Interactions between indazole derivative and magnesium cations-NMR investigations and theoretical calculations

Article abstract of DOI:10.1016/j.molstruc.2013.05.011

The paper describes a new NMR approach for the determination of interaction sites between a heterocyclic ligand, like indazole, and magnesium ions. The method is based on the comparison of chemical shifts of indazole derivative before and after complexati

Full text of DOI:10.1016/j.molstruc.2013.05.011

Journal of Molecular Structure 1047 (2013) 292–301
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Interactions between indazole derivative and magnesium cations – NMR
investigations and theoretical calculations
b,c
a
a
a
a
Jacek Kujawski a, , Marek Doskocz , Hanna Popielarska , Anna Myka , Beata Drabinska , Joanna Kruk ,
⇑
´
Marek K. Bernard a
a
´
Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6 str., 60-780 Poznan, Poland
b Foundation for Development of Science and Business on Medical and Exact Sciences Area, Legnicka 65 str., 54-206 Wrocław, Poland
c RootInnovation sp. z oo, Piłsudskiego 95 str., 50-016 Wrocław, 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
ꢀ
ꢀ
ꢀ
A new pyrazolylindazole derivative of
potential anticancer activity was
synthesized.
Magnesium complexation by the
compound was studied by NMR and
DFT calculations.
A new general approach was
proposed for the determination of the
complexation site.
a r t i c l e i n f o
a b s t r a c t
Article history:
The paper describes a new NMR approach for the determination of interaction sites between a heterocy-
clic ligand, like indazole, and magnesium ions. The method is based on the comparison of chemical shifts
of indazole derivative before and after complexation with magnesium salt and is supported by DFT
(B3LYP/6-31G(d,p) level of theory, solvent model PCM, IGLO-II basis set) theoretical calculations.
Ó 2013 Elsevier B.V. All rights reserved.
Received 3 March 2013
Received in revised form 1 May 2013
Accepted 1 May 2013
Available online 16 May 2013
Keywords:
Indazole derivatives
Magnesium cation complexation
NMR spectroscopy
DFT calculations
1. Introduction
spectroscopy has found its use for the determination of interac-
tions of some ligands with proteins and allows to assess quantita-
Interactions between metal ions and organic molecules, partic-
ularly xenobiotics and macromolecules, are important for many
biological processes. Investigations on these interactions are
carried out by a variety of analytical methods, including: MS [1],
IR [1–3], RAMAN [3], scanning tunnelling spectroscopy (STS) [4],
and NMR spectroscopy. Worthy of mention is the fact that NMR
tively these interactions if the equilibrium constants and changes
in chemical shifts are taken into account [5]. It is also helpful in
studying ion complexation phenomena including the complexa-
tion of magnesium ions by small molecule ligands, e.g. EDTA [6].
In general, the NMR method is useful for study of such interactions
by recording of two important parameters: chemical shift (d) and
coupling constant (J). Although these parameters are not signifi-
cantly affected when the interactions metal ion–molecule are
weak, these interactions may result in a change of molecular
geometry (energy) that, in turn, can be measured by NMR.
⇑
Corresponding author. Tel.: +48 618546670; fax: +48 618546680.
E-mail address: jacekkuj@ump.edu.pl (J. Kujawski).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.05.011

J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
293
Moreover, the conformational alterations involve changes of the
dihedral angle H–C–C–H that influence the coupling constant.
However, the investigations and understanding of the relationship
between molecular structure and NMR parameters can sometimes
be quite difficult, and therefore are often supported by theoretical
calculations [7,8]. The information gathered from quantum-chem-
ical calculations, especially on solvation model and ion solvation
sphere, is very valuable and allows for a rapid visualization of
the results [9,10]. This especially relates to the processes proceed-
ing in solvents.
Although pyrazole and indazole derivatives rarely occur in nat-
ure, they are bioisosters of very important compounds: pyrrole, in-
dole, and purines. Pyrazole derivatives and their condensed
analogues have received considerable attention due to their broad
spectrum of activity including cytostatic and DNA intercalating ac-
tion [11,12], as well as antibacterial, antifungal, antiviral, antiin-
flammatory, antihypertensive, anticonvulsant and antidepressive
activity [13]. Moreover, these compounds demonstrate affinity
for serotonin HT receptors [14] and are useful in hormone replace-
ment therapy as well as in erectile dysfunction treatment [13].
