosalicylaldehyde.
3

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The present article involves a complete experimental and theoretical characterization of the four
hydrazones and the comparative analyses with the hydrazone of o-vanillin (2-hydroxy-3-
methoxybenzaldehyde, o-HVa) and INH, INHoVa, already reported by our group (Figure 3) [2].
Figure 3. Structure of the obtained hydrazones.
For the compounds which afforded suitable single crystals the solid state structures were
determined by X-ray diffraction methods. A detailed analysis of inter- and intra-molecular H-
bonds is conducted to explain their effects on structure stabilization. FTIR, NMR and electronic
absorption spectra were recorded and assigned with the assistance of results from computational
methods based on the Density Functional Theory (DFT).
2
2
. Experimental section
.1. General procedure for the synthesis of isonicotinoyl Schiff bases
The compounds were synthesized according to an adaptation of the reported procedure for the
analytical determination of Isoniazid with Va [4]. Twenty milliliters of an ethanolic solution
containing 3 mmol of the aldehyde (Sigma) was drop-wise added to a solution of 1.5 mmol of
Isoniazid (Sigma) in ten mL of ethanol (Merck), with continuous stirring and slight heating.
Excess of aldehyde has been already employed in previous work [2] as allows obtaining the
products in milder experimental conditions, avoiding reflux or long-time heating at high
temperature, and have shown to improve the yield. Drops of concentrated hydrochloric acid
(Merck) were added to reach a pH value of 5. Upon standing, the products were obtained and the
solids were filtered out from the solution, washed repeatedly with cold ethanol and then dried in a
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desiccator. Melting points were determined using a Bock monoscop “M” instrument as an
additional criteria of the products.
It is worth mentioning at this point that the hydrazone obtained by the condensation reaction of
INH with vanillin (INHVa) has been previously reported by other authors [5] following a
different synthetic method and the crystal structure of a different conformer was already reported
in the form of a monohydrate [6]. Furthermore, the compound resulting from the condensation
reaction of INH with ClSal was already obtained by other authors in different conditions of
synthesis [7, 8], but no crystal structure was reported.
INHVa (1): (E)-N’-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide.
Orange solid precipitated immediately, it was further recristalized from ethanol and yellow
prismatic crystals were obtained after a week (m. p. 260-263 °C).
INHBrVa (2): (E)-N’-(3-bromo-4-hydroxy-5-methoxybenzaldehyde)isonicotinohydrazide.
Orange solid not suitable for structural X-ray diffraction study precipitated after two days (m. p.
2
85-287 °C).
INHClSal (3): (E)-N’-(5-chloro-2-hydroxybenzaldehyde)isonicotinohydrazide.
Colorless thin prismatic crystals suitable for X-ray diffraction were obtained after eleven days
(m. p. 246-248 °C).
INHBrSal (4): (E)-N’-(5-bromo-2-hydroxybenzaldehyde)isonicotinohydrazide.
Yellow crystals suitable for structural X-ray diffraction study were obtained after 24h (m. p. 259-
2
61 °C).
2
.2. X-ray diffraction data
The measurements were performed on an Oxford Xcalibur Gemini, Eos CCD diffractometer with
graphite-monochromated CuKα (λ=1.54178 Å) radiation. X-ray diffraction intensities were
collected (ω scans with ϑ and κ-offsets), integrated and scaled with CrysAlisPro [9] suite of
programs. The unit cell parameters were obtained by least-squares refinement (based on the
angular setting for all collected reflections with intensities larger than seven times the standard
deviation of measurement errors) using CrysAlisPro. Data were corrected empirically for
absorption employing the multi-scan method implemented in CrysAlisPro. The structures were
solved by direct methods with SHELXS of the SHELX package [10] and the molecular model
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developed by alternated cycles of Fourier methods and full-matrix least-squares refinement with
SHELXL of the same suit of programs. The H-atoms were found at approximated positions in a
difference Fourier map phased on the heavier atoms. However, all but the hydroxyl and water
hydrogen atoms were located stereo-chemically and refined with the riding model. The hydroxyl
and the water hydrogen atoms were refined at their found positions with isotropic displacement
parameters.
The closely related chemical formula, the same space group and almost identical cell constant
values strongly suggested that the bromine-containing solid was isomorphic to the chlorine-
containing one. In fact, an initial molecular model assuming the same atom positions as in the
chlorine crystal with the identity of the ring halogen atom changed from chlorine to bromine, lead
to smooth convergence of the structural parameters for the bromide crystal during the least-
squares refinement against the corresponding X-ray data set. Interestingly, the Flack’s absolute
structure parameter [11] (x) turned out to be x=1.01(3) thus, indicating that the bromine crystal
was the chiral counterpart of the chlorine one. In fact, by inverting the molecular model, the x-
value dropped to -0.03(2). The Flack’s parameter is the fractional contribution to the diffraction
pattern due to the molecule’s racemic twin and for the correct enantiomeric crystal it should be
zero to within experimental error [10].
Crystal data and structure refinement results are summarized in Table 1.
2
.3. Spectroscopic analysis
The FTIR spectra were obtained with a Bruker EQUINOX 55 spectrometer in KBr discs with 4
-1
-1
1
13
cm resolution and 60 scans, in the 4000-400 cm range. H-NMR and C-NMR spectra were
recorded as DMSO-d6 solutions, on a Bruker Advance II spectrometer at 500 MHz using
tetramethylsilane as the internal reference. Electronic absorption spectra were measured on
-5
ethanolic 3 x 10 M solutions in the 200-800 nm region, with a Hewlett-Packard 8452-A diode
array spectrometer using 10 mm quartz cells.
2
.4. Computational methods
The conformational space of the compounds was explored with the aid of the semiempirical PM6
method [12]. Several starting geometries derived from selected variations in C14-C10-C9-N2,
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C10-C9-N2-N1, N2-N1-C8-C1 and N1-C8-C1-C6 torsion angles were optimized (see Figure 3
for atom labelling). Geometries obtained from X-ray diffraction were also used as a starting point
for the optimizations. The geometries were further re-optimized both in gas phase and including
solvent effects using the hybrid meta-GGA M06–2X exchange–correlation density functional
[13] with a triple-zeta 6–311G(d,p) basis set [14,15]. Solvent effects (ethanol) were included
implicitly through the Conductor-like Polarizable Continuum Model [16,17]. Numerical
integrations were carried out using a grid containing 96 radial points and 590 angular points
around each atom. For compounds that involve Br atom [INHBrVA (2) and INHBrSal (4)], a grid
