Experimental and computational studies on the tautomerism of N-substituted 3-amino-5-oxo-4-phenyl-1H-pyrazolo-1-carboxamides with antibacterial activity

Article abstract of DOI:10.1016/j.molstruc.2013.08.010

The tautomerism of N-substituted 3-amino-5-oxo-4-phenyl-1H-pyrazolo-1- carboxamides with antibacterial activity is studied with X-ray crystallography, IR, ¹H and ¹³C NMR (including NOESY spectra) and quantum chemical calculations. It

Full text of DOI:10.1016/j.molstruc.2013.08.010

Journal of Molecular Structure 1051 (2013) 188–196
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Experimental and computational studies on the tautomerism
of N-substituted 3-amino-5-oxo-4-phenyl-1H-pyrazolo-1-carboxamides
with antibacterial activity
Agnieszka A. Kaczor a,b, Tomasz Wróbel a, Zbigniew Karczmarzyk c, Waldemar Wysocki c,
Ewaryst Mendyk d, Antti Poso b, Dariusz Matosiuk a, Monika Pitucha e,
⇑
a Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modeling Lab, Faculty of Pharmacy with Division of Medical Analytics, 4A
´
Chodzki St., PL-20093 Lublin, Poland
b School of Pharmacy, University of Eastern Finland, Yliopistoranta 1, P.O. Box 1627, FI-70211 Kuopio, Finland
c Department of Chemistry, Siedlce University, 3 Maja 54 St., PL-08110 Siedlce, Poland
d Analytical Laboratory, Faculty of Chemistry, M. Curie-Skłodowska University, 3 M. Curie-Skłodowska Sq., PL-20031 Lublin, Poland
e
´
Department of Organic Chemistry, Faculty of Pharmacy with Division of Medical Analytics, 4A Chodzki St., PL-20093 Lublin, Poland
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
Tautomerism of N-substituted
3-amino-5-oxo-4-phenyl-1H-
pyrazolo-1-carboxamides.
The form with the keto group is the
preferred one in the crystalline state
and DMSO.
Some fraction with the corresponding
hydroxy group also occurs in both
states.
This finding was related to the
antibacterial activity of the studied
compounds.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2013
Received in revised form 9 August 2013
Accepted 9 August 2013
Available online 14 August 2013
The tautomerism of N-substituted 3-amino-5-oxo-4-phenyl-1H-pyrazolo-1-carboxamides with antibac-
terial activity is studied with X-ray crystallography, IR, 1H and 13C NMR (including NOESY spectra) and
quantum chemical calculations. It is found that the form with the keto group at position 5 is the preferred
one in the crystalline state and in DMSO, although some fraction with the corresponding hydroxy group
also occurs in both states. This finding was related to the antibacterial activity of the studied compounds
as the energetic stabilization of the keto group may determine their proper hydrogen bond interactions
with the bacterial enzyme.
Keywords:
Antibacterial compounds
Pyrazolones
Ó 2013 Elsevier B.V. All rights reserved.
Quantum chemical calculations
Tautomerism
X-ray structure determination
1. Introduction
play an important role in the transmission of genetic information
and cell division. It is a part of the purine which forms nucleic
Azoles are medicinally important compounds with a wide range
of activity. Azole ring occurs in many biological molecules, which
acids, DNA and RNA. There are many reports about the activity
and possible practical application of pyrrole, imidazole, oxadiazole
and triazole derivatives [1–4]. However, among azoles pyrazoles
deserve special attention. They are known as herbicides, fungi-
cides, insecticides, MDR modulators and also antibacterial,
⇑
Corresponding author. Tel.: +48 815357374.