Magnesium ions participate in more than 300 biochemical pro-
cesses in human organism, particularly these involved in prolifer-
ation, differentiation, angiogenesis and apoptosis [15].
rupole moment of 14N renders the nitrogen NMR spectroscopy
impractical [19,20]. Considering the low utility of 13C NMR spec-
troscopy for the investigations of interactions between metal ions
and small molecule organic compounds [5] as well as the low nat-
ural abundance of 13C, we decided to use 1H NMR for the studies
depicted in our paper. The NMR measurements were carried out
at three different temperatures, namely 25, 40 and 60 °C. The shifts
in the signal positions refer to the experiments involving sepa-
rately compound 7 and the same molecule in the presence of mag-
nesium hydrate (Figs. 1–3). The water signal (3.32 ppm) is omitted
from the diagrams due to relatively large drifting that equals to
ꢂ0.080 and ꢂ0.174 ppm at 40 and 60 °C, respectively, or to
0.355, 0.259, and 0.140 ppm at 25, 40 and 60 °C, respectively, after
the addition of magnesium salt (the values are given with refer-
ence to the water signal in DMSO). These significant shifts are
caused by an increase of the water concentration connected with
the use of magnesium hydrate and by a small pH change. The ten-
dencies in the signal shifts (Fig. 4) upon temperature change ap-
pear to be identical. Concurrently with the temperature increase,
the remaining signals are shifted upfield. The protons B (Scheme 1)
signal is shifted considerably (
the protons signal remains practically unchanged
= 0.001 ppm). These shifts confirm that there are significant
D = 0.011 ppm) while the position of
E
(D
The goal of this paper is to develop a fast and cheap protocol for
the estimation of interactions between metal ions and small to
medium size molecules. The approach should be characterized by
the following features:
interactions with the solvent in the neighbourhood of the tosyl
group. Moreover, the pyrazole proton F is also notably shifted
(D
= 0.015 ppm) that indicates a possibility of interaction between
the pyrazole ring and solvent. The proton signal A is shifted only
weakly ( = 0.003 ppm) probably due to steric reasons – this pro-
ton is shielded by the neighbouring tosyl and pyrazole moieties.
The indazole protons C and D are significantly shifted ( = 0.010
D
(i) short time of the experiment, preferably not longer than
1.5 h for two NMR samples;
D
(ii) introduction of a control step allowing for repeatability of
results under different conditions.
and 0.017 ppm, respectively) that suggests strong interactions
with the environment. The alterations of the methyl singlets are
largest and are connected with the high susceptibility of the
methyl group to interaction with the medium. The above conclu-
sions are correct assuming that the DMSO reference signal remains
unchanged due to the high concentration of the solvent (14 mol/
dm3). However, the analysis of the drifts in all proton signals of
the molecule shows that the reference line is not locked when tem-
perature increases. Due to the above problem we had to elaborate a
new method based on the analysis of relationship between the sig-
nals of the analyte. In order to identify the signals with the highest
sensibility to temperature, we focused on the interval changes be-
tween the signals. The signals that have similar tendencies in the
chemical shifts changes will have the same interval between the
signals. If one signal drifts downfield and the second stays at the
same position or moves less than the first one, then the distance
between signals will increase. Eleven signals in the NMR spectrum
recorded at 25 °C were analysed, namely for protons A, B, B1 (for
checking the method, two multiplets of the same proton were ana-
lysed), C, D, E, F, as well as for DMSO and three methyl groups (Me1,
Me2, Me3). The next step involved construction of an 11 ꢁ 11
matrix for the intervals between signals (MAT). The results of the
analysis of NMR spectra registered at 40 and 60 °C were elaborated
identically. Then, the matrices were subtracted for the visualiza-
tion of the interval changes in chemical shifts influenced by tem-
perature. As a result a MAT40–MAT20 matrix (Table 1) and a
MAT60–MAT40 matrix (Table 2) were obtained for temperatures
40 and 25 °C as well as 60 and 40 °C, respectively. As the analysis
of tabulated numerical values would be difficult, a matrix was pro-
posed in which colours were used instead of numbers (Figs. 5 and
6). Both diagrams are similar. Nearly full convergence in the posi-
tion of multiplets B and B1 proves that the assumption is correct. In
the diagram, the signals corresponding to protons D and E have a
deeper colour, characteristic for the central ring protons; this dem-
onstrates thermal sensibility of these protons. The signals E and
Me1 correspond to the C–C(CH3)–C part of the tosyl group. Upon
2. Results and discussion
2.1. NMR measurements
The procedure was tested using the pyrazole derivative ob-
tained by our group, namely 5-(3,5-dimethyl-1H-pyrazol-1-yl)-3-
[(4-methylphenyl)sulfonyl]-1H-indazole (compound 7, Scheme 1),
and the results were verified by theoretical calculations.
Compound 7 was obtained in a four-step synthesis (Scheme 2).
4-Nitrophenyl derivative of pyrazole 3 was subjected to the vicar-
ious nucleophilic substitution of hydrogen (VNS) [16,17] and the
nitro group was hydrogenated using hydrazine hydrate as a source
of hydrogen and palladium as a catalyst to give amine 6. Diazotiza-
tion of the latter compound proceeded with annulation and affor-
ded indazole derivative 7 [16,18].
