Synthesis, spectroscopic and computational characterization of the tautomerism of pyrazoline derivatives from chalcones

Article abstract of DOI:10.1016/j.saa.2015.07.041

In the present study a series of novel pyrazolines derivatives has been synthesized, and their structures assigned on the basis of FT-Raman, ¹H and ¹³C NMR spectral data and computational DFT calculations. A joint computational study using B3LYP/6-311G(2d,2p) density functional theory and FT-Raman investigation on the tautomerism of 3-(4-substituted-phenyl)-4,5-dihydro-5-(4-substituted-phenyl)pyrazole-1-carbothioamide and 3-(4-substituted-phenyl)-4,5-dihydro-5-(4-substituted-phenyl)pyrazole-1-carboxamide are presented. The structures were characterized as a minimum in the potential energy surface using DFT. The calculated Raman and NMR spectra were of such remarkable agreement to the experimental results that the equilibrium between tautomeric forms has been discussed in detail. Our study suggests the existence of tautomers, the carboxamide/carbothioamide group may tautomerize, in the solid state or in solution. Thermodynamic data calculated suggests that the R(CS)NH₂ and R(CO)NH₂ species are more stable than the R(CNH)SH and R(CNH)OH species. Additionally, results found for the ¹H NMR shifting, pointed out to which structure is present.

Full text of DOI:10.1016/j.saa.2015.07.041

Accepted Manuscript
Synthesis, spectroscopic and computational characterization of the tautomerism
of pyrazoline derivatives from chalcones
Fábio Balbino Miguel, Juliana Arantes Dantas, Stefany Amorim, Gustavo F.S.
Andrade, Luiz Antônio Sodré Costa, Mara Rubia Costa Couri
PII:
S1386-1425(15)30090-1
http://dx.doi.org/10.1016/j.saa.2015.07.041
SAA 13940
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 March 2015
3 June 2015
7 July 2015
Please cite this article as: F.B. Miguel, J.A. Dantas, S. Amorim, G.F.S. Andrade, L.A.S. Costa, M.R.C. Couri,
Synthesis, spectroscopic and computational characterization of the tautomerism of pyrazoline derivatives from
chalcones, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/
10.1016/j.saa.2015.07.041
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1
Synthesis, spectroscopic and computational characterization of the
tautomerism of pyrazoline derivatives from chalcones
Fábio Balbino Miguela, Juliana Arantes Dantasa, Stefany Amorimb, Gustavo F. S. Andradeb,
Luiz Antônio Sodré Costac, Mara Rubia Costa Couria,*
aNUPEQ – Núcleo Multifuncional de Pesquisas Químicas, Departamento de Química,
Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora-
MG, Brazil.
bNEEM – Laboratório de Nanoestruturas Plasmônicas, Núcleo de Espectroscopia e Estrutura
Molecular, Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de
Juiz de Fora, 36036-900, Juiz de Fora- MG, Brazil.
cNEQC – Núcleo de Estudos em Química Computacional, Departamento de Química,
Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora-
MG, Brazil.
*Corresponding author: mara.rubia@ufjf.edu.br

2
ABSTRACT: In the present study a series of novel pyrazolines derivatives has been
1
synthesized, and their structures assigned on the basis of FT-Raman, H and 13C NMR
spectral data and computational DFT calculations. A joint computational study using
B3LYP/6-311G(2d,2p) density functional theory and FT-Raman investigation on the
tautomerism of 3-(4-substituted-phenyl)-4,5-dihydro-5-(4-substituted-phenyl)pyrazole-1-
carbothioamide and 3-(4-substituted-phenyl)-4,5-dihydro-5-(4-substituted-phenyl)pyrazole-1-
carboxamide are presented. The structures were characterized as a minimum in the potential
energy surface using DFT. The calculated Raman and NMR spectra were of such remarkable
agreement to the experimental results that the equilibrium between tautomeric forms has been
discussed in detail. Our study suggests the existence of tautomers, the
carboxamide/carbothioamide group may tautomerize, in the solid state or in solution.
Thermodynamic data calculated suggests that the R(C=S)NH2 and R(C=O)NH2 species are
more stable than the R(C=NH)SH and R(C=NH)OH species. Additionally, results found for
the 1H-NMR shifting, pointed out to which structure is present.
KEYWORDS: Synthesis, Tautomerism, Chalcones, Pyrazolines, Raman, DFT