with 160 radial points and 974 angular points was used. The critical points found after
optimization were characterized by the sign of the eigenvalues of the Hessian matrix of the total
electronic energy with respect to the nuclear coordinates. When the critical point corresponded to
a minimum on the potential energy surface, the eigenvalues were converted to harmonic
vibrational frequencies. Vibration frequencies were scaled by a factor of 0.982 to ease the
comparison with experimental values [13]. Electronic transitions were calculated within the
framework of the Time-Dependent DFT (TD-DFT) [18,19] using the PBE0 functional [20] and
the triple zeta 6-311+G(d,p) basis set [14,15], with solvent effects, as was previously stated.
Geometry optimizations, Hessian matrix calculation and diagonalization, and electronic transition
calculations were performed with the GAMESS-US program [21]. Isotropic magnetic shieldings
1
3
1
of C and H were calculated at the B3LYP/6–311+G(2d,p)//B3LYP/6–31G* level of theory as
suggested by Cheeseman and co-workers, using the Gauge-independent Atomic Orbital method
[22-24]. Isotropic magnetic shieldings were turned into chemical shifts by subtracting the
corresponding isotropic magnetic shieldings of TMS, which were calculated at the same level of
theory as above. Solvent effects (DMSO) were included implicitly through the Integral Equation
Formalism version of the Polarizable Continuum Model [25]. Isotropic magnetic shieldings were
calculated with the Gaussian 03 package [26]. The animation of the vibrational modes was done
with Gabedit [27] and MO’s figures were done with wxMacMolPlt [28]. Potential energy
distribution analyses were carried out with the program vibca [29]. Marvin was used for drawing
chemical structures [30]. Chloride counter-ion and water molecules present in the crystals were
not considered in the calculations.
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3
3
. Results and Discussion
.1. Crystallographic structural data
Crystal data and structure refinement results are summarized in Table 1 for all compounds which
were suitable for crystallographic analyses.
Table 1. Crystal data and structure refinement results for (E)-N’-(5-X-2-hydroxybenzaldehyde)
isonicotinohydrazide chloride hydrate (X
methoxybenzylidene) isonicotinohydrazide hydrate.
=
Cl, Br) and (E)-N-(4-hydroxy-3-
Compound
INHVa (1)
INHClSal (3)
INHBrSal (4)
Empirical formula
Formula weight (g/mol)
Temperature (K)
Wavelength (Å)
Crystal system
Space group
unit cell dimensions
a (Å)
C H N O
C H Cl N O
C H BrClN O
3
1
4
15
3
4
13 13
2
3
3
13 13
3
289.29
293(2)
1.54184
330.16
374.62
295(2)
295(2)
1.54184
1.54184
Monoclinic
P2 /n
Orthorhombic
P2
Orthorhombic
P2
1
1
2
1
2
1
1 1 1
2 2
8.3741(6)
13.103(1)
12.683(2
99.02(1)
1374.4(2)
6.7061(2)
13.5765(6)
15.8941(5)
6.7021(1)
13.6568(3)
15.9769(3)
b (Å)
c (Å)
β (º)
3
1447.08(9)
4, 1.515
1462.35(5)
4, 1.702
volume (Å )
Z, calc. dens. (Mg/m )
3
4, 1.398
-
1
0.874
4.171
5.648
Abs. Coeff. (mm )
F(000)
608
680
752
Crystal shape/color
Plate / yellow
0.269 x 0.178 x 0.015
4.88 to 70.96
Prism / colorless
0.793 x 0.148 x 0.074
4.28 to 73.31
-3≤h≤8,
Prism / yellow
3
0.581 x 0.086 x 0.066
4.26 to 71.52
-8≤h≤5,
Crystal size (mm )
θ-range(º) for data collection
-
10≤h≤10,
-16≤k≤14,
15≤l≤13
Index ranges
-15≤k≤16,
-19≤l≤17
-16≤k≤16,
-
-19≤l≤16
Reflections collected
5778
3768
4195
Independent reflections
Observed reflections [I>2σ(I)]
2638 [R(int) = 0.0417]
1320
2610 [R(int) = 0.0216]
2329
2426 [R(int) = 0.0161]
2308
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9
9.5
98.4
99.1.
Completeness (%)
(
to ϑ=70.96°)
(to ϑ=73.31°)
0.7589 and 0.5735
(to ϑ=73.31°)
0.6932 and 0.1472
Max. and min. transmission
Refinement method
Full-matrix least-squares onFull-matrix least-squares onFull-matrix least-squares on
2
2
2
F
F
F
Data / restraints / parameters
2638 / 3 / 203
1.018
2610 / 0 / 202
1.041
2426 / 0 / 203
1.047
2
Goodness-of-fit on F
a
Final R indices [I>2σ(I)]
R indices (all data)
R1 = 0.0578, wR2 = 0.1171R1 = 0.0402, wR2 = 0.1075R1 = 0.0289, wR2 = 0.0762
R1 = 0.1217, wR2 = 0.1597R1 = 0.0452, wR2 = 0.1123R1 = 0.0304, wR2 = 0.0776
Absolute structure parameter
0.00(2)
-0.03(2)
-
3
0.146 and -0.191
0.217 and -0.265
0.306 and -0.272
Larg. diff. peak & hole (e.Å )
a
2
2 2
2 2 1/2
R =Σ||F |-|F ||/Σ|F |, wR =[Σw(|F | -|F | ) /Σw(|F | ) ]
1
o
c
o
2
o
c
o
3
.1.1.(E)-N’-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide hydrate: INHVa (1).
Figure 4 shows an ORTEP [31] drawing of the solid state structure and corresponding intra-
molecular bond distances and angles are listed in the Supplementary Material, Table S1a. The
organic molecule is nearly planar [rms deviation of non-H atoms from the best least-squares
molecular plane of 0.044 Å] and closely related to the isomorphic pair (3) and (4). In fact, it can
be obtained from these latter molecules by replacing the halide-substituents for a methoxy group
and changing the location of the hydroxyl group from 2- to 4- position in the ring. The pyridine
moiety is deprotonated, rendering the conformer electrically neutral. The main structural
differences between (1) and the pair (3) and (4) include: (i) a shortening of the mean pyridine C-
N bond length of 0.011 Å upon deprotonation and (ii) the removal of the intra-molecular OH···N
bond in the isomorphic compounds and the formation of a bent OH···O(methoxy) new bond in
the conformer [d(OH···O)=2.237 Å and ∠ (O-H···O)=108.0º].
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Figure 4. View of (E)-N’-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide hydrate
crystal (1), showing the labelling of the non-H atoms and their displacement ellipsoids at the 30%
probability level. Intra- and inter-molecular H-bonds are indicated by dashed lines.
The molecular structure of a quite different conformer, also crystallized as monohydrate in the
same space group P2 /n, having nearly the same volume but in a different unit cell, has been
1
reported [6]. In fact, the conformers are nearly related to each other through a rotation of about
1
80º around the Ph-C σ-bond (C1-C8 bond in Figure 4). Though showing a good agreement in
bond distances and angles, the reported one further differs from the presented here in its
appreciable deviation from planarity. As expected, the conformer crystals also differ from each
other in their packing.
The mentioned differences between both conformers can be a consequence of different
experimental conditions in the preparation. In the present work the crystals were obtained by
letting the recrystallization solution slowly cool to room temperature and then keeping it for at
least one week. The previously reported procedure describes a faster cooling followed by the
separation of the product from the solution. The anhydrate of this latter conformer has been
reported to crystallize in the Cc space group [32] and the structure of yet another closely related
conformer that crystallizes in the P2 /c space group with a methanol solvent molecule has also
1
been published [33].
Hydrogen bond distances and angles are detailed in Table S2a of the Supplementary Material.
The crystal is further stabilized by inter-molecular H-bonds. Neighbouring molecules are linked
1
0