In order to analyse the interactions between azole 7 and a metal
ion, compound 7 was dissolved in DMSO-d6 and separately in
DMSO-d6 with the admixture of Mg(NO3)2ꢁ6H2O. Albeit com-
pound 7 has four nitrogen atoms within its structure, the low
abundance of the 15N isotope in comparison with the 1H isotope
as well as the significant signals broadening due to the large quad-
N
B
HN
O
S
O
N
HN
E
O
S
O
C
D
Me1
Me
B
A
E
Me3
N
N
Me
N
N
Me2
F
Me
Scheme 1. Two possible conformers of 5-(3,5-dimethyl-1H-pyrazol-1-yl)-3-[(4-
methylphenyl)sulfonyl]-1H-indazole (7).

294
J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
Me
Me
Me
Me
N
N
N
N
Me
Me
Me
Me
N
3
N
N
N
[H]
iii
[HNO2]
iv
1
2
+
i
ii
Ts
N
NO2
NO2
NH2
N
Ts
Ts
H
6
7
4
5
Scheme 2. Synthesis of 5-(3,5-dimethyl-1H-pyrazol-1-yl)-3-[(4-methylphenyl)sulfonyl]-1H-indazole (7); 1 – p-nitrophenylhydrazine, 2 – 2,4-pentadione; i = EtOH, reflux,
30 min; ii = DMSO/KOH, rt, 1 h; iii = H2N–NH2ꢁH2O/10% Pd-C/EtOH, reflux, 8 h; iv = isopentyl nitrite/AcOH, 0–5 °C, 2 h.
Fig. 1. 1H NMR spectra of aromatic region of compound 7 with and without magnesium salt taken at 25, 40 and 60 °C.
Fig. 2. 1H NMR spectra of aliphatic region of compound 7 with and without magnesium salt taken at 25, 40 and 60 °C. Signal 2.50 ppm belongs to DMSO.
Fig. 3. 1H NMR spectra (pyrazole 4-H signal) of compound 7 with and without magnesium salt taken at 25, 40 and 60 °C.
the temperature rise, the tosyl group rotates faster leading to a
greater contribution of higher energetic conformers in the struc-
ture analysed by NMR. The above assumption allows us to elabo-
rate a very good method for assessment of binding sites in the

J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
295
ton C chemical shift results also in a small alteration of the signal
of proton A placed para to proton C, thus conjugated with the latter
one. The DMSO multiplet also changes its position after the addi-
tion of magnesium salt. Thus, DMSO solvates magnesium cation
similarly to water, forming a hexametric complex. Herein it must
be added that the use of analogously constructed matrices basing
on 15N NMR would result in serious errors. This substantiates once
more the assumption that the use of proton NMR spectroscopy is
correct for the studies described in the present paper.
2.2. Theoretical investigations
The theoretical calculations of the chemical shifts for indazole 7
were executed for all energy minima found during the alteration of
the N4–N3–C6–C5 and C7–S1–C8–C9 dihedral angles connected
with the rotation of the dimethylpyrazole moiety and tosyl frag-
ment, respectively; conformers IꢂVIII are shown in Fig. 8. The cal-
culated proton chemical shifts for the optimized conformers are
accurate and consistent with the experimental values apart from
two protons (Tables 3–5). The relative error for the NH group is al-
most 3 ppm due to the NH proton exchangeability and dynamic
dissociation equilibrium in the solvation sphere. A large error is
associated with the position of proton D (H2) – the relative error
is about 0.4 ppm for the structure at global minimum. A likely
explanation for this inaccuracy is the solvation effect that deter-
mines spatial arrangement of the dimethylpyrazole moiety. It
should be emphasized that tautomerism involving protonation of
the nitrogen atoms N1 and N3 can be neglected in the above case
and possible tautomeric variants of conformers IꢂVIII of com-
pound 7 are identical. The calculations according to the literature
method [21] has shown that the energies of different tautomers
of the same conformer are practically the same, e.g. for the con-
former VIII (Fig. 8), the energy of each tautomer is ꢂ1502.93 Har-
tree, i.e. ꢂ943102.1 kcal/mol. For conformers IꢂIV with the
hydrogen atom D in close proximity to the pyrazole nitrogen, the
errors are less than 0.09 ppm whereas for the conformers with this
hydrogen neighbouring the methyl group, the errors are about
0.4 ppm. The computations involving populations of individual
conformers are more accurate for the cases where conformation
is crucial for proton shielding while in other cases they are similar
to the results obtained for the predominating conformer in the
population, e.g. 17% for the conformer VI (Table 3). One limitation
concerning the changes in chemical shifts in the theoretical spec-
trum of the compound 7-magnesium ion complex is difficulty to
combine conformational analysis with the correct estimation of
both various interaction points and solvation sphere for magne-
sium ions. With this in mind, we carried out optimization of the
compound 7-magnesium ion complex using the following models
of conformers (Fig. 9): 7-Mg(OH)2ꢁ4H2O (IX), 7-Mg(OH)2ꢁ4H2O
(X, second isomer), 7-Mg(OH)+ꢁ5H2O (XI), 7-Mg2+ꢁ6H2O (XII).