3
1. INTRODUCTION
The interest in obtaining chalcones and their pyrazolines analogues has grown in the
past ten years because of numerous pharmacological properties have been discovered,
namely: antimicrobial [1-6], anti-tubercular [1,7,8], anticancer [8,9], anti-inflammatory [8]
and antioxidant [5] activities. The wide range of pharmacological activities of these
compounds shows the importance of this family of heterocyclic compounds in the area of
medicinal chemistry. Consequently, the use of known methods for the synthesis and
characterization of this class of compounds constitutes an important field of synthetic organic
chemistry [2,10,11].
In general, 2-pyrazolines derivatives are obtained by condensation between α,β-
unsaturated carbonyl compound and hydrazine derivatives [10,12,13]. In the case of
compounds having a large number of unsaturations, an effect known as tautomerism may
occur. The study of the tautomerism may contribute to the understanding of the
structural/biological activity of this family of compounds [14,15]. Among the available
spectroscopic tools, Raman spectroscopy has been suggested as a powerful technique to
identify tautomers. The possibility of understanding the substances fingerprints in a Raman
spectrum, which may be assigned to characteristics frequencies for each tautomeric form, has
led to the development of this spectroscopic technique as an important tool for the
characterization of tautomeric forms [16-19].
On the other hand, nuclear magnetic resonance (NMR) spectroscopy was introduced
into organic chemistry to determine carbon skeleton of the organic compounds, plays an
enormously important role in studying various chemical interactions, tautomeric
rearrangement, purity and authenticity of molecules. In this context, NMR is a widely used
and very powerful tool for the characterization of tautomeric equilibrium for some important
compounds such as histidine and D-fructose, among others [20,21]. The use of NMR
techniques for the description of tautomeric equilibrium relies on chemical systems that
present slow enough proton exchange for both systems to be measured in solution [22,23].
Additionally, the support from quantum chemistry predictions is usually essential for the
interpretation of NMR results [22,24]. Molecular structure, conformational stability and
vibrational frequencies have been studied by ab initio and DFT methods [22,25-28]. The
support of quantum chemistry methods, mostly based on density functional theory (DFT), has
been essential for the interpretation of both NMR and Raman spectroscopy results for the

4
assignment of spectra from both techniques, as well as on the calculation of total energy
changes between different tautomeric species. Recently, Hadda’s research group has
published a benchmark paper featuring the tautomerism of some curcumin derivatives which
have several biological significance [29].
In this report, a series of pyrazolines derivatives was synthesized and the structures
1
assigned on the basis of FT-Raman, H and 13C NMR spectral data and computational DFT
calculations. Furthermore, theoretical calculations were also used to check on the stability of
such compounds. Here, the spectroscopic techniques have been used along with
computational calculation for the evaluation of the tautomerism. Based on the spectroscopic
data experimentally obtained, full quantum mechanical calculations have been applied to
pyrazoline derivatives (Figure 1).
2. MATERIAL AND METHODS
All procedures for the synthesis of pyrazoline derivatives 18-47 are presented in
Supporting Information as well as the structural data collected. NMR spectra and other details
about some procedures related to computational details are also presented.
2.1. Synthesis
In order to synthesize O-alkylated chalcone derivatives 1-17, 4-hydroxybenzaldehyde
was initially alkylated (79–99% yield), using a Williamson ether synthesis [38-40], with
K2CO3 and butyl, hexyl, octyl, nonyl, decyl, dodecyl or tetradecyl bromide in DMF.
Commercial
aldehydes
4-bromobenzaldehyde,
4-methoxybenzaldehyde,
4-
(dimethylamino)benzaldehyde, 4-methylbenzaldehyde, 4-chlorobenzaldehyde, benzaldehyde
and O-alkylated aldehydes were then treated with equimolar quantities of the acetophenone or
1-(4-fluorophenyl)ethanone and NaOH (1.5 eq.) in ethanol (Scheme 1), using Claisen–
Schmidt reaction [40-43]. All the compounds were purified by recrystallization using a
suitable solvent and the assignment of the structures is fully supported by their characteristic
chemical shift values.