E-mail address: monika.pitucha@umlub.pl (M. Pitucha).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.08.010

A.A. Kaczor et al. / Journal of Molecular Structure 1051 (2013) 188–196
189
anti-inflammatory, anti-tumor antihyperglycemic, antipyretic and
analgesic agents [5–14]. Among their range of properties, some
compounds containing the pyrazole scaffold were shown to exhibit
HIV-1 reverse transcriptase and IL-1 synthesis inhibitory activity
[14].
and treated as riding on their parent C atoms with NAH distance
of 0.86 Å and CAH distances of 0.93 Å (aromatic), 0.97 Å (CH2)
and 0.96 Å (CH3). All H atoms were refined with isotropic displace-
ment parameters taken as 1.5 times those of the respective parent
atoms. All calculations were performed using WINGX version
1.64.05 package [35]. CCDC – 924496 contains the supplementary
crystallographic data for this paper. These data can be obtained
free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html (or
from the Cambridge Crystallographic Data Centre (CCDC), 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email:
deposit@ccdc.cam.ac.uk). Crystal data of 5 are collected in Table 1.
The activity of pyrazoles as potential pharmacological agents
depends naturally on the chemical structure, including the tauto-
meric form of the molecule. It is well known that the tautomerism
of biologically important compounds may determine their activity
as it contributes to their interactions with the molecular target.
The tautomerism of pyrazolones is a well-defined problem of pyr-
azole chemistry and thus it has been the subject of a significant
number of studies [15–28]. Regarding the structural aspects of
these derivatives, it was established that they may be presented
in many forms as a result of tautomeric proton migration between
the carbonyl group and the nitrogen atom. The existence of C@O,
ANH, ACOH and AN tautomers is possible. Although this is a com-
mon type of isomerism, it is difficult to determine the structure of
the investigated compounds [29,30]. The electronic effects and the
position of the substituent, pH and solvent polarity are major fac-
tors that influence tautomerization.
2.2. NMR spectra
All NMR spectra were obtained on Bruker AVANCE III 600 MHz
spectrometer with a BBO probe equipped with Z-gradient. Solvents
were used as received from a commercial supplier. Tetramethylsil-
ane was used as an internal standard for proton and carbon NMR
spectra.
2.3. IR spectra
Searching for novel pharmacologically active compounds, we
obtained N-substituted 3-amino-5-oxo-4-phenyl-1H-pyrazol-1-
carboxamides 1–10 (Scheme 1) with antibacterial activity [31].
As the biological properties of chemical compounds are clearly
connected with the compounds’ structure, the elucidation of struc-
tural parameters of novel compounds is essential to rationalize
their activity. The aim of this study is to determine the tautomer-
ism of pyrazolone moiety with experimental (X-ray crystallogra-
phy, 1H and 13C NMR, IR) and quantum chemical approaches as
we have previously found that this phenomenon determines phar-
macological activity in similar systems [32,33]. The studied com-
pounds 1–10 are characterized by different substituents at the
nitrogen atom and thus may be expected to exhibit different rela-
tive stability of the hydroxy and the keto form.
IR spectra were recorded on a Thermo Nicolett 6700 FTIR spec-
trometer using ATR diamond Orbit stage and IR spectra in DMSO
were recorded on a Perkin–Elmer1725X FTIR spectrometer.
2.4. Computational details
The molecular structure of 1–10 in the ground state (in vacuo)
was optimized with the B3LYP DFT (the variant of the DFT method
using Becke’s three parameter hybrid functional (B3) [36] with cor-
relation functional such as the one proposed by Lee, Yang, and Parr
(LYP) [37]) using 6-31G(d,p) as included in Gaussian09 [38]. Fur-
thermore, frontal molecular orbital (FMO) analysis was performed
with Gaussian09 on the 6-31G(d,p)/B3LYP level of theory. The en-
ergy for both tautomers of 1–10 was calculated for isolated mole-
cules (gas phase) and molecules in DMSO solutions. The population
of both tautomeric forms was estimated using non-degenerate
Boltzmann distribution. 6-31G(d,p)/HF and 6-31G(d,p)/B3LYP cal-
culations with Gaussian09 were also used to calculate the Contin-
uous Set of Gauge Transformations (CSGT) [39–41] 1H and 13C NMR
chemical shifts as well as vibrational frequencies and infrared
intensities. Calculations were performed using the Polarizable Con-
tinuum Model (PCM) [42]. This method creates a solute cavity via a
set of overlapping spheres.