Analysis of the chemical shifts for protons C, D, and A in systems
Fig. 4. Diagram of changes in signals chemical shifts (the left three-point lines
represent signals of compound 7 at 25, 40 and 60 °C; the right three-point lines
represent signals 7-Mg ions complex at 25, 40 and 60 °C.
analysed molecule. A comparison of the chemical shifts of the sig-
nals before and after the addition of magnesium salt under variable
thermal conditions can be analysed in a classical way, i.e. by tabu-
lation of the numerical values, or by graphical visualization. The
latter one is an easier and faster way to get the results (Figs. 5
and 6). In order to determine the magnesium salt impact on the
change in chemical shifts as a temperature function, the following
equations were used: (a) (MATG40–MAT40) ꢂ (MATG20–MAT20), (b)
(MATG60–MAT60) ꢂ (MATG40–MAT40). The results are gathered in a
graphical form (Figs. 5–7). Both graphics lead to a conclusion that
the chemical shift of proton C changes the most after the addition
of magnesium salt. It means that magnesium is chelated in the
close proximity to this proton (Scheme 3). The change in the pro-
Table 1
Differences in distances (in ppm) between signals in the 1H NMR spectra registered at 40 and 20 °C (MAT40–MAT20).
Signals
A
B
B1
0.0035
0.0000
–
0.0008
ꢂ0.0026
0.0062
ꢂ0.0017
0.0054
0.0126
0.0045
0.0094
C
D
E
F
DMSO
Me1
Me2
Me3
A
B
B1
C
D
–
0.0035
–
0.0000
0.0008
0.0027
0.0061
0.0026
0.0026
0.0034
–
0.0088
0.0009
0.0080
0.0152
0.0071
0.0120
ꢂ0.0027
ꢂ0.0062
ꢂ0.0062
ꢂ0.0054
ꢂ0.0088
–
ꢂ0.0079
ꢂ0.0008
0.0064
0.0052
0.0017
0.0017
0.0025
ꢂ0.0019
ꢂ0.0054
ꢂ0.0054
ꢂ0.0046
ꢂ0.0080
0.0008
ꢂ0.0071
–
ꢂ0.0091
ꢂ0.0126
ꢂ0.0126
ꢂ0.0118
ꢂ0.0152
ꢂ0.0064
ꢂ0.0143
ꢂ0.0072
–
ꢂ0.0010
ꢂ0.0045
ꢂ0.0045
ꢂ0.0037
ꢂ0.0071
0.0017
ꢂ0.0062
0.0009
0.0081
–
ꢂ0.0059
ꢂ0.0094
ꢂ0.0094
ꢂ0.0086
ꢂ0.0120
ꢂ0.0032
ꢂ0.0111
ꢂ0.0040
0.0032
ꢂ0.0035
ꢂ0.0035
ꢂ0.0027
ꢂ0.0061
0.0027
ꢂ0.0008
ꢂ0.0008
–
ꢂ0.0034
0.0054
ꢂ0.0025
0.0046
0.0118
0.0037
0.0086
ꢂ0.0026
0.0062
ꢂ0.0017
0.0054
0.0126
0.0045
0.0094
ꢂ0.0009
0.0079
–
E
F
ꢂ0.0052
DMOS
Me1
Me2
Me3
0.0019
0.0091
0.0010
0.0059
0.0071
0.0143
0.0062
0.0111
0.0072
ꢂ0.0009
0.0040
ꢂ0.0017
ꢂ0.0081
ꢂ0.0049
0.0032
ꢂ0.0032
0.0049
–

296
J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
Table 2
Differences in distances (in ppm) between signals in the 1H NMR spectra registered at 60 and 40 °C (MAT60–MAT40).