5
1: R1 = H; R2 = O(CH2)3CH3 (79%)
2: R1 = H; R2 = O(CH2)5CH3 (99%)
3: R1 = H; R2 = O(CH2)7CH3 (95%)
4: R1 = H; R2 = O(CH2)8CH3 (52%)
5: R1 = H; R2 = O(CH2)9CH3 (95%)
6: R1 = H; R2 = O(CH2)11CH3 (96%)
7: R1 = H; R2 = O(CH2)13CH3 (99%)
8: R1 = F; R2 = O(CH2)7CH3 (66%)
9: R1 = F; R2 = O(CH2)8CH3 (72%)
10: R1 = F; R2 = O(CH2)9CH3 (51%)
11: R1 = F; R2 = O(CH2)13CH3 (74%)
12: R1 = F; R2 = Br (42%)
13: R1 = F; R2 = OMe (38%)
14: R1 = F; R2 = NMe2 (28%)
15: R1 = F; R2 = Me (55%)
16: R1 = F; R2 = Cl (69%)
17: R1 = F; R2 = H (72%)
O
NaOH
EtOH, t.a.
O
CH3
1)
R1
R2
CHO
(1-17)
2)
R1
R2
Scheme 1. General synthesis of chalcone derivatives.
The pyrazolines 18-47 were prepared by treatment of chalcone derivatives in ethanol,
hydrazine derivatives under basic conditions (Scheme 2). All the compounds were purified by
recrystallization using a suitable solvent. After purification procedures, pyrazolines 18-47
were characterized by 1H NMR, 13C NMR spectral data.

6
O
H2N
O
H2N
O
.
HN NH2
HCl
N N
R1
R2
R1
R2
NaOH, EtOH, reflux
(1-17)
(18-34)
1: R1 = H; R2 = O(CH2)3CH3
2: R1 = H; R2 = O(CH2)5CH3
3: R1 = H; R2 = O(CH2)7CH3
4: R1 = H; R2 = O(CH2)8CH3
5: R1 = H; R2 = O(CH2)9CH3
6: R1 = H; R2 = O(CH2)11CH3
7: R1 = H; R2 = O(CH2)13CH3
8: R1 = F; R2 = O(CH2)7CH3
9: R1 = F; R2 = O(CH2)8CH3
10: R1 = F; R2 = O(CH2)9CH3
11: R1 = H; R2 = O(CH2)13CH3
12: R1 = F; R2 = Br
18: R1 = H; R2 = O(CH2)3CH3 (30%)
19: R1 = H; R2 = O(CH2)5CH3 (11%)
20: R1 = H; R2 = O(CH2)7CH3 (34%)
21: R1 = H; R2 = O(CH2)8CH3 (25%)
22: R1 = H; R2 = O(CH2)9CH3 (16%)
23: R1 = H; R2 = O(CH2)11CH3 (22%)
24: R1 = H; R2 = O(CH2)13CH3 (15%)
25: R1 = F; R2 = O(CH2)7CH3 (23%)
26: R1 = F; R2 = O(CH2)8CH3 (24%)
27: R1 = F; R2 = O(CH2)9CH3 (22%)
28: R1 = F; R2 = O(CH2)13CH3 (25%)
29: R1 = F; R2 = Br
(9%)
13: R1 = F; R2 = OMe
30: R1 = F; R2 = OMe (15%)
31: R1 = F; R2 = NMe2 (17%)
32: R1 = F; R2 = Me (13%)
14: R1 = F; R2 = NMe2
15: R1 = F; R2 = Me
16: R1 = F; R2 = Cl
33: R1 = F; R2 = Cl
34: R1 = F; R2 = H
(15%)
(10%)
17: R1 = F; R2 = H
H2N
S
O
H2N
S
N N
HN
NH2
R1
R2
NaOH, EtOH, reflux
R1
R2
(2-14)
(35 - 47)
2: R1 = H; R2 = O(CH2)5CH3
3: R1 = H; R2 = O(CH2)7CH3
4: R1 = H; R2 = O(CH2)8CH3
5: R1 = H; R2 = O(CH2)9CH3
6: R1 = H; R2 = O(CH2)11CH3
7: R1 = H; R2 = O(CH2)13CH3
8: R1 = F; R2 = O(CH2)7CH3
9: R1 = F; R2 = O(CH2)8CH3
10: R1 = F; R2 = O(CH2)9CH3
11: R1 = H; R2 = O(CH2)13CH3
12: R1 = F; R2 = Br
35: R1 = H; R2 = O(CH2)5CH3 (29%)
36: R1 = H; R2 = O(CH2)7CH3 (27%)
37: R1 = H; R2 = O(CH2)8CH3 (14%)
38: R1 = H; R2 = O(CH2)9CH3 (29%)
39: R1 = H; R2 = O(CH2)11CH3 (22%)
40: R1 = H; R2 = O(CH2)13CH3 (37%)
41: R1 = F; R2 = O(CH2)7CH3 (22%)
42: R1 = F; R2 = O(CH2)8CH3 (25%)
43: R1 = F; R2 = O(CH2)9CH3 (23%)
44: R1 = F; R2 = O(CH2)13CH3 (26%)
45: R1 = F; R2 = Br
(21%)
13: R1 = F; R2 = OMe
14: R1 = F; R2 = NMe2
46: R1 = F; R2 = OMe (23%)
47: R1 = F; R2 = NMe2 (30%)
Scheme 2. General synthesis of pyrazoline derivatives.
NMR results suggests that only the R(C=S)NH2 and R(C=O)NH2 species, more stable,
1
are present. The H NMR spectra of the compounds showed three doublet of doublets in the
regions of δ 3.0 and δ 6.0 ppm with Ja,b ~ 17.7 Hz, Ja,x ~ 4.8 Hz, and Jb,x ~ 12.0 Hz, confirming
the nonequivalence of hydrogen at Hx (ABX system). NH2 protons were observed at δ 6.51
ppm (compound 32) or δ 7.92-8.03 ppm (compound 46) as singlet. In the 13C NMR spectra