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to each other through OH···Npyr bonds [d(OH···N)=1.806 Å and ∠ (O-H···N)=149.6º] giving
rise to a nearly planar, ribbon-like, polymeric structure that extends along the crystal [-3, 0, 1]
direction. In turn, neighbouring polymers in the lattice are bridged by the water molecule (nearly
onto the molecular plane) through N-H···Ow-H1···O’carb [d(NH…Ow)=2.135 Å and ∠ (N-
H···Ow)=166.7º; d(OwH1···O’carb)=1.997 Å and ∠ (Ow-H1···O’carb)=168.2º] and NH.Ow-
H2···O”ox [d(OwH1···O”ox)=2.247 Å and ∠ (Ow-H1···O”ox)=143.3º] bonds (see Figure4).
3
.1.2. (E)-N’-(5-X-2-hydroxybenzaldehyde)isonicotinhydrazide (X: Cl, Br) chloride
hydrate: INHClSal (3) y INHBrSal (4).
As detailed in the experimental section, the solid state compounds are isomorphic and chiral
counterparts of each other, differing only in the interchange of chlorine and bromine atoms in the
aldehyde ring. In fact, the rms separation between homologous non-H atoms in the best least-
squares structural fitting of the organic molecules, calculated by the Kabsh’s procedure [34] is
0
.037 Å (0.018 Å excepting the ring halogen atoms) and involves an improper relative rotation
(determinant equal to -1) around the common molecular centroid. Figure 5 is an ORTEP [31]
drawing of the chlorine-containing crystal. The corresponding intra-molecular bond distances and
angles for both compounds are listed in the Supplementary Material, Tables S1b,c.
Due in part to delocalized π-bonding, the 5-X-2-hydroxyphenylidene (X=Cl, Br) fragments are
nearly planar [rms deviation of non-H atoms from the best least-squares plane is less than 0.035
Å for (3) and 0.036 Å for (4)]. As shown in Fig. 5, the planarity affords the formation of an intra-
molecular OH···N bond [OH···N bond distances of 2.012 Å (3) and 2.064 Å (4), O-H···N angles
of 147.2º (3) and 145.41º (4)]. As expected, the positively charged pyridine moiety is also planar
[rms deviation of atoms from the plane of 0.015 Å (3), 0.014 Å (4)] and therefore the only
degrees of freedom left to define the molecular conformation are un-hindered rotations around
the C1-C8, N1-N2, N2-C9, and C9-C10 σ-bonds. In fact, the chiral organic conformers are
related to each other mainly through a change of 32.9(3)º in the torsion angle around C9-C10
bond.
1
1

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Figure 5. View of the chlorine-containing member of the (E)-N’-(5-X-2-
hydroxybenzaldehyde)isonicotinohydrazide (X: Cl, Br) chloride hydrate isomorphic pair (3) and
(4).
Observed intra-molecular bond distances and angles are in agreement with the Organic
Chemistry’s rules. In particular, C-C distances within the phenyl ring [in the 1.368(4)-1.414(4) Å
(
3) and 1.375(4)-1.402(4) Å (4) ranges] are as expected for phenyl resonant-bond structures. C-X
bond distances are 1.743(3) Å (3) and 1.901(3) Å (4). The short imino C8-N1 length [1.267(4) Å
3) and 1.268(4) Å (4)] contrasts with the longer amido C9-N2 value [1.339(4) Å (3) and
.345(4) Å (4)], clearly confirming the formally double and single bond character for these links,
(
1
respectively. The distance of N-N single bond linking the 5-X-2-hydroxyphenylidene and
pyridincarbonylic molecular fragments is 1.388(3) Å (3) and 1.396(3) Å (4). C-OH and C=O
bond distances of 1.349(4) Å (3) and 1.355(4) Å (4), and 1.223(4) Å (3) and 1.217(4) Å (4) are
the expected for the single and double character of these bonds, respectively. C-N bond lengths
within the pyridinum group are 1.340(4) and 1.330(4) Å (3), and 1.336(4) and 1.329(4) Å (4).
Bond distances in the bromide-containing organic cation are in general agreement with the
corresponding ones reported in the literature for the neutral (deprotonated) moiety [35, 36].
+
-
-
The crystals are further stabilized by (pyr)NH ···Cl and OwH···Cl bonds. The water molecule
also bridges neighboring organic molecules acting as donor in a OwH···O(carb) bond and as
acceptor in a PhOH···Ow bond with a given organic molecule and as acceptor in a NH···Ow
bond with another one (see Figure 5). Hydrogen bond distances and angles for both compounds
are detailed in the Supplementary Material, Tables S2b,c.
1
2

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3
.2. Conformational analysis and geometry optimization
As it was previously mentioned, calculations were done using M06-2X functional and 6-
11G(d,p) basis set in the gas phase and including solvent effects, as described in the
3
experimental section.
In the case of (1), the conformational searching shows that one structure is more stable than other
-1
conformations by more than 4 kcal mol , both in the gas phase and after including solvent
effects. Even the conformation optimized from the X-ray structure is found to be well above in
energy than the lowest-energy conformation. For (3), the conformational searching in the gas
-
phase shows that one conformation is more stable than other conformations by about 3 kcal mol
1
,
whereas the conformation optimized from the X-ray structure becomes the more stable one by
-1
about 1 kcal mol after including solvent effects. Interestingly, (4) exhibits a very similar
-1
behavior. A conformation is more stable than others by more than 1 kcal mol in the gas phase,
whereas the conformation optimized from the X-ray structure becomes the more stable one by
-1
about 2 kcal mol when solvent effects are taken into account. Finally, in the case of (2), for
which no experimental structure is known, the conformational searching leads to optimized
-1
geometries that become the more stable ones by more than 3 kcal mol both in the gas phase and
after including solvent effects. From now on, the lowest-energy conformations obtained after
including solvent effects are used both for the comparison of geometrical parameters with those
structures obtained by X-ray diffraction methods and for the calculation of electronic transitions
that aid in the assignment of the electronic spectra. On the other hand, gas-phase lowest-energy
conformations are used to calculate harmonic vibrational frequencies that are useful in the
assignment of experimental vibrational spectra.
A selection of calculated geometrical parameters is listed in Table 2. Experimental values for
those structures that were characterized by X-ray diffraction methods and structural data
calculated for (2) are included for comparison. It is appreciated from the table that the intra-
molecular N1···HO1 bond is overestimated by the calculations in both salicylaldehyde
derivatives (3) and (4). In addition, C-H distances are also slightly overestimated by the
calculations. Nonetheless, there is excellent agreement between experimental and theoretical
results regarding C-O, C-Cl and C-Br bond distances. It is worth noting that interatomic distances
obtained by optimizing with solvent effects for (2) agree very well with those obtained for (1)
1
3