Signals
A
B
B1
0.0042
C
D
E
F
DMSO
Me1
Me2
0.0004
Me3
A
B
B1
C
D
–
0.0045
–
0.0003
0.0049
0.0004
0.0007
–
0.0083
0.0038
0.0041
0.0034
–
0.0092
0.0007
0.0091
0.0168
0.0079
0.0131
ꢂ0.0009
ꢂ0.0054
ꢂ0.0051
ꢂ0.0058
ꢂ0.0092
–
ꢂ0.0085
ꢂ0.0001
0.0076
0.0076
0.0031
0.0034
0.0027
ꢂ0.0008
ꢂ0.0053
ꢂ0.0050
ꢂ0.0057
ꢂ0.0091
0.0001
ꢂ0.0084
–
ꢂ0.0085
ꢂ0.0130
ꢂ0.0127
ꢂ0.0134
ꢂ0.0168
ꢂ0.0076
ꢂ0.0161
ꢂ0.0077
ꢂ
ꢂ0.0048
ꢂ0.0093
ꢂ0.0090
ꢂ0.0097
ꢂ0.0131
ꢂ0.0039
ꢂ0.0124
ꢂ0.0040
0.0037
ꢂ0.0045
ꢂ0.0042
ꢂ0.0049
ꢂ0.0083
0.0009
ꢂ0.0003
ꢂ0.0041
ꢂ0.0038
ꢂ0.0045
ꢂ0.0079
0.0013
ꢂ0.0072
0.0012
0.0089
–
–
ꢂ0.0004
ꢂ0.0038
0.0054
ꢂ0.0031
0.0053
0.0130
0.0041
0.0093
ꢂ0.0007
ꢂ0.0041
0.0051
ꢂ0.0034
0.0050
0.0127
0.0038
0.0090
ꢂ0.0034
0.0058
ꢂ0.0027
0.0057
0.0134
0.0045
0.0097
ꢂ0.0007
0.0085
–
E
F
ꢂ0.0076
DMOS
Me1
Me2
Me3
0.0008
0.0085
0.0084
0.0161
0.0072
0.0124
0.0077
ꢂ0.0012
0.0040
ꢂ0.0004
ꢂ0.0013
ꢂ0.0089
ꢂ0.0037
ꢂ0.0052
0.0048
0.0039
0.0052
–
(a)
(b)
Fig. 5. Visualization of data given in Tables 1 and 2: (a) red colour: d = 0.0152 ppm, white ꢂ0.0152 ppm; and (b) red: d = 0.0186 ppm, white ꢂ0.0186 ppm. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(a)
(b)
Fig. 6. Difference of chemical shifts changes between NMR signals recorded at different temperatures: (a) red: d = 0.0156 ppm, white ꢂ0.0156 ppm; and (b) red:
d = 0.0186 ppm, white ꢂ0.0186 ppm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
IX–XII before and after complexation (Table 6) shows the most sig-
nificant alteration for proton C. It should be added that the use of
computation method is tantamount with making assumptions
concerning the ligand structure. A hypothesis could be formulated

J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
297
(a)
(b)
Fig. 7. Comparison of range changes between NMR signals of compound 7 before and after addition of magnesium salt: (a) red d = 0.0049 ppm, white ꢂ0.0049 ppm; and (b)
red: d = 0.0044 ppm, white ꢂ0.0044 ppm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
that conformers in which magnesium ion is close to the central
indazole ring are the most appropriate. If the total charge corre-
sponds to Mg2+ ion, simple energetic arguments predict that the
solvated magnesium ion will prefer proximity of the dimethylpyr-
azole ring. Moreover, to check accuracy of the computation model,
the energy of the interaction N–Hꢃ ꢃ ꢃO–Mg for conformers IꢂVIII
(Fig. 8) was determined using the basis set superposition error
(BSSE) [22]. This energy amounted to ca 27 kcal/mol and is indica-
tive of hydrogen bonds formed with water molecules; it is located
within the energy limits for strong hydrogen bonds (15–40 kcal/
mol). This correlates well with the significant solvation of magne-
sium ions observed in our experiment. On the other hand, possibil-
ity of the direct contact of magnesium ions with the N1 nitrogen
atom was ruled out due to the scarce value of the calculated cou-
pling constant JN–Mg (0.07 Hz) which was below the experimental
error. However, prediction of the accurate solvation sphere, confor-
mation and site of the azole–ligand interaction is a complicated
process, and we intend to improve the procedure in future. Never-
theless, the depicted herein theoretical calculations have provided
an interesting visualization of compounds complex from the quan-
tum chemistry standpoint.
3. Experimental part
The reactions progress and purity of the products were moni-
tored by TLC using silica gel plates Kieselgel 60F254 (Merck).
Melting points were determined on a Boetius apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on a Var-
ian Gemini 300VT (300 and 75 MHz) or a Varian V NMR-S (600 and
150 MHz) spectrometer using TMS as internal standard. The spec-
tra were obtained using zg30 (30° pulse angle) pulse sequence. The
acquisitions were carried out on non-spinning samples at 298, 313
and 333 K. The spectra were recorded for a solution of compound 7
in DMSO-d6 as well as a mixture of compound 7 and Mg(NO3)2
ꢁ6H2O dissolved in the same solvent. The spectrum was refer-
enced to the residual DMSO signal. The number of scans was 64.
Polynomial base line correction was applied. Line broadening
was set up at 0.3. The assignment of the methyl group signals of
the compounds 5 and 7 was performed based on NOE experiments.
In order to discriminate protons, they were marked according to
Scheme 1 (values in ppm): positions: A 7.97, dd, J = 2 and 0.7 Hz;
B 7.93–7.89, m AA0XX0; C 7.78, dd, J = 8 (9) and 0.7 Hz; D 7.62,
dd, J = 9 and 2 Hz; E 7.44–7.40, m AA0XX0; F 6.12, s; Me1 2.35, s;
Me3 2.30, s; Me2 2.21, s; NH 14.13, bs. Low resolution MS spectra
were taken on an AMD 402 instrument. IR spectra were recorded
on a Specord 75-IR apparatus. Elemental analyses were performed
on a Vario EL III instrument.