7
signals in the δ 43.0 and δ 59.0 ppm corresponding to C4 and C5 carbons (for details see
Supporting Information).
2.2. Spectroscopy techniques
The Raman spectra were recorded in the 50-3500 cm-1 region at a resolution of 4 cm-1
using a FT-Raman Bruker RFS-100 spectrometer. The Nd:YAG laser line at 1064 nm
wavelength has been used as exciting radiation for the Raman measurements. The laser power
has been kept at 20 mW, and 1000 or 2000 scans have been averaged for each solid sample.
The hydrogen (1H) and carbon (13C) NMR spectra were recorded on Bruker Advance
DRX300 (300 MHz) spectrometer. The chemical shifts values (δ) have been reported in parts
per million (ppm) with reference to tetramethylsilane (TMS) as internal reference. NMR
experiments have been carried out in deuterochloroform (CDCl3) or deuterodimethylsulfoxide
(DMSO-d6). The following abbreviations are used for the multiplicities for proton spectra: s
(singlet); d (doublet); dd (double dublet); m (multiplet). Coupling constants (J) are reported in
Hertz (Hz).
2.3.
Computational Methodology
In order to get a full comparison with the spectroscopic data experimentally obtained,
full quantum mechanical calculations have been applied to compounds 21, 29, 30, 31, 32, 37,
45 and 46.
H2N
H2N
N N
O
S
N N
R1
R2
21: R1 = H; R2 = O(CH2)8CH3
R1
R2
37: R1 = H; R2 = O(CH2)8CH3
45: R1 = F; R2 = Br
46: R1 = F; R2 = OMe
29: R1 = F; R2 = Br
30: R1 = F; R2 = OMe
31: R1 = F; R2 = NMe2
32: R1 = F; R2 = Me
Figure 1. Chemical structure of the pyrazolines analogues.

8
Firstly, the compounds were fully unconstrained optimized to a global minimum point
at the potential energy surface using B3LYP functional [30,31] with 6-311G(2d,2p) basis set
[32] in the polarizable continuum method IEFPCM [33] as implemented in Gaussian 09
program package [34]. The solvent used was the DMSO since the synthesized compounds are
soluble in this solvent. Vibrational harmonic frequencies calculations were performed for
each compound in order to obtain the calculated Raman spectra. Those frequencies were
scaled by different factors for a better comparison with experimental data. Thermal
contributions to Gibbs free energy and other state functions were calculated at 298.15 K and 1
atm. NMR shifts were calculated using the Gauge-Independent Atomic Orbital (GIAO)
method [35]. TMS was used as reference and all NMR calculations were performed
considering the DMSO dielectric constant.
Then, for a better comparison a scan calculation have been performed in order to
obtain the most accurate geometry related to the dihedral which turns the functional group
N−CO(NH2) as it has been done for pyrazinamide ligands [36]. The best (lowest) calculated
structure in energetic terms was obtained as displayed in Figure 2 for compound 32. The
optimization of all structures was then based on the general geometry obtained through this
scan as depicted in Figure 2.