2. Experimental
2.1. X-ray studies
X-ray data of 5 were collected on the Kuma KM4 diffractometer;
crystal sizes 0.40 ꢁ 0.30 ꢁ 0.20 mm, Mo K
a (k = 0.71073 Å) radia-
ꢂ2h scans. The structure was solved by direct methods
tion,
x
using SHELXS97 [34] and refined by full matrix least squares with
SHELXL97 [34]. In the absence of significant anomalous scattering,
Friedel pairs were merged and the S absolute configuration was
arbitrarily chosen. The H atoms were positioned geometrically
Table 1
Crystal data and structure refinement for the compound 5.
Empirical formula
Formula weight
Crystal system
C18H18N4O2
322.36
Monoclinic
P21
Space group
Unit cell parameters
a = 12.384(2)
b = 7.7515(14)
c = 17.032(3) Å
b = 101.470(15)°
1602.3(5) Å3
4
Volume, V
Molecular multiplicity, Z
Density (calculated)
F(000)
1.336 g/cm3
680
Radiation
l(Mo Ka
) = 0.090 mmꢂ1
Temperature
293 K
No. of measured reflections
h Range for data collection
No. of independent reflections
Extinction coefficient
Final R indices: R, wR(F2)
Goodness-of-fit on F2, S
10,319
2.26–30.10°
5014
0.026(5)
Final R = 0.072, wR = 0.169
0.882 for 1252 reflections with I > 2
r(I)
Scheme 1. The studied compounds 1–10.

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A.A. Kaczor et al. / Journal of Molecular Structure 1051 (2013) 188–196
Table 2
Hydrogen-bond geometry (Å, °) of 5.
DAHꢃ ꢃ ꢃA.
DAH
Hꢃ ꢃ ꢃA
Dꢃ ꢃ ꢃA
DAHꢃ ꢃ ꢃA
N22AAH22A...O3A
N22BAH22Bꢃ ꢃ ꢃO3B
N1AAH1Aꢃ ꢃ ꢃO21Bi
N5AAH51Aꢃ ꢃ ꢃO21Bi
N1BAH1Bꢃ ꢃ ꢃO21Aii
N5BAH51Bꢃ ꢃ ꢃO21Aii
N5AAH52Aꢃ ꢃ ꢃO3Aiii
0.86
0.86
0.86
0.86
0.86
0.86
0.86
2.05
2.04
2.17
2.28
2.27
2.42
2.36
2.733(9)
135
137
137
142
139
143
131
2.731(10)
2.858(10)
3.003(10)
2.974(11)
3.145(11)
2.990(11)
Symmetry code: (i) = 1ꢂx, ꢂ½ + y, 1ꢂz; (ii) = 1ꢂx, ½ + y, 1ꢂz, (iii) = 1ꢂx, ꢂ½ + y, ꢂz.
Scheme 2. Synthesis of compounds 1–10.
1.75 g (0.01 mol) of 1-cyanophenylacetic acid hydrazide and
0.01 mol of appropriate isocyanate in 10 ml of acetonitrile was
kept for 24 h at room temperature; then the compound formed
was filtered off, washed with acetonitrile and crystallized from a
methanol–acetonitrile (1:1) mixture (Scheme 2) [31].
In the case of NMR chemical shifts DMSO was used as a solvent.
The 1H and 13C NMR chemical shifts were converted to the TMS
scale by subtracting the calculated absolute chemical shielding of
TMS. These are 30.67 ppm and 182.72 ppm for 6-31G(d,p)/B3LYP
for hydrogen and carbon atoms, respectively. The vibrational band
assignments were made using ChemCraft [43], Pymol v. 0.99 [44],
Discovery Studio v. 3.1 [45], Yasara structure [46], Mercury [47]
and ArgusLab [48] were also used for visualization of results.