Starting materials 3 and 4 were obtained according to known
methods [17,23].
H
H
H
Mg2+
H
O
H
N
N
O
3.1. Synthesis and spectroscopic data
O
S
H
3.1.1. 3,5-Dimethyl-1-[3-(4-methylphenyl)sulfonyl)methyl)-4-
nitrophenyl]-1H-pyrazole (5)
CH3
O
Finely powdered KOH (2.0 g) was added in one portion to a stir-
red solution of carbanion precursor 1 (1.02 g, 5 mmol) and nitroa-
rene 4 (1.08 g, 5 mmol) in dry DMSO (10 mL). The resulting dark
coloured mixture was vigorously stirred for 60 min at 22 °C,
quenched with 2% HCl (100 mL) and extracted twice with CH2Cl2.
The extract was washed with water, dried over MgSO4 and the sol-
vent was removed under vacuum. The residue was crystallized
from ethanol to give 1.04 g (54%) of compound 5 as white needles,
N
CH3
N
H3C
Scheme 3. Possible structure of Mg-compound
7
complex (7-Mg) from the
experimental data.

298
J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
Fig. 8. Visualization of the selected optimized conformers of compound 7. The structures I–VIII were optimized at the B3LYP/6-31G(d,p) level of theory with PCM solvent
model.
Table 3
List of geometric parameters for conformers I–VIII including dihedral angles C–S–C–C and N–N–C–C.
Dihedral angle (degree)
Conformer
I
II
V
VI
96.46
140.79
17.2
III
VII
IV
VIII
C–S–C–C
N–N–C–C
Distribution [%]
97.49
98.27
99.20
39.90
9.9
ꢂ99.21
ꢂ39.89
9.9
ꢂ97.48
141.01
15.3
ꢂ98.52
38.24
8.6
ꢂ97.48
140.99
15.3
ꢂ141.00
ꢂ38.25
15.3
8.6
m.p. 142–143 °C. 1H NMR (300 MHz, acetone-d6) d: 2.20 (s, 3H, 3-
CH3), 2.36 (s, 3H, 5-CH3), 2.42 (s, 3H, CH3, Ts), 5.17 (s, 2H,CH2), 6.11
(s, 1H, 4-H), 7.38–7.43 (m, 2H, 300-H i 500-H), 7.59–7.63 (m, 3H, 20-H,
200-H i 600-H), 7.84 (dd, J = 9 Hz and 2 Hz, 1H, 60-H), 8.18 (d, J = 9 Hz,
1H, 50-H). 13C NMR (75 MHz, acetone-d6) d: 13.52; 21.50; 30.32;
58.73; 110.00; 124.15; 125.83; 127.54; 128.48; 129.20; 130.73;
136.51; 141.08; 144.34; 146.10; 147.65; 151.03. MS m/z (%): 385
(67), 339 (9), 230 (100), 185 (22), 139 (18), 91 (42). Anal. Calcd.
for C19H19N3O4S (385.4): C 59.21; H 4.97; N 10.90. Found: C
59.01; H 5.05; N 11.02.

J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
299
Table 4
Theoretical and experimental values of chemical shifts for conformers I–VIII.
Locant
Conformer
I
II
2.31
III
2.37
IV
2.32
V
VI
2.35
VII
2.31
VIII
2.35
Average
Experimental
Me1
Me2
Me3
A
B
C
D
E
F
NH
2.35
2.22
2.42
7.99
8.19
8.04
8.02
7.60
6.25
2.37
2.34
2.24
2.38
8.12
8.18
8.04
7.90
7.59
6.24
11.30
2.35
2.21
2.3
7.97
7.91
7.78
7.62
7.42
6.12
2.26
2.29
8.36
8.15
8.03
7.71
7.56
6.22
11.36
2.26
2.33
8.32
8.21
8.04
7.66
7.64
6.24
11.35
2.21
2.46
7.97
8.15
8.05
8.02
7.55
6.23
11.26
2.26
2.33
8.32
8.21
8.04
7.66
7.64
6.24
11.35
2.22
2.42
7.99
8.19
8.04
8.02
7.60
6.25
11.27
2.26
2.29
8.36
8.15
8.04
7.71
7.55
6.22
11.36
2.22
2.42
7.99
8.19
8.04
8.02
7.60
6.25
11.27
11.27
14.3
Table 5
Relative errors in theoretical chemical shifts for conformers I–VIII.