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and that C-Br bond distance in (2) shows almost the same value as the ones found both
experimentally and theoretically for (4). Calculated bond and torsion angles are also in agreement
with those obtained by X-ray diffraction methods and calculated values for (2) are very close to
those obtained for the other hydrazones. Regarding bond angles, calculated values are in
excellent accordance with experimental results (see Table 2). It is worth noticing that the angle
formed by –OH with the oxygen atom from OCH substituent in vanillin derivatives, is well
3
reproduced by calculated results, being also very close to the value found in (2) compound, for
which there is no experimental structural data. Also, in salicylaldehyde derivatives, O-H···N1
and C2-O-H bond angles are well reproduced by calculated results, in accordance with the inter-
molecular hydrogen bond between N1 and the OH group. Torsion angle values obtained by DFT
methods and, in particular, those involved in these inter-molecular hydrogen bonds, show a good
agreement with those obtained by diffraction experiments.
Table 2. Selected experimental and calculated (M06-2X/6-311G(d,p)) structural parameters. See
Figures 3-5 for atom labeling and substituents nature. For compound (1), experimental data in the
first column were extracted from Ref. 6.
INHVa (1)
INHBrVa (2)
INHClSal (3)
INHBrSal (4)
a
b
Exp.
Exp.
Calc.
Calc.
Bond lengths (Å)
---
Exp.
Calc.
Exp.
Calc.
N1···HO1
C2-R4
---
---
2.015
1.349
0.930
0.930
1.743
1.330
1.801
1.345
1.084
1.083
1.749
1.346
2.024
1.356
0.930
0.930
1.901
1.330
1.804
1.353
1.083
1.083
1.890
1.346
0.929
1.367
1.358
0.930
1.321
1.046
1.362
1.365
0.999
1.326
1.085
1.367
1.369
1.084
1.335
1.082
C3-R3
1.363
C4-R2
1.358
C5-R1
1.900
C13-N3
1.334
Bond angles (°)
---
O-H-N1 (R4)
C-O-C3 (R3)
C2-O-H (R4)
C4-O-H (R2)
O-H (R2)-O(R3)
C1-C2-R4
---
---
---
147.2
---
142.5
---
143.7
---
142.5
---
116.9
---
117.0
---
122.3
---
123.5
---
107.3
---
109.3
---
107.8
---
109.4
---
110.9
111.8
120.1
125.5
111.1
107.9
120.1
125.0
113.6
112.1
120.8
113.3
112.0
103.6
---
---
---
---
121.1
123.1
119.4
123.1
118.4
123.3
119.3
123.1
118.4
C2-C3-R3
113.1
1
4

ACCEPTED MANUSCRIPT
C3-C4-R2
C4-C5-R1
121.4
120.0
122.8
117.8
127.3
116.7
126.3
120.2
119.7
120.3
119.5
120.6
119.0
120.1
119.9
118.0
Torsion angles (°)
C14-C10-C9-N2
C10-C9-N2-N1
N2-N1-C8-C1
N1-C8-C1-C6
C3-C2-O-H
174.1
175.5
-178.3
-170.5
---
6.69
-143.5 -141.9
10.7 10.7
-180.0 -179.7
163.9
-177.5
-176.0
177.3
-176.6
---
140.2
-180.0
179.9
178.1
-179.1
---
-163.2
177.4
175.3
-177.9
-179.9
---
-142.1
179.8
178.6
-178.8
179.7
---
-179.5
179.6
-4.53
---
4.50
---
0.918
---
C5-C4-O-H
178.1
11.8
-178.7
-3.03
-178.6 178.3
-177.4 -179.9
C2-C3-O-C
---
---
---
---
3
.3. Vibrational spectroscopy
-1
The infrared spectra of the compounds were measured in the 400-4000 cm spectral range. The
assignments were accomplished with the help of data obtained from Density Functional Theory
calculations at the M06-2X/6–311G(d,p) level using both visualization tools and potential energy
distribution analyses
, taking into account the internal coordinates that mainly contribute to each
calculated frequency. Recorded spectra of INH, Va and (1) in the most relevant spectral range,
-1
namely 1800-400 cm , are depicted in Figure 6. IR spectra of (2), (3) and (4) are included as
Supplementary Material. (Figures S1a-S1d). The analyses based on the potential energy
distribution are available as Supplementary Material.
In order to illustrate the behavior in the high frequencies spectroscopic region, FTIR spectra of
one hydroxialdehyde (Va) and one hydrazone showing protonated pyridine N atom (3) in the
-1
4
000-400 cm range, are included as Supplementary Material (Figures S1-e and S1-f,
respectively)
A selection of relevant vibrational modes denoting the formation of the hydrazones are listed in
Table 3, together with calculated values and proposed assignments. The complete list of
vibrational modes and assignments are available as Supplementary Material Tables S3a-S3e. The
assignments are in agreement with data reported previously [2,3,6-8,34,37]. Results for the
already previously reported hydrazone from INH and o-vanillin (INHoVa) [2] are included in the
Table for the purpose of comparison. For atom numbering in the following discussion see Figure
3
.
1
5

ACCEPTED MANUSCRIPT
Figure 6. Experimental IR spectra of INH (red, dashed), Va (blue, dotted) and INHVa (1) (black,
-1
solid) in the 1800–400 cm spectral range.
Table 3. Selected experimental bands of the studied Schiff bases IR spectra. Values for INH and
the respective aldehydes are included for comparison. Complete assignment of relevant bands
and calculated frequencies are available as Supplementary Material (Tables S3a-S3e).
Assignment INH o-HVa INHoVa Va
INHVa BrVa INHBrVa ClSal INHClSa BrSal INHBrSal
1) (2) (3)l (4)
3014 w 3014 vvw 3018 w 3020 sh 3018 vw 3037 m 3047 vw 3048 w 3042 vw 3044 w
2590 m,b -- --- 2517 m,b 2410 m,b
(
ν OH
ν N(Py)H
ν C=O 1667 vs 1645 vs 1688 vs 1665 vs 1665 vs 1676 vs 1676 s 1663 vs 1674 vs 1672 vs 1676 vs
ν C=N
1606 vs
1599 s,b
1598 s,b
1603 s,b
1605 vs,b
δ OH
1388 s 1392 sh 1430 s 1398 m 1426 s 1418 m 1378 s 1363 m,b 1373 m 1361 m-w
1327 s 1324 m-w 1300 m 1287 s 1354 s 1297 s 1304 m 1301 m 1305 mw 1301 m
νAr-OH
ν N-N 1192 vw
γ OH
1148 w
838 m 839 m 813 m 816 m
437 w 453 vw 456 vw
1172 m-w
1188 m
854 s 840 w,b 831 s 836 m-s 892 m 834 m-s
454 vw 448 vw 447 vw
1182 w,b
1197 vw
γ NH
Symmetrical and unsymmetrical
νH O modes are found in similar frequencies in all the
2
-1
compounds. The Ar-X (X= Cl, Br) stretching modes appear around 500 cm for the bromine
-1
Schiff bases, while for the chlorine Schiff base are located at 715 cm .
1
6