9
Figure 2. Highlighted at bottom right, the obtained structure in the scan procedure. This
structure corresponds to the lowest point in the graphic.
For compounds 21 and 37, those with a long aliphatic carbon chain, a simulated
annealing has been performed in order to achieve a structure with the lowest energy. The
method was basically the same used before in Rey’s work [37]. Nine geometries in a range of
2 kcal mol-1 were chosen in order to perform full DFT calculations using the same level of
theory as used for structure optimization. The results point out to the same geometry, in
general. All simulated annealing details can be found in the Supplementary Material.
3. RESULTS AND DISCUSSION
Scientists in biosciences field must understand and take into consideration the
tautomerism effect and the spatial conformers as a relevant aspect for biological activity. In
this sense, the characterization of the pyrazoline derivatives of the representative compounds
32, and 46 (see scheme 2) is presented in details by NMR, FT-Raman techniques and DFT
methods. The results for the compounds 21, 29, 31, 37 and 45 have been subjected to a

10
similar analysis, and are presented as Supporting Information. The structural characterization
of the cited compounds could be regarded as representative of the behavior of the entire
series. In the case of the pyrazolines synthesized, a tautomerism effect may occur; namely, the
carboxamide/carbothioamide group (R(C=O)NH2/R(C=S)NH2) may tautomerize to the
corresponding (R(C=NH)OH/R(C=NH)SH) species, in the solid state or in solution (see
Scheme 3).
3.1.
Raman characterization of the tautomeric structures
Raman spectroscopy, due to the sensitivity to the molecular structure and to the well-
recognized ability of the Raman effect to detect more than one tautomeric form
simultaneously [19], has been chosen to characterize the tautomeric behavior of the
synthesized series of pyrazolines derivatives. The pyrazoline derivatives could present a
tautomerism as the one presented in Scheme 3.
R
R
tautomerization
N
R'
N
N
R'
N
X
XH
H
N
HN
H
X= S tautomer II
X= O tautomer IV
X= S tautomer I
X= O tautomer III
Scheme 3. Tautomeric forms of pyrazoline derivatives.
It should be mentioned that characteristic bands of the tautomers may be found in the
Raman spectra: for the carboxamide derivatives, the changes in intensity of the band assigned
to C-OH groups compared to C=O would be enough to characterize the presence of different
tautomer; similar information would be available from the analysis of the bands assigned to
C-SH/C=S in the Raman spectra of the carbothioamides. For the characterization of the
tautomeric species, one representative compound of each carboxamide and carbothioamide
series are discussed in detail. Figure 3 presents the experimental and DFT calculated Raman

11
spectrum of compound 46. An assignment of the Raman spectrum, based on the literature [44]
and on theoretical calculations of vibrational wavenumbers, is presented in Table 1.
Figure 3. (a) Experimental Raman spectra of the compound 46 in the solid-state; the inset
present a zoom in the 500-1250 cm-1 and 2400-2700 cm-1 regions of the spectrum. (b) Raman-
DFT calculated spectrum of compound 46 in DMSO dielectric; the inset represents the DFT-
optimized structure for 46, used for the calculation of the Raman spectrum.

12
Table 1. Assignment of selected experimental and theoretical Raman band positions for the
compound 46 (solid-state).
Reference to
Figure 3
Experimental
wavenumber / cm-1
Theoretical
wavenumber / cm-1
Theoretical scaled
wavenumbera / cm-1
Assignment
605 b
731-858
1030
A
B
C
D
E
F
601
700-915
1018
1094
1158
1228
1266
1334
1577
1604
2593
599
723-867
1020
νC-S, (NCNrocking)
δoopC-H
νtriCC aromatic
νC=S
1351
1422
1043, 1096
1298, 1322
1368
1098, 1154
1233, 1256
1300
νCN, νCC
βCH
G
H
I
νCC, νCN, νC-NH2
νCN
1489
1414
1614
1582
νCC aromatic + νsC=N
νCC aromatic + νsC=N
νS-H
J
1614
1582
c
c
K
2836, 2910, 2965,
2976
2873, 2908, 2929,
2955, 2962, 2988
3024, 3061, 3083,
3111, 3118, 3145
L
νC-H aliphatic
3168, 3175, 3193,
3199, 3201, 3212,
3214, 3216
3009, 3016, 3033,
3039, 3040, 3051,
3053, 3055
3014, 3036, 3052,
3064, 3078
M
N
νC-H aromatic
νSNH2
3334
3586
3407
aScaled fators: A, B, C = 0.99; D, E, F, G, H = 0.95; I, J = 0.98; L, M, N = 0.95. Most bands are
b
related to conjugated modes, i.e., no simply assignment can be made. The band at 601 cm-1 has been
found to be assignable to a different mode in the carbothioamide tautomer. cThe νS-H band cannot be
identified in this calculation since the structure of compound 46 has been optimized as a thioamide.
The bands assigned in Table 1 are characteristic of the chemical moieties present in
compound 46, but one can observe three bands, two at 601/2593 cm-1 assigned to C-SH group
and one at 1094 cm-1 assigned to the C=S group (bold in Table 1). The presence of the two