I
II
III
0.017
IV
V
VI
0.001
VII
VIII
Average
0.001
0.014
0.116
0.021
0.284
0.262
0.400
0.185
0.132
ꢂ0.036
0.053
ꢂ0.007
0.393
0.238
0.254
0.087
0.136
0.100
ꢂ2.945
ꢂ0.029
0.002
0.158
ꢂ0.001
0.238
0.267
0.404
0.131
0.109
ꢂ3.038
0.017
ꢂ0.037
0.052
ꢂ0.008
0.387
0.236
0.255
0.086
0.130
0.100
ꢂ2.940
0.001
ꢂ0.007
0.026
0.084
0.146
0.272
0.261
0.276
0.173
0.120
ꢂ2.999
0.050
0.026
0.352
0.304
0.257
0.039
0.218
0.122
ꢂ2.951
0.050
0.026
0.352
0.304
0.257
0.039
0.218
0.122
ꢂ2.951
0.014
0.117
0.022
0.284
0.262
0.401
0.185
0.132
ꢂ3.027
0.014
0.116
0.021
0.284
0.262
0.400
0.185
0.132
ꢂ3.027
ꢂ3.027
Fig. 9. Binding modes of Mg2+ cations to compound 7. The structures IX–XII were optimized at the B3LYP/6-31G(d,p) level of theory with PCM solvent model.
3.1.2. 1-[4-Amino-3-(p-toluenesulfonylmethyl)phenyl]-3,5-
dimethylpyrazole (6)
anol (120 mL). The resulting mixture was refluxed for 8 h, cooled,
and filtered through Celite. The solvent was distilled off and the
Hydrazine hydrate (4.63 g, 8.2 mmol) and Pd/C (0.21 g) were
added to a warm solution of nitroarene 5 (2.70 g, 7 mmol) in eth-
residue was crystallized from ethanol to give compound
6
(1.74 g, 72%) as white plates, m.p. 72–74 °C. 1H NMR (300 MHz,

300
J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
Table 6
Calculated and experimental data for 1H NMR spectra of indazole 7-magnesium ions complex; the following parameters were determined for the proton groups of the complex 7-
Mg: experimental (Exp.) and calculated values of the chemical shifts (conformers IX–XII), percentage relative errors (E1–E4), mean values if the absolute and relative errors (
D d
and E, respectively); calculated Href = 31.54833 ppm.
Locants
Exp.
IX
X
XI
XII
E1
E2
E3
E4
D
d
E
B/B1
A
D
C
E
7.88
7.93
7.78
7.61
7.41
6.1
2.32
2.26
2.18
8.18
8.176
8.25
8.296
8.084
8.034
7.875
7.657
6.215
2.416
2.366
2.226
8.194
8.251
7.804
7.845
7.584
6.241
2.39
4
4
0
24
2
2
1
3
3
4
4
2
3
2
2
6
4
2
5
2
3
3
3
2
4
5
2
4
7
0
3
2
2
3
2
6
8.21
8.27
7.87
8.31
7.59
6.23
2.4
4
4
1
8
2
2
3
3
3
8.232
7.766
9.687
7.554
6.223
2.345
2.33
7.905
7.834
7.562
6.228
2.453
2.359
2.226
F
Me1
Me3
Me2
2.3
2.31
2.34
2.25
2.23
acetone-d6) d: 2.00 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.42 (s, 3H, CH3,
Ts), 4.58 (s, 2H, CH2), 5.03 (bs, 2H, NH2, exchangeable with D2O),
5.93 (s, 1H, 4-H), 6.84 (d, J = 2 Hz, 1H, 20-H), 6.91 (d, J = 8 Hz, 1H,
50-H), 7.15 (dd, J = 9 and 2 Hz; 1H, 60-H), 7.17–7.41 (m, 2H, 300-H i
500-H, Ts), 7.72–7.45 (m, 2H, 200-H i 600-H, Ts). MS m/z (%): 355
(20), 290 (0,9), 275 (0,5), 200(100), 117 (2), 91 (4). Anal. Calcd
for C19H21N3O2S (355.4): C 64.20; H 5.95; N 11.82. Found: C
63.00; H 5.07; N 12.27.
pound 7 were optimized using the density functional theory (DFT)
[25] with the B3LYP [26] functional and standard basis 6-31G(d,p)
[27]. The solvent effect on the geometry of 7 and complex 7ꢂMg
was determined using a polarized continuum (PCM) model of
water [28]. Vibrational frequencies and thermodynamic properties
were calculated applying the ideal gas, rigid rotor, and harmonic
oscillator approximations [29]. Energy minimum was confirmed
by the frequency calculation for all conformers. The initial struc-
tures of magnesium complex 7ꢂMg were designed by a change
in the CA–C–N–N torsion angle and rotation of the tosyl group.
The NMR parameters, were calculated using the coupled perturbed
density functional theory (CP–DFT) [30] method with B3LYP func-
tional. This functional has been found to be in good agreement
with experimental data [31]. The calculations were performed
using the NMR dedicated IGLOII [32] basis for H, C, N, O, S atoms
and standard base cc-pVTZ [33] for magnesium atom. The same
solvent models as employed for the geometry optimization were
used in the calculation of scalar couplings [34]. The proton chem-
ical shifts were referenced to the central signal of residual DMSO
(2.50 ppm). The DMSO geometry was determined in a similar man-
ner by theoretical methods. The proton shift for B3LYP/6-
31G(d,p)//B3LYP/IGLO-II method equalled 31.54833 ppm. The
NMR theoretical calculations were done including populations of
individual conformers at the local minimum according to the nor-
malized Boltzmann distribution equation for compound 7 as well
as for the selected conformers of the compound 7-magnesium
ion complex (7ꢂMg).