3.2. X-ray and computed structure of 5
Crystal structure determinations of 5 were undertaken in order
to confirm the synthesis pathway and to identify its tautomeric
form in the solid state. The X-ray structure analysis of 5 revealed
that the asymmetric part of the unit cell of its crystal contains
two independent molecules in the same arbitrarily chosen abso-
lute configuration, denoted as A and B in Fig. 1A. Both molecules
exist in N22-amino/O21-keto tautomeric form, as evidenced by
the C21AO21 bond lengths of 1.235(11) and 1.214(10) Å for A
and B, respectively, which are typical for the carbonyl group. The
3. Results
3.1. Chemistry
N-substituted 3-amino-5-oxo-4-phenyl-1H-pyrazol-1-carbox-
amides 1–10 were obtained as reported earlier: a mixture of
Fig. 1. ORTEP drawing of 5 with numbering of non-H atoms (A); the overlay of two crystallographic independent molecules A and B of 1 by least-squares fitting of the atoms
0
of the pyrazole ring [RMS = 0.0682 ÅA] (B).

A.A. Kaczor et al. / Journal of Molecular Structure 1051 (2013) 188–196
191
Fig. 2. The molecular packing with the net of intermolecular hydrogen bonds in crystal of 5.
Fig. 3. Overlap of crystal (molecules A and B) and computed structures of 5 on the
pyrazolone system. Hydrogen atoms not shown for clarity.
Fig. 4. HOMO (A) and LUMO and (B) orbitals for the keto form of 5.
molecules A and B differ in conformation of the phenyl and ethyl-
phenyl substituents in relation to the pyrazole-carboxamide
system, as can be seen in Fig. 1B. The conformation of these sub-
stituents is described by the torsion angles C3AC4AC41AC42 of
128.6(11) and ꢂ51.9(16)°, C21AN22AC23AC31 of ꢂ84.1(12) and
ꢂ105.8(11)° and N22AC23AC31AC32 of ꢂ24.5(11) and 67.9(11)°
NAHꢃ ꢃ ꢃO hydrogen bonds connecting pairs of molecules A and B
related by the 21 axis into molecular dimers. These dimers form
molecular chains parallel to the b crystallographic axis via the
intermolecular N5AAH52Aꢃ ꢃ ꢃO3A hydrogen bond (Table 2). The
net of intermolecular hydrogen bonds in the crystal of 5 is shown
in Fig. 2.
The structure of 5 computed with the B3LYP DFT approach and
6-31G(d,p) basis set of Gaussian09 was aligned to the molecules A
and B of 5 from crystallographic studies. The alignment on the pyr-
azolone system of molecules A and B from the X-ray structure of 5
and its computed structure is presented in Fig. 3. It can be seen that
for A and B, respectively. The pyrazole-carboxamide part of 5 is
0
planar to within 0.083(9) and 0.094(11) ÅA in molecule A and B,
respectively. In both molecules, the carbonyl group of the carbox-
amide moiety adopts a cis conformation with respect to the NAN
bond in the pyrazole ring giving the possibility to form an intramo-
lecular N22AH22ꢃ ꢃ ꢃO3 hydrogen bond (Table 3). The amino and
carbonyl groups characteristic for the tautomeric form of 5 ob-
served in its crystal are involved in two bifurcated intermolecular

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A.A. Kaczor et al. / Journal of Molecular Structure 1051 (2013) 188–196
Table 5
Experimental and computed NMR chemical shifts for 1.
Atom
Experimental
Keto
Hydroxy
H1
10.45
5.33
9.58
H10 H14
H11 H13
H12
7.55
7.31
7.11
6.85
6.73
6.5
6.95
6.73
6.51
H15
H16
6.48
8.79
3.31
3.25
4.1
5.24
Fig. 5. Electron density distribution in the keto form of 5.
H17
H18–H22
C3
C4
C5
C6
C9
C10 C14
C11 C13
C12
3.58–3.71
1.20–1.92
162.87
85.93
154.48
149.08
131.99
126.24
127.99
124.39
47.50
3.67
1.07
3.65
1.19
174
168.12
94.84
160.38
157.61
138.18
130.48
133.45
129.99
54.94
33.62
25.43
24.09
Table 3
95.75
161.39
158.19
139.84
132.32
136.38
131.75
57.39
43.79
29.31
30.17
The stabilization energy
DE (kcal/mol) for the keto forms of 1–10 in relation to
antibacterial activity [31].