13
groups of bands strongly suggests the presence of both tautomeric forms indicated in Scheme
2. It should be noticed, however, that the relative intensity of the ν(SH) in the Raman
spectrum of compound 46 (inset of Figure 3) is considerably lower than what is usually
expected for this mode [29]. This unexpected low relative intensity of the band at 2593 cm-1
suggests that an additional resonance structure, that presents the C-S group, but deprotonated
(C-S-), may have an important contribution to the structure of the compound 46 in the solid
state (see Scheme 4).
F
F
OCH3
OCH3
+
N
N
N
N
S-
S
H
N
H
N
H
H
Scheme 4. Resonance structure of the pyrazoline derivatives.
Raman spectra of the carbothioamide compounds 37, 40, 45 and 46 have been
obtained and a similar analysis can be performed. The intensity ratio between the band
assigned to ν(C-S) to the band assigned to ν(C=S), presented in Table 2, changes as a function
of the substituent, and one can additionally observe a change when the Raman spectra is
acquired for the compounds in chloroform solution.
Table 2. Relative Intensity of the Raman bands νC=S (thioamide tautomer) versus νC-S (thiol
tautomer) for carbothioamide compounds in the solid state and in chloroform solution.
Compound
Solid-state
0.88
In solution
1.8
2.0
1.6
2.5
(37)
(40)
(45)
(46)
0.87
1.52
0.98

14
One can notice that for the substances in the solid state, compounds 37, 40 have a
similar value of the I(νC=S)/ I(νC–S) intensity ratio, and for compound 46 there is an increase
in the contribution of the thioamide tautomer, which increases even further for compound 45.
This change may be related with electron withdrawing properties of the substituents;
compounds 37 and 40 present moderately donating O-atom connected directly to the aromatic
rings, but compound 46 presents a moderately withdrawing and one moderately donating
group, while compound 45 presents two moderately withdrawing substituents. These
statements are summarized in Scheme 5. For the compounds in solution the I(νC=S)/ I(νC–S)
relative intensity has changed in the direction of the carboxithioamide tautomer (see Table 3),
indicating that interactions with solvent plays an important role in the tautomeric equilibrium
of this family of compounds.

15
for electron-donating substituents
O(CH2)8CH3
O(CH2)8CH3
N
N
N
N
SH
S
HN
H2N
(37)
O(CH2)13CH3
O(CH2)13CH3
N
N
N
N
SH
S
HN
H2N
(40)
for two electron-withdrawing substituents
F
F
Br
Br
N
N
N
N
S
SH
H2N
HN
(45)
for electron-donating and electron-withdrawing substituents
F
F
OCH3
OCH3
N
N
N
N
S
SH
H2N
HN
(46)
Scheme 5. Tautomerization of the thioamide to the thiol form.
For the carboxamides a case study is presented in Figure 4, which presents the
experimental Raman spectrum of compound 32 in the solid state, along with the DFT-
calculated spectrum in DMSO solution (the optimized DFT structure is presented as inset);
the vibrational assignment for the Raman spectrum is presented in Table 3. The Raman
spectrum in Figure 4(a) presents reasonable resolution and high signal-to-noise ratio, and one
can notice from the assignment in Table 3 a medium-weak intensity band assigned to νC=O;
however, it is not straightforward to assign specific bands to the C-OH group present in
tautomer II (Scheme 3) of this series of compounds, because the bands assignable to C-O and
C-N groups are assigned to quite similar regions of the Raman spectrum. One can notice, on