3.1.3. 5-(3,5-dimethyl-1H-pyrazol-1-yl)-3-[(4-
methylphenyl)sulfonyl]-1H-indazole (7)
A solution of amine 6 (0.11 g, 0.3 mmol) in anhydrous acetic
acid (19 mL) was cooled to 0 °C and isopentyl nitrite (0.04 g,
0.33 mmol) was added dropwise at 0–5 °C. The reaction mixture
was stirred at this temperature for 2 h, then it was neutralized with
25% ammonia and extracted with CH2Cl2 (5 ꢁ 50 mL). The organic
extracts were combined, washed with water, and dried over
MgSO4. The solvent was distilled off and the residue was crystal-
lized from ethanol to afford compound 7 (0.09 g, 80%) as white
plates, m.p. 189–192 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.21
(s, 3H, 3-CH3, pyrazole), 2.30 (s, 3H, 5-CH3, pyrazole), 2.35 (s, 3H,
CH3, Ts), 6.12 (s, 1H, 4-H), 7.40–7.44 (m, 2H, 300-H i 500-H, Ts),
7.62 (dd, J = 9 and 2 Hz, 1H, 60-H), 7.78 (dd, J = 8 i 0.7 Hz, 1H, 70-
H), 7.89–7.93 (m, 2H, 200-H i 600-H, Ts), 7.97 (dd, J = 2 and 0.7 Hz,
1H, 40-H), 14.13 (bs, 1H, NH, exchangeable with D2O). MS m/z
(%): 366 (100), 324 (6), 227 (5), 211 (29), 143 (8), 91 (17). 13C
NMR (150 MHz, DMSO-d6) d: 12.0 (CH3-50), 13.2 (CH3-30), 20.9
(CH3-400), 107.1 (C-40), 112.3 (C-7), 114.0 (C-4), 119.6 (C-8), 124.8
(C-6), 127.1 (C-200, C-600), 130.1 (C-300, C-500) 135.3 (C-9), 138.0
(C-400), 139.4 (C-50), 139.7 (C-5), 143.6 (C-3), 144.5 (C-100) 148.0
(C-30). The signals were assigned using the HSQC and HMBC spec-
tra. Anal. Calcd for C19H18N4O2S (366.4): C 62.28; H 4.95; N 15.29.
Found: C 62.99; H 4.67; N 15.00.
4. Conclusions
The above discussed method is fast and cheap ꢂ in our experi-
ment we registered only six 1H NMR spectra ꢂ and enables to fol-
low changes in chemical shifts with no need for time-consuming
use of reference compounds and different solvents. We have
shown that reliable results can be obtained from the comparative
analysis of the changes in chemical shifts in complexes at different
temperatures.
3.2. Procedure for the preparation of sample solutions for the NMR
analyses
Solution1: (Sol1): 0.30 mg of compound 7 in 0.2 mL of DMSO.
Solution2: (Sol2): 0.041 g of Mg(NO3)2ꢁ6H2Oin 0.4 mL of
DMSO-d6 (concentration: 0.4 mol/dm3).
Sample1: (the reference sample): 0.2 mL of Sol1 in 0.4 mL of
DMSO-d6.
A potential application of the method has been checked on a
model NMR analysis at 25, 40 and 60 °C for indazole derivative 7
and its complex with magnesium ion. Basing on the analysis of
changes in chemical shifts and matrix operations we have identi-
fied the sites susceptible to interactions with magnesium ions.
The interactions have also been investigated by DFT calculations
(B3LYP/6-31G(d,p)//B3LYP/IGLO-II) performed for several con-
formers of compound 7 before and after complexation. Comparison
of the proton chemical shifts for the system IX–XII before and after
complexation shows the greatest change of the proton C signal that
is in agreement with the experimental data.
Sample2: 0.2 mL of Sol1 + 0.4 mL of Sol2.
The 1H NMR spectra were registered for Sample1 and Sample2
at 25, 40 and 60 °C.
3.3. Theoretical calculations
The above approach can be used for the analysis of interactions
ligand–protein, between a couple of organic compounds, biofluids
with metal salts and biofluids with metabolites. Thus, it can be
The theoretical calculations were executed using Gaussian G09
suite code [24]. The structures of the different conformers of com-

J. Kujawski et al. / Journal of Molecular Structure 1047 (2013) 292–301
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Acknowledgements
The experimental studies were supported by the Polish
National Science Centre Grant No. N N401 642340, as well as the
Poznan WUP scholarship (2011-2012, PO IG 8.2.2; Mr. Jacek
Kujawski). The computations were supported by PCSS (Poznan
Supercomputing Center) Grant No. 92/2011.
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2013.
05.011.
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