ACCEPTED MANUSCRIPT
N–H Vibrations
Stretching -NH vibrations of the hydrazone moiety (N2), appear in the range of 3209-3113 cm ,
-1
-1
in agreement with the corresponding ones observed for INHoVa (3205 cm ).
It is found in the crystal structures of salicylaldehyde derivatives (3) and (4) that the pyridinic
-1
-1
nitrogen is protonated. The broad intense band at 2517 cm and 2410 cm , respectively,
observed for these Schiff bases can be assigned to the pyridinic N-H stretching mode. Regarding
-1
these results, we can propose that the band with similar shape at 2590 cm in INHoVa spectra,
corresponds to this mode [5]. It is worth mentioning that Malhotra et al [37] report the (1)
hydrazone protonated at the pyridinic nitrogen. Despite this fact, they do not report N-H
vibrations.
-1
Unsurprisingly, the bands related to the NH
2
group in INH (at 3111, 1634 and 1321 cm ) are not
present in the hydrazones spectra. A very weak band, corresponding to NH(hyd) out-of-plane
-1
-1
deformation (γ) is found between 447 cm and 456 cm . The NH bending mode is found to be
coupled with other modes in the hydrazones studied here and in INHoVa, showing a non-
distinctive tendency along the family of compounds.
-1
-1
N-N stretching band, on the other hand, appears between 1172 cm and 1197 cm for the studied
-1
-1
compounds, while for INHoVa and INH is at 1148 cm and 1192 cm , respectively.
O–H Vibrations
The IR spectra of the studied compounds show weak bands corresponding to OH stretching
-1
modes in the 3020-3048 cm range. Values obtained from DFT methods are much higher than
the experimental ones as H-bond interactions, present in the crystal packing of the hydrazones
and the aldehydes, are neglected in the calculations. This stretching vibration is observed at 3014
-1
cm in INHoVa Schiff base. It also remains observable at almost the same frequency after
condensation for all compounds except (2).
Regarding
δOH mode, it can be seen from the tables that it shifts to lower frequencies upon
condensation for all Schiff bases. Notwithstanding, INHoVa compound shifts this mode to a
-1
-1
higher value, from 1388 cm in o-HVa to 1392 cm in the hydrazone. With the aid of
computational methods, it is also observed that this vibration is in general present in combination
with other modes, making it difficult to analyze the trend in the frequency values.
1
7

ACCEPTED MANUSCRIPT
Comparing the hydrazones Ar-OH stretching mode, including INHoVa, with the corresponding
aldehyde, it can be appreciated from the table that it is conserved except for (1). This behaviour
can be explained by the OH group position together with the presence of deprotonated pyridinic
nitrogen atom. As can be seen in the packing of (1), the OH is linked to the referred N atom of a
neighboring molecule by H-bond interactions, hence, leading to a red shift of this mode.
Moreover, as it was expected, computational methods underestimate OH out-of-plane
deformation (
γ), which is conserved in all cases, but shows a slight shift in both Va derivatives.
C=O and C=N stretching
-1
-1
The strong bands found from 1663 cm to 1676 cm are characteristic of the C=O stretching of
the Schiff bases. It is seen that this mode is slightly blue-shifted in all the hydrazones, except for
(1) in which is conserved.
-1
A strong band is observed in the 1599–1605 cm range, characteristic of the C=N stretching
mode, which appears due to the formation of the hydrazone. Considering INHoVa [2] (C=N
-1
stretching band at 1606 cm , see Table 3) and other related compounds [3], it can be inferred that
this mode is insensitive to the nature and position of the substituents in the aldehyde ring.
In addition, it is observed that DFT calculations overestimate OH, C=O and C=N stretching
modes, possibly due to the lack of restrictions in the gas phase that are present in the solid (see
Supplementary Material). It must be noted that the hydrogen atom from the OH group, the
oxygen atom of C=O moiety and the nitrogen atom of C=N group are all involved in H-bonds in
the crystals (see Figures 4 and 5).
OCH Vibrations
3
3
Ar-OCH stretching vibrations are present as broad bands in vanillin derivatives. For (1) it
-1
-1
-1
appears at 1254 cm and for (2) from 1250 cm to 1264 cm , coupled with other modes. It is
-1
worth mentioning that INHoVa compound has similar bands in the 1256-1283 cm range.
-1
Regarding CH vibrations, the asymmetrical δCH strong band is observed at 1510 cm in (1)
3
3
-1
-1
and at 1498 cm in (2), being both in agreement with the value of 1464 cm found for INHoVa.
-1
-1
In the three compounds, this band is accompanied by a weak band in the 1440 cm - 1471 cm
range, corresponding to the symmetrical mode.
1
8

ACCEPTED MANUSCRIPT
C–H Vibrations
The IR spectra of the compounds show weak bands corresponding to C8H stretching vibrations
-1
-1
between 2842 cm and 2926 cm in the Schiff bases. These modes appear shifted to lower
frequencies in comparison with those of the aldehydes, in most cases, except for INHoVa.
Calculations overestimate frequency values in all cases.
It is also seen that symmetric and antisymmetric CH(methyl) stretching vibration modes are
conserved in all hydrazones after condensation, in comparison with the same mode of the
aldehyde. Calculated values of these stretching frequencies are higher than the observed ones. It
is worth mentioning that these modes have a similar behaviour than those of INHoVa.
-1
The band assigned to rings
ν CH modes appear in the 3157-3050 cm region in INHoVa; 3156-
-
1
-1
-1
3
3
046 cm region in (1); 3148-3094 cm region in (2); 3089-3027 cm region in (3) and in the
-1
091-3017 cm region in (4). It is worth noticing that for Va derivatives, the highest frequency
value is originated in the aldehyde fragment, contrary to the salicylaldehyde derivatives, in which
it corresponds to the INH fragment of the molecules. It is observed that the bands related to the
C-H stretching mode in the hydrazide fragment are blue-shifted in comparison with those of INH.
The bands corresponding to CH stretching modes in the aldehydes are overlapped with broad
bands due to H O stretching in some cases (see Tables S3a-S3e). As a consequence, a
2
comparison between these modes and those of the hydrazones could not be made.
3
.4. NMR spectroscopy
1
13
H NMR and C NMR spectra of (2), (3) and (4) were registered as DMSO-d6 solutions (see
experimental section for detailed information). Selected spectral data and their assignments are
listed in Table 4, together with previously reported INHoVa and (1) data [2, 37]. Recorded
spectra of compound (4), for which no crystal structure has been determined, are shown in Figure
1
7
. It can be seen, with more clarity in the H spectrum, that signals have satellite weaker ones that
conserved the ratio of integrated area with respect to the main signals. This fact could be a
consequence of tautomeric equilibrium in solution.
1
9