16
the other hand, that the bands assigned to C-N groups, present both in carboxamide and
carbothioamide compounds, are more intense in the carboxamide compounds (comparing, for
example Figure 1 and 3); although not conclusive, the change in intensity may be an
indication of the presence of the tautomer II for this series of compounds. In Figure 4(b), the
DFT calculated Raman spectrum can be seen for the compound 32 and shows an impressive
correspondence with the experimental one.
Figure 4. (a) Experimental Raman spectra of the compound 32 in the solid-state; the inset
presents a zoom in to the 1100 to 1800 cm-1 spectral range. (b) Raman-DFT calculated
spectrum of compound 32 in DMSO dielectric; the inset represents DFT-optimized structure
for 32, used for the calculation of the Raman spectrum.
Table 3. Assignment of selected experimental (solid-state) and theoretical Raman band
positions for the compound 32.
Theoretical scaled
to Figure 4 wavenumber / cm-1 wavenumber / cm-1 wavenumbera / cm-1
Reference
Experimental
Theoretical
Assignment

17
734; 741; 744;
831; 857
907
727; 734; 737;
823; 848
898
A
683; 717; 812; 877
δC−Harom
B
C
D
E
F
897
1158
νtri(CC)
νCN, νCC
νCN
1156
1144
1454
1250
1248
1295
νCN, νCC, νC-OHb
βC−Harom
1357
1276
1436; 1448; 1454
1613; 1616
1350; 1361; 1367
1516; 1519
1354; 1376; 1412
1518
G
H
I
νC=N
1576
νC=N + νCCarom
νCCarom
1636; 1648; 1652
1538; 1549; 1553
1608
1712
1678
J
1673
νC=O
3031; 3063; 3076;
3079; 3099; 3108
3159; 3175; 3178;
3196; 3199; 3210;
3212
2879; 3910; 2922;
2925; 2944; 2953
3001; 3017; 3019;
3036; 3039; 3050;
3051
K
L
2922; 2941; 2951
3012; 3034; 3059
νC−Haliph
νC−Harom
aScaled fators: A, B, C = 0.99; D = 0.86; E, F, G, H, I = 0.94; J = 0.98; K, L = 0.95. Most bands like
the one in 1454 cm-1 (non-scaled theoretical value) are related to conjugated modes, i.e., no simply
b
assignment can be made. The νC-OH band cannot be identified in this calculation since the structure
of compound 32 has been optimized as a ketone, for clarifying.
3.2. Computational studies on the tautomeric structures
In order to check on the stability of the series of pyrazoline derivatives, and also to get
as much spectroscopic information as it could be provided by the unconstrained
optimizations, NMR calculations were performed in addition to the Raman spectrum. All
structures were characterized as a minimum in the potential energy surface.
The Raman spectra obtained for compounds 32 and 46 were of remarkable agreement
in comparison with the experimental ones as already discussed (see Figure 3(b) for compound
46 and Figure 4(b) for compound 32). This noteworthy agreement between experiment and

18
calculation enabled the assignment of the Raman spectra with a great contribution from the
calculated spectra, as one can notice in Tables 1 and 3, which showed the most important
bands with the respective assignments.
The main bands of the calculated Raman spectrum of compound 46 are in good
agreement with the experimental one. Some scaled factors were applied in order to improve
this agreement for all bands, even for those bands that it would not be necessary, in order to
comply a statistical meaning. Since the highest the scaled factor, better the description of the
band wavenumber, it can be seen that only the theoretical value found for the band labeled as
“D” in Tables 2 is far from the experimental data. This specific spectral region is quite
convoluted to be assigned by the DFT calculations, once different conjugated modes have
been found in this region. The very same statement could be done for the Raman
spectroscopic data found for compound 32, if one looks closely to Table 3. Once again the
calculated bands are in a good agreement to the band positions form the experimental
spectrum. The assignments of some normal vibrational modes might be very difficult to
determine since in most theoretical calculations many vibrational modes are conjugated with
others and the displacement vectors imply in different attribution when comparing with
experimental data; this has not been observed in the data obtained for the pyrazoline
derivatives presented in this work. Therefore, the data show in Tables 1 and 3 are in fact good
for this level of calculations.
In order to identify the tautomeric equilibrium, the NMR-GIAO calculations have
been performed for compound 32 and 46. From these calculations the differences between the
protonated and non-protonated structures can be stablished. Such results are showed in Figure
1
5. The results found for the H-NMR shifting seems to provide a good linear fit between
experimental and DFT calculations, and so the calculation results could be used for deciding
which tautomeric structure is present.