Compound Staphylococcus aureus, ATCC25923, MIC
g/ml)
Gas
phase
DMSO
(l
1
2
3
4
5
6
7
8
9
10
15.63
62.5
125
250
500
500
500
>1000
ND
21.18
15.10
21.72
10.76
11.13
8.15
16.56
15.10
14.76
16.15
12.14
9.02
12.0
7.30
5.44
6.45
6.97
5.56
5.23
5.44
C17
C18 C22
C19 C21
C20
32.41
24.98
23.88
Naturally, the highest electron density occurs on both oxygen
atoms and on the amino nitrogen atom.
ND
ND – not determined.
3.3. Boltzmann distribution studies in relation to antibacterial activity
Table 4
The energy for both the keto and hydroxyl tautomers of 1–10
was calculated for isolated molecules (gas phase) and molecules
in DMSO solutions. The results of the calculations are presented
in Table 3. It can be seen from Table 3 that the keto form is the
one existing in the crystalline state and this form is the most ener-
getically stable in the DMSO solution. The population of the hydro-
xy form estimated using non-degenerate Boltzmann distribution is
less than 0.01% for 1–10 in the crystalline and 1–4 and 6–7 in
DMSO whereas it is about 0.01% for 5 and 8–10 in DMSO at
25 °C. The energy differences between the keto and hydroxy forms
change in the relatively narrow range of 5.23–21.72 kcal/mol. The
results of the calculations are in good agreement with the experi-
mental data obtained from X-ray analysis, NMR and IR spectros-
copy without a solvent but do not confirm the presence of the
hydroxyl form for all compounds. However, the stabilization en-
ergy is much lower in DMSO, so a general tendency is shown.
The stabilization energy of the keto form of compounds 1–10
may be used to address their antibacterial activity. Table 3 shows
the antibacterial activity of 1–10 in relation to their stabilization
energy [31]. It can be seen that the higher stabilization of the keto
form generally promotes antibacterial activity, as is the case for 1
and 3 and to the lesser extent for 2. By contrast, the low energy
barrier decreases activity in 5, 6, 7 and 8. Thus the keto group is
probably one of the pharmacophore groups for these compounds
and might be involved as an acceptor in the formation a hydrogen
bond with the appropriate residues of the bacterial enzyme.
Experimental and computed IR frequencies involved in the tautomerism of com-
pounds 1–10.
R
IR
NAH (cmꢂ1
)
C@O (cmꢂ1
)
OH (cmꢂ1
Exp. Calc.
)
Exp.
Calc.
Exp.
Calc.
1
2
3
4
5
6
7
8
9
10
ATR
DMSO
3461
3427
3489
3517
1692
1703
1778
1728
3130
2932
3312
3261
ATR
DMSO
3487
3316
3479
3511
1678
1703
1828
1771
3381
3179
3447
3448
ATR
DMSO
3469
3260
3476
3513
1694
1703
1775
1728
3328
3167
3332
3288
ATR
DMSO
3483
3314
3449
3492
1678
1703
1768
1737
3301
3178
3335
3289
ATR
DMSO
3374
3303
3462
3494
1681
1704
1763
1733
3279
3175
3321
3275
ATR
DMSO
3466
3306
3477
3512
1684
1703
1732
1784
3328
3165
3330
3278
ATR
DMSO
3475
3443
3453
3492
1683
1703
1787
1749
3131
2994
3375
3334
ATR
DMSO
3446
2970
3451
3484
1701
1708
1787
1741
3154
2900
3361
3337
ATR
DMSO
3461
3261
3449
3490
1684
1711
1790
1743
3128
3166
3393
3369
ATR
DMSO
3453
3435
3445
3484
1689
1709
1790
1741
3128
2995
3367
3344
conformation of the benzyl group is different for both molecules A
and B but for the phenyl group it is different for only one of them.
Further conformational analysis studies may help to obtain a more
accurate structure. Fig. 4 presents HOMO and LUMO orbitals for 5
whereas Fig. 5 presents the distribution of electron density for 5.