ACCEPTED MANUSCRIPT
1
13
6
Table 4. Selected H and C (500 MHz) NMR chemical shifts, in ppm, for the Schiff bases (d -
DMSO). Calculated chemical shifts and data reported by other authors are also shown.
INHoVa
INHVa (1)
INHBrVa (2)
INHClSal (3)
INHBrSal (4)
Exp. [2] Calc. [2] Exp. [37] Calc. Exp.
Calc.
Exp.
Calc.
Exp
Calc.
H-O
H-C=N 8.76
H-N ---
C=N-H 10.70
12.46
11.56
8.91
5.08
8.34
---
6.15
8.12
---
4,92
8.50
---
6.50
7.98
---
12.98
8.81
11.48
8.60
12.95 10.78
8.81
11.11 11.21
9.05 9.10
8.45
12.48
10.30
11.07
8,98
11.26
9.51
11.86
8.71
12.68
8.74
13
C
C=O
160.10 167.4
55.87 57.19
163.42
55.89
175.52 159.84 175.48 160.09 166.50 159.81 166.00
58.67 55.99 59.05 --- --- --- ---
158.4| 148.97 154.83 155.59 168.55 156.6 166.27
O-CH
3
C-OH
148.00 158.00 151.94
1
13
Figure 7: H NMR (a) and C NMR (b) of INHBrVa (2) in d
6
-DMSO solution
The spectra of the hydrazones showed a signal at δ 4,92 -12.95 ppm characteristics of phenolic
OH proton, sensitive to their position in the ring. Calculations underestimate these chemical shifts
for (3) and (4) hydrazones in wich OH···N H-bonds give rise to pseudo-rings. On the contrary,
values for (1) and (2) having the OH group in para-possiton, are overestimated. This could be
explained by considering that calculations were done for a static conformation, while in solution
atoms can freely rotate along a single bond and thus, observed chemical shifts are average values
2
0

ACCEPTED MANUSCRIPT
of different conformations. A singlet observed at δ 8.50-8.81 ppm is assigned to the azomethine
proton. The signals at δ 8.98-12.68 ppm are proposed to be due to the C=N-H hydrazine protons.
Different values observed in the protonic spectra are proposed to be due to the variation of
substituents in the hydrazone’s rings and to the effect of protonation or deprotonation of the
pyridinic N in the molecules. It is interesting to see that despite the fact that Malhotra et al [37]
report the hydrazone to be protonated in the pyridinic nitrogen, the authors do not report the
1
respective signal in the H NMR spectrum. In the case of (3) and (4) a very broad signal of
difficult evaluation at δ 11 ppm approximately, can be assigned to this proton.
Besides the selected signals listed in the table, two well-defined aromatic spin systems can be
observed. As expected, pyridine hydrogens appear at higher δ values than those of the phenolic
ring
.
1
3
Regarding the C spectra, the presence of a peak at δ 159.81-160.1 ppm is assigned to the
carbonyl carbon. The methoxy carbon is proposed to show a signal at 55.89 ppm for (1) and at
5
5.99 ppm for (2), both vanillin derivatives, in agreement with the reported for o-vanillin
derivative at δ 55.87 ppm. The carbon atom linked to the OH group is, as Table 4 shows, in the
13
1
48.0-156.60 ppm range. Calculations were of aid to the assignment of the C chemical
shieldings, despite the fact that they generally overestimate values of the carbon spectra.
3
3
.5. Electronic Spectroscopy
.5.1. Spectra analysis
The electronic absorption spectra of INH, hydroxy-aldehydes and of the hydrazones were
measured in the 200-800 nm spectral range. Figure 8 shows the electronic spectrum of the
hydrazones in the most relevant spectral range, as an illustrative example. INHoVa spectrum is
included for comparison.
2
1

ACCEPTED MANUSCRIPT
Figure 8. Electronic absorption spectra of the hydrazones in ethanolic solution: INHVa (1):
dashed, blue; INHBrVa (2), dotted, blue; INHoVa, solid black; INHClSal (3), dashed black and
INBrSal (4) dotted black.
The studied Schiff bases contain two aromatic rings, one belonging to the INH and the other to
the aldehyde. For simplicity, the descriptions of the electronic transitions involving these rings
are done mentioning INH or ALD referring to the respective fraction of the molecule.
Experimental absorption bands and calculated electronic transitions are listed in Table 5, together
with the proposed assignments. Only those calculated electronic transitions relevant for the
assignments are listed. The MO’s mainly involved in the electronic transitions used to assign
observed bands are available as Supplementary Material (Figures S2a-d). It can be seen from
Table 5 that the calculated transitions describe very well the experimental electronic spectra.
-5
Table 5. Electronic absorption spectra of 3 x 10 M ethanolic solutions of the studied
isonicotinoyl hydrazones. Results from TD-DFT are also shown. Percentage contributions of
calculated transitions are given in parentheses. Absorption maxima are given in nm. Oscillator
strengths, shown in parenthesis, are in a.u.
INHVa (1)
Exp.
Calc.
Assignment
2
2
08 sh 210 (0.0485) HOMO – 4 → LUMO + 1 (72%)
14
215 (0.1011) HOMO – 1 → LUMO + 4 (61%)
HOMO → LUMO + 4 (31%)
2
32 sh 240 (0.6052)
HOMO – 1 → LUMO + 1 (55%)
2
2

ACCEPTED MANUSCRIPT
2
2
80 (0.2491) HOMO – 1 → LUMO (58%)
89 (0.1193) HOMO → LUMO + 1 (85%)
2
3
76
32
328 (0.3620) HOMO → LUMO (98%)
INHBrVa (2)
Exp.
Calc.
Assignment
HOMO – 3 → LUMO + 1 (34%)
HOMO – 1 → LUMO + 2 (34%)
HOMO – 4 → LUMO + 1 (29%)
HOMO – 3 → LUMO + 1 (23%)
HOMO – 1 → LUMO + 1 (23%)
HOMO → LUMO + 1 (20%)
2
10 sh 215 (0.0802)
2
19 (0.1027)
35 (0.1369)
2
22
2
2
40 (0.2914) HOMO → LUMO +2 (33%)
HOMO – 3 → LUMO (20%)
2
42 sh
2
41 (0.1885) HOMO → LUMO + 2 (56%)
3
3
07 sh 278 (0.2373) HOMO – 1 → LUMO + 1 (88%)
30
316 (0.4116) HOMO → LUMO (98%)
INHClSal (3)
Exp.
Calc.
Assignment
2
2
2
2
3
3
10 sh 210 (0.1039) HOMO – 2 → LUMO + 3 (20%)
20
46
92
231 (0.3940) HOMO → LUMO + 3 (76%)
245 (0.0265) HOMO – 3 → LUMO (84%)
260 (0.4151) HOMO – 1 → LUMO + 1 (83%)
03 sh 307 (0.1913) HOMO → LUMO + 1 (93%)
45
404 (0.1661) HOMO → LUMO (98%)
INHBrSal (4)
Exp.
Calc.
Assignment
HOMO – 1 → LUMO + 3 (39%)
HOMO → LUMO + 6 (38%)
2
08 sh 212 (0.2034)
2
2
2
3
3
20
48
91
234 (0.4460) HOMO → LUMO + 3 (76%)
239 (0.1112) HOMO – 6 → LUMO (63%)
260 (0.4147) HOMO – 1 → LUMO + 1 (83%)
04 sh 308 (0.1668) HOMO → LUMO + 1 (94%)
44 407 (0.1580) HOMO → LUMO (98%)
sh = shoulder
3
.5.2. Description of the MO’s involved in the transitions
For all hydrazones (see Figures S2a-S2d), HOMO and HOMO–1 are π in character in the
aldehyde ring, and non-bonding in the OH, C-N, C=O and C=N groups. In addition, they have
some contributions from the different electrophilic substituents of the aldehydes, namely OCH3
2
3