19
R2
Hb Ha
Hx
N
N
R1
O,S
H2N
(c)
Figure 5. 1H-NMR shifts experimental-DFT comparison for compounds (a) 32 and (b) 46. (c)
Labelled scheme for hydrogens Ha, Hb, Hx and NH2 to better correlate the data in plots (a)
and (b).
1
As can be seen in Figure 5, the theoretical results for H-NMR calculated under the
DMSO dielectric constant are in very good agreement with the experimental ones. The R2
data from fitting linear regression is equal to 0.96 for both comparisons in Figures 5(a) and
5(b). The largest disagreement regards to the NH2 hydrogen atoms for both compounds 32
and 46, which is in fact interesting due to the mixture of tautomers that exists in real solution.
The other hydrogen signals are in almost perfect accordance to those found in solution. Here,
is interesting to highlight the signals of hydrogens Ha, Hb and Hx, according to the labels
presented in Figure 5(c). For compound 32, the NMR experimental signals found were 3.02
(dd, 1H, Ha, Ja,x= 5.1 Hz, Ja,b= 18.0 Hz); 3.76 (dd, 1H, Hb, Jb,x= 12.0 Hz, Jb,a= 18.0 Hz);
5.36 (dd, 1H, Hx, Jx,a= 5.1 Hz, Jx,b= 12.0 Hz); 6.51 (sl, 2H, N-H). For compound 46, the
NMR experimental data are 3.12 (dd, 1H, Ha, Ja,x= 3.0 Hz, Ja,b= 18.0 Hz); 3.71 (s, 3H, CH3);
3.85 (dd, 1H, Hb, Jb,x= 12.0 Hz, Jb,a= 18.0 Hz); 5.87 (dd, 1H, Hx, Jx,a= 3.0 Hz, Jx,b= 12.0
Hz); 7.92-8.03 (m, 4H, Ar e N-H). All other NMR data can be found at the Supporting
Information.

20
Another important factor correlated to the differences between these two tautomers is
the thermodynamics. Through the frequencies calculations the ∆G values between the
protonated and non-protonated species suggests that the R(C=S)NH2 and R(C=O)NH2 species
are more stable than the R(C=NH)SH and R(C=NH)OH species. The calculated difference
between compounds 46 and its protonated counterpart is 21.34 kcal mol-1, while for
compound 32 and its tautomer one can find a difference of 22.50 kcal mol-1. Both values are
in the same order and point out to the original compounds 32 and 46 as the most stable in this
comparison. From such Gibbs energies results is possible to suggest that both species can be
found in solution but probably there would be a large amount of 32 and 46 than the
protonated ones. This calculation results for the pyrazoline compounds in solution agrees with
the observed for the Raman of the carbothioamide compounds in solution (Table 1), in which
it has been verified that tautomer I structures presented larger Raman intensity than those for
tautomer II.
4. CONCLUSIONS
In this work the synthesis of a series of thirty new pyrazolines has been successfully
1
performed, and the compounds have been characterized by H and 13C NMR. In order to
understand in more details the tautomeric behavior in the solid and solution states, a joint
experimental-computational study using density functional theory, FT-Raman and NMR
investigation were carried on the tautomerism of pyrazole-carbothioamide and pyrazole-
carboxamide compounds.
The Raman spectra of selected compounds were conclusive for the carbothioamide
compounds and indicated that the tautomeric distribution changes significantly from solid to
solution, as tautomer I seems to be more stable in solution than in the solid state. Density
functional theory calculations have played an important role in this analysis, and presented a
good accordance that allowed a vibrational assignment of the Raman spectra. Such analysis
might assist the experimentalists to further discuss some assignments. Also, a very good
agreement was achieved by the NMR calculations in DMSO solution. The results indicates
that in fact in a real solution the tautomers must be present since only the NH2 hydrogen shift
have a large disagreement between experiment and calculation. Furthermore, the calculated

21
thermodynamics indicates the stability of the compounds in the tautomer I form, in agreement
with the Raman spectroscopy indicated for the carbothioamide compounds.
ACKNOWLEDGMENTS
The authors gratefully acknowledge UFJF, CAPES, CNPq and FAPEMIG for fellowships.
GFSA thanks CNPq for a research fellowship. SA thanks FAPEMIG for a fellowship. This
work is a collaboration research project of members of the Rede Mineira de Química (RQ-
MG) supported by FAPEMIG.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and spectroscopic data for the
compounds, computational data and experimental Raman data and assignment on selected
compounds are available.
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

Synthesis of a series of new pirazolines derived from chalcones is described
NMR and Raman spectroscopy study on the tautomerization equilibrium in the series
of compounds

Energy and vibrational computational DFT calculations allowed understanding the
tautomerization equilibrium.