3.4. Experimental and computed IR spectra
IR spectra were recorded without a solvent and in DMSO as well
as computed with the B3LYP DFT method. The IR data for the
frequencies connected with the tautomerism of the investigated

A.A. Kaczor et al. / Journal of Molecular Structure 1051 (2013) 188–196
193
Fig. 6. An expanded fragment of the NOESY spectrum for 1 (A) and 6 (B).
compounds are presented in Table 4. The data indicate that some
fraction of the hydroxyl tautomer occurs in both the crystalline
state and DMSO for all the compounds; a fact that is not fully con-
firmed by the computational data discussed above.
10.5/6.5 ppm (Fig. 6) was found to have the same phase as the
diagonals, confirming chemical exchange but not close vicinity of
imine and amine protons which would correspond to the keto
form. Thus we tried the 1H–15N HSQC approach to detect any cou-
pling present when the proton is directly attached to nitrogen.
Fig. 7 represents the N-HSQC spectrum where the three cross
peaks represent three protonated nitrogens with the very weak
one at 10.5/121 ppm corresponding to the keto form. This confirms
the presence of a keto form, although it is difficult to say whether it
is a dominant form as broad signals can fail to show up clear
coupling.
3.5. Experimental and computed 1H and 13C NMR spectra
The NMR data of the compounds investigated are presented in
Table 5. All proton and carbon resonances were assigned by the
combined application of standard 1D and 2D NMR techniques
(1H-decoupled 13C NMR, 1HA13C multiplicity edited HSQC with
gradient selection[49–54], 1HA13C HMBC with gradient selection
using 3-fold low pass filter for better 1 J suppression [55] and
Phase sensitive NOESY [56,57]).
As chemical shifts of exchangeable protons are seldom diagnos-
tic, the NOESY experiment which correlates protons through dipo-
lar couplings was used instead. This was chosen to check whether
the origin of the signal around 10.5 ppm comes from either the
proton on pyrazole nitrogen or enolic oxygen. The cross-peak at
Furthermore, we investigated the correlation of predicted 13C
chemical shifts with those measured experimentally in order to
confirm the structure. These are spread over a wide spectral range,
and can therefore be used to identify putative structures by corre-
lating with predicted values. As proposed by Rychnovsky for revi-
sion of the structure of hexacyclinol [58], we utilized this approach
to identify whether the keto or hydroxy form is preferred. The
values of experimental and computed 13C chemical shifts are

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A.A. Kaczor et al. / Journal of Molecular Structure 1051 (2013) 188–196
Fig. 7. N-HSQC expanded spectrum of 1.
Fig. 8. The differences in 13C experimental and computed chemical shifts for the keto and hydroxyl forms of 1.
presented in Table 5. The average difference for a keto form is 4.94
whereas for a hydroxyl form it is 8.21. As the standard deviation for
this data set is 3.01, this difference is statistically significant. The
differences in experimental and computed chemical shifts for the
keto and hydroxyl form are shown in Fig. 8.
In order to find whether the equilibrium between the hydroxyl
and keto forms shifts as a function of temperature, 1H NMR of 1
was recorded from ꢂ50 to 50 °C in increments of 10 °C. The exper-
imental spectrum is shown in Fig. 9. Downfield peaks visible in
lower temperatures may be responsible for deshielded protons in-
volved in hydrogen bonds of the hydroxy or keto forms but in the
light of the IR results they may be interpreted as those of the hy-
droxyl form. These peaks are almost entirely absent in higher tem-
peratures indicating quick chemical exchange and breaking of
hydrogen bonds.
4. Discussion
The presented experimental and computational investigations
on the tautomerism of N-substituted 3-amino-5-oxo-4-phenyl-
1H-pyrazolo-1-carboxamides agree that the keto form of 1–10 is
the preferred one in the crystalline form and in DMSO solution
and that the fraction of the hydroxyl form is minor. The results
obtained constitute a solid contribution to the chemistry of
pyrazoles, in particular the tautomeric equilibrium of this class of
compounds.