ACCEPTED MANUSCRIPT
(1), Br [(2) and (4)] and Cl (3). HOMO–2 in (3) is non-bonding in Cl atom, the hydrazone group
and oxygen atoms; it is also π in character in both INH and ALD rings. HOMO–3, involved in
electronic transitions of (2) and (3), is π in character in the aldehyde ring for the salicylaldehyde
derivative and in INH ring for the vanillin derivative; it is also located in oxygen and halogen
atoms, C=N and C-N groups. HOMO–4 is involved in electronic transitions of (1), (2) and (4). In
(1) is π in character in the aldehyde ring and it is also located in oxygen atoms, pyridinic nitrogen
and hydrazone group. In (2), is π in character in the INH ring with some contributions from
nitrogen and oxygen atoms of C=N and C=O groups. In (4), it is distributed among the Br atom,
oxygen atoms and the hydrazone group; it is also π in character in the rings, mostly INH ring,
being very similar to HOMO–6.
The LUMO is mostly π anti-bonding in the INH ring for the four hydrazones; with some
contributions from oxygen atoms of the C=O group, atoms in the hydrazone moiety and ALD
ring. LUMO+1 is similar to LUMO, but involving also oxygen atoms from the OCH , the OH
3
group and the aldehyde ring. LUMO+2 is involved in electronic transitions of (2), it is π* in
character in both rings and it is also localized in Br atom and O atom of OCH substituent.
3
LUMO+3, on the other hand, participates in electronic transitions of salycilaldehyde derivatives,
(3) and (4). It is π* in character in ALD ring and non-bonding in the halogen atom, O atom of
OH group and C (8). LUMO+4 is π* in character in ALD ring of (1). LUMO+6 is π* in character
in both rings, with some contributions from C-N, C=N and C=O groups of (4).
3
.5.3. Electronic absorption spectrum of INHVa (1)
According to the above description and the data from Table 5, the observed shoulder at 208 nm is
calculated at 210 nm and mainly assigned to a π → π* transition from ALD ring to the INH ring.
The observed band at 214 nm, which is calculated at 215 nm, is proposed to be due to a transition
involving ALD ring, with some contributions of C-N and C=N groups. A π → π* electronic
transition mainly from ALD ring to INH ring and with minor contributions from C=N, C=O and
OH groups is calculated at 240 nm to describe the observed shoulder at 232 nm. Two close
electronic transitions calculated at 280 nm and 289 nm, correspond to the band measured at 276
nm; this band is due to π → π* transitions from the ALD fragment of the molecule to the INH
2
4