X-ray structure determination of 5 made it possible to find that
the keto form is preferred in the crystalline state. The bond dis-
tances and angles in molecules A and B of 5 are in normal ranges
[59] and are comparable to the corresponding values observed in
closely related structures of N-ethoxycarbonylmethyl-3-amino-5-
oxo-4-phenyl-1H-pyrazole-1-carboxamide and N-butyl-3-amino-
5-oxo-4-phenyl-1H-pyrazole-1-carboxamide [31]. The computed
structure of 5 was a relatively good approximation but was differ-
ent in the flexible fragments of the molecule, as was also found for
similar systems [32,33].
The presence of a urea fragment and thus the existence of the
O7 keto form is evident as the H16 amide proton couples clearly
to
a nearby methine in the cyclohexane ring and the C6
chemical shift is in the range of carbonyl carbon in urea
derivatives.

A.A. Kaczor et al. / Journal of Molecular Structure 1051 (2013) 188–196
195
Fig. 9. 1H NMR spectrum of 1 recorded in the range of temperatures: from ꢂ50 °C to 50 °C in increments of 10 °C.
The Boltzmann distribution studies confirmed that the keto
5. Conclusions
form is much more stable than the hydroxy tautomer and made
it possible to predict that for compounds 5 and 8–10 some fraction
of the hydroxyl form occurs in DMSO solution.
A valuable insight into the tautomerism of 1–10 was made by
recording IR spectra which made it possible to detect a tiny
fraction of the hydroxyl form in the solid state and in the DMSO
solution. This confirms our earlier results [32] and results by other
authors obtained with IR [26] and Raman spectroscopy [20]
approaches, namely that vibrational spectra are a reliable tool to
study tautomeric equilibriums.
Further data on the tautomerism of 1–10 were supplied by N-
HSQC spectra which confirmed couplings between protons and
nitrogens. The tautomerism in similar systems was investigated
by Holzer et al. [25]. They found that apart from chemical shift
considerations and NOE effects the magnitude of the geminal
2 J[pyrazole C-4,H3(5)] spin coupling constant permits the
unambiguous differentiation between 1H-pyrazol-5-ol (OH) and
1,2-dihydro-3H-pyrazol-3-one (NH) forms. Such an experiment
was not possible in our case due to the lack of protons directly at-
tached to the pyrazole ring and thus an impossibility of measuring
relevant 2 J CAH couplings. The NMR approach was also used by
Abood and Al-Shlhai [28] who found that the keto form is the
preferred one for pyrazol-5-one and its derivatives, which is in
accordance with our data. Similar results were obtained by means
of computations with semiempirical methods by Enchev and
Neykov [17].
In this study we found that the keto form of N-substituted
3-amino-5-oxo-4-phenyl-1H-pyrazolo-1-carboxamides is the pre-
ferred form in the crystalline state and in DMSO solution, although
a tiny fraction of the hydroxyl tautomer occurs in both considered
states. Importantly, we related these findings to the antibacterial
activity of the investigated compounds and proposed that the
energetic stabilization of the keto form promotes antibacterial
activity, enabling proper hydrogen bond interactions with the bac-
terial enzyme. This finding is crucial for future QSAR and molecular
modeling studies.
Acknowledgments
The presented research was partially performed during the
postdoctoral stay of Agnieszka A. Kaczor at the University of
Eastern Finland, Kuopio, Finland, under a Marie Curie fellowship.
Calculations were performed under a computational grant from
the Interdisciplinary Center of Mathematical and Computational
Modeling (ICM) in Warsaw, Poland, Grant Number G30-18. The
paper was developed using equipment purchased under the pro-
ject ‘‘The equipment of innovative laboratories doing research on
new medicines used in the therapy of civilization and neoplastic
diseases’’ within the Operational Programme Development of East-
ern Poland 2007–2013, Priority Axis I Modern Economy, operations
I.3 Innovation promotion.
The results of the studies on the tautomerism of 1–10 were re-
lated to their antibacterial activity. It seems that the keto form is
the pharmacologically active one and its stabilization promotes
antibacterial activity. The importance of tautomerism for biological
activity is a common pattern for ligand–protein interactions [60–
63]. We have previously found that the stabilization of the keto
form is responsible for the anticancer activity of triazole deriva-
tives, acting probably through EGFR kinase [33]. Indeed, the results
presented in this study are of high importance for subsequent
structure–activity and molecular docking studies.
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