ACCEPTED MANUSCRIPT
one, involving also OH, C-N, C=N and OCH
3
groups. The band observed at 332 nm is calculated
at 328 nm and assigned mostly to a π → π* transition from ALD ring to the INH ring.
3
.5.4. Electronic absorption spectrum of INHBrVa (2)
The bands are assigned on the basis of the description of the MO’s and the data of Table 5. The
shoulder observed at 210 nm is calculated at 215 nm and assigned mostly to a π → π* transition
involving INH ring with minor non-bonding contributions from Br atom, oxygen atoms and C-N
and C=N groups. The band at 222 nm is described by two calculated transitions, one at 219 nm
and the other at 235 nm; it is assigned to π → π* transitions from both aromatic rings to INH
ring. The shoulder at 242 nm is also described by two calculated values, at 240 nm and at 241
nm; it is assigned to π → π* transitions involving both rings, with minor contributions from the
electrophilic substituents of the aldehydes. The shoulder found at 307 nm and the band at 330 nm
are calculated at 278 nm and 316 nm, respectively. These bands are assigned to π → π*
transitions from ALD ring to the INH ring, with minor contributions from OH, C-N, C=O and
C=N groups.
3
.5.5. Electronic absorption spectrum of INHClSal (3)
On account of the provided descriptions of the MO’s and the electronic spectra described in
Table 5, the shoulder observed at 210 nm is mainly assigned to an electronic transition from both
rings of (3) to the ALD fragment of the molecule, including non-bonding contributions of its
susbtituents. The band at 220 nm is calculated at 231 nm and assigned to a π → π* transition in
the ALD ring and its substituents. The band at 246 nm is due to a π → π* transition from ALD
ring to INH ring, with some contributions of nitrogen and chlorine atoms. The band at 292 nm
and the shoulder at 303 nm are calculated at 260 nm and 307 nm, respectively; they are assigned
to electronic transitions from ALD ring to both rings with contributions from Ar-C, C-N and
C=N bonds. Interestingly, chlorine atom is not involved. The band measured at 345 nm is
calculated at 404 nm and assigned to a π → π* transition from ALD ring and substituents to INH
ring, with minor contributions from C-N and C=N bonds, OH group and oxygen atom from C=O.
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3
.5.6. Electronic absorption spectrum of INHBrSal (4)
According to the analysis of the MO’s and the data from Table 5, the shoulder at 208 nm can be
mainly assigned to one transition in ALD ring and its substituents, with minor contributions from
oxygen and nitrogen atoms; and to another from ALD ring to both rings, C and O atom from
C=O group and nitrogen atom form hydrazone group. The band at 220 nm, calculated at 234 nm,
is assigned to π → π* transitions in ALD ring, with non-bonding contributions from bromine,
oxygen and nitrogen atoms. The band found at 248 nm, is calculated at 239 nm and assigned to π
→
π* transitions in both ALD and INH rings, with non-bonding contributions from bromine,
oxygen and nitrogen atoms. The shoulders at 280 nm and 304 nm are assigned to transitions from
ALD ring, including its substituents, to both rings with minor contributions from nitrogen atoms.
The band at 291 nm is calculated at 293 nm and assigned mainly to a transition from both rings,
bromine and oxygen atom from C=O, to INH ring. The band at 344 nm is described by two
calculated values, one at 333 nm and the other at 407 nm; it is assigned to a π → π* transition
from ALD ring to INH ring, with minor contributions from bromine, nitrogen and oxygen atoms.
3
.6 A theoretical analysis of the reactivity of the hydrazones
The interesting application of hydrazones as good chelating agents, has been extensively studied
see [1] and references therein). This capacity arises from the available electronic density in the –
(
C=N-N-C=O group, suitable for metal coordination [5].
H
Furthermore, it has been stated before [38] that HOMO energies (E ) are associated with the
electron donating ability of a molecule. In particular, high values (that is, less negative ones) of
are likely to indicate a tendency of a molecule to donate electrons to appropriate receptors,
E
H
such as transition metals. Thus, E could indicate the tendency of ligands to coordinate such
H
metals.
L
On the other hand, the LUMO energy, E , is equivalent to electron affinity, according to
Koopmans’ theorem [39]. Moreover, LUMO energies are also associated with electron transfer
rates [38]. Regarding the well-known anti-oxidant properties of this kind of ligands, often
enhanced upon metal complex formation, the analysis of the electron affinity gains interest. This
property has been employed to account for the electron transfer rate from superoxide anion to
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L
copper atom [40-42]. The lower the electron affinity (that is, less negative values of E ), the
higher the electron transfer rate, leading to higher superoxide radical scavenging activity, being
the superoxide dismutase-mimic activity one of the main interests in this kind of copper
complexes. To gain more insight into the reactive behaviour of this family of hydrazones, we
calculated these properties, which are listed in Table 6.
Table 6. HOMO (E ) and LUMO (E ) energies (in eV) for the hydrazones, calculated at the M06-2X/6–
H
L
3
11G(d,p) level of theory.
EH
EL
INHVa (1)
-6.49 -1.05
INHBrVa (2) -7.31 -1.45
INHClSal (3) -6.70 -3.11
INHBrSal (4) -6.70 -3.11
It can be seen in Table 6 that the E values of the vanillin hydrazone, (1), and the salicylaldehyde
H
derivatives (3) y (4) are the less negative ones and, moreover, they are close each other. Thus,
they would be better candidates to form complexes with metal ions than the hydrazine (2). It is
interesting to note from Figures 2 and 3 that the OH group is in para position in vanillin
derivatives, whereas it is in ortho position in the salicylaldehyde derivatives. The OH group in
ortho position, together with the azomethine group, might favour the role of polydentate
chelating agent for salicylaldehyde derivatives. Therefore, it is proposed that (3) and (4)
hydrazones would be better candidates to form complexes with metal ions than (1) or (2).
Therefore, we propose that hydrazones (3) and (4) would have a better chance of being chelated
than (1) or (2).
Calculated E values, shown in Table 6, indicate that (1) would have the highest superoxide
L
radical scavenging activity of the studied hydrazones, closely followed by (2).
MEP-derived atomic charges (ESP charges, from now on) can be a useful index to identify
potential sites for coordination with metals, as they predict possible sites for nucleophilic or
electrophilic attacks [43]. We calculated them, in an attempt to identify these reactivity sites, as
we have already done in previous work [44]. Selected ESP charges of the compounds are listed in
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Table 7.
Table 7. MEP-derived atomic charges of selected atoms, in units of |e|, at the M06-2X/6–
11G(d,p) level of theory. See Figure 3 for atom numbering and structures.
3
INHVa (1)
INHBrVa (2) INHClSal (3) INHBrSal (4)
O (OH)
-0.5029
-0.5029
-0.2497
-0.1780
-0.3453
-0.6379
-0.5057
-0.0307
------
-0.5691
------
-0.5680
------
O (OCH3) -0.2436
N1
-0.2285
-0.3222
-0.6601
-0.5134
------
-0.1393
-0.4125
-0.1761
-0.4591
------
-0.1265
-0.4131
-0.1534
-0.4572
-0.0196
------
N2
N3
O (C=O)
Br1
Cl1
------
-0.0679
It can be seen from the table that the oxygen atoms belonging to OH and C=O groups present
large negative ESP charges for all the molecules under study suggesting that they are prone to
undergo electrophilic attacks. It is interesting to note that although the oxygen atom in the OCH3
group exhibits a small negative ESP charge, it could participate in the coordination sphere of a
complex together with the oxygen atom from the OH group, as it was determined in the vanillin-
copper complex [45]. Also, both nitrogen atoms in the –C=N-N-C=O moiety seem to be good
candidates for an electrophilic attack, although N2 presents a considerably most negative ESP
charge than N1. As expected, N3 shows a small negative charge when is protonated in ClSal and
BrSal Schiff bases, whereas large negative charges are found for that atom in (1) and (2), in
which it is not protonated. The pyridinic nitrogen atom could participate in a coordination
compound, forming complex structures, as it was reported previously [46]. These findings agree
with those found above when the magnitude of E
molecules under study to form complexes.
H
was correlated with the ability of the
4
. Conclusions
Four hydrazones derived from isoniazid (INH), a well-known antituberculous agent, were
prepared. They were obtained with good yield upon condensation with a group of
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hydroxybenzaldehydes, namely Va, BrVa, ClSal, BrSal.
The crystal structures of three of them were determined and compared with reported data for the
related Schiff base INHoVa. Hydrazones (3) and (4) are isomorphic and chiral counterparts of
each other. Structure of compound (1) differs from the already reported by other authors,
probably as an effect of the crystallization rate during the synthesis process.
DFT-based calculations carried out on the family of compounds showed a very good agreement
with experimental data. These results were of relevance in the interpretation and assignment of
the bands in the spectroscopic analysis. The few cases in which the calculated values differed
significantly from the experimental results were attributed to molecular interactions which were
neglected in the calculations. Moreover, calculated geometrical parameters are in very good
agreement with experimental data, even in the cases involving atoms that participate in
intermolecular H-bonds.
The FT-IR of solid samples and UV-vis of ethanol solutions spectra of the four hydrazones were
recorded together with those of INH and the respective aldehydes. They were analysed and
discussed in comparison with INHoVa hydrazone. The vibrational behaviour is in perfect
accordance with the expected for azomethine moiety formation and the observed crystallographic
details. Strong coupling among different modes was observed in the assignment of various bands.
The effect of the nature and location of substituent groups in the ring and of protonation or
1
13
deprotonation of the pyridinic N atom are also evidenced in the H and C NMR spectra.
In the electronic spectra the bands predicted in the calculations for the hydrazones could be
observed and assigned. In most cases, more than one electronic transition was involved in each
signal.
The calculation of some reactivity properties indicate that the Schiff bases (3) and (4) are good
candidates to act as polydentate ligands in the formation of coordination compounds. On the
other hand, the vanillin hydrazone (1) shows the highest superoxide radical scavenging activity,
calculated as the energy of the LUMO, which could be related to electron transfer processes
present in superoxide radical anion dismutation.
Acknowledgments
This work was supported by CONICET (CCT-La Plata), UNLP and ANPCyT, Argentina.
G.A.E., O.E.P., R.P.D. and A.C.G.B. are members of the Research Career of CONICET. The
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