Synthesis, three-dimensional structure, conformation and correct chemical shift assignment determination of pharmaceutical molecules by NMR and molecular modeling

Article abstract of DOI:10.21577/0103-5053.20160248

This work includes the synthesis of phenanthrenequinone guanylhydrazone and phenanthro[9,10-e][1,2,4]triazin-3-amine to be tested as intercalate with DNA for treatment of cancer. The other synthesized product, 2-[(4-chlorophenylamino)methylene]malononitrile, was designed for future determination of its activity against leishmaniasis. A common problem about some articles on the literature is that some previously published compounds display error of their molecular structures. In this article it is shown the application of several procedures of nuclear magnetic resonance (NMR) to determine the complete molecular structure and the non questionable chemical shift assignment of the synthesized compounds, and also their analysis by molecular modeling to confirm the NMR results. To determine the capacity of pharmacological compounds to interact with biological targets is determined by docking. This work is to motivate the application of NMR and molecular modeling on organic synthesis, being a process that is very important for the study of the prepared compounds as interactions with biological targets by NMR.

Full text of DOI:10.21577/0103-5053.20160248

http://dx.doi.org/10.21577/0103-5053.20160248
J. Braz. Chem. Soc., Vol. 28, No. 6, 975-984, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Article
Synthesis, Three-Dimensional Structure, Conformation and Correct Chemical
Shift Assignment Determination of Pharmaceutical Molecules by NMR and
Molecular Modeling
Sirlene O. F. de Azeredo, Edijane M. Sales and José D. Figueroa-Villar*
Grupo de Ressonância Magnética Nuclear e Química Medicinal, Departamento de Química,
Instituto Militar de Engenharia, Praça General Tibúrcio 80, 22290-270 Rio de Janeiro-RJ, Brazil
This work includes the synthesis of phenanthrenequinone guanylhydrazone and
phenanthro[9,10-e][1,2,4]triazin-3-amine to be tested as intercalate with DNA for treatment
of cancer. The other synthesized product, 2-[(4-chlorophenylamino)methylene]malononitrile,
was designed for future determination of its activity against leishmaniasis. A common problem
about some articles on the literature is that some previously published compounds display error
of their molecular structures. In this article it is shown the application of several procedures of
nuclear magnetic resonance (NMR) to determine the complete molecular structure and the non
questionable chemical shift assignment of the synthesized compounds, and also their analysis by
molecular modeling to confirm the NMR results. To determine the capacity of pharmacological
compounds to interact with biological targets is determined by docking. This work is to motivate
the application of NMR and molecular modeling on organic synthesis, being a process that is
very important for the study of the prepared compounds as interactions with biological targets
by NMR.
Keywords: synthesis, phenanthrenequinone derivatives, NMR, molecular modeling, chemical
shift determination, definitive molecular structure
Introduction
available for their complete ligand-target complex structure
determination, but in this case the NMR can be used to
determine the interaction of the ligand with the target,
especially discovering which areas of the ligand are the
One of the most important information from compounds
with biological activity is the determination of their
complete three-dimensional and conformational structure,
and also their definitive chemical shift assignment. This
information is necessary to determine the interaction
process of these pharmacological compounds with the
biological targets, as enzymes, nucleic acids (DNA or
RNA) and membrane receptors by nuclear magnetic
resonance (NMR).
The molecular structure determination of
pharmacological agents with biological targets is usually
executed by X-ray diffraction, but sometimes, when
crystallization of the complex is not possible, this method
is not available. On the other hand, when the ligand-target
complex possess mass lower than 70 kD, it is possible
to determine their complete structure and its dynamic
condition by NMR. However, if their mass targets are
more than 100 kD, the NMR method is generally not
4
,5
most effective for interacting sessions with the target,
being this the most effective method to study the interaction
of pharmacological ligands with any type of targets. In
order to determine by NMR which is the target site of
interaction with the ligand it is necessary to execute the
ligand competition with the previously described inhibitors,
which is a process that confirms its interaction with the
specific regions of the target, especially with the active
6-9
sites of the enzymes.
1
The normal NMR interaction analysis of
pharmacological agents with biological targets are usually
executed using water as solvent, and for this type of analysis
the NMR method requires the complete information of
the conformational structure and the definitive chemical
shift of these pharmacological agent, being a process that
allows the determination of quality and interaction with this
2,3
1
agent. These NMR methods are based on the H chemical
*e-mail: jdfv2009@gmail.com
shift, relaxation times (T and T ), diffusion gradients and
1
2

9
76
Synthesis, Three-Dimensional Structure, Conformation and Correct Chemical Shift Assignment Determination
J. Braz. Chem. Soc.
7
-9
nuclear Overhauser effects (NOE). For example, one of
the most NMR used methods for this type of interaction
Synthesis and spectra of phenanthrenequinone
guanylhydrazone (3)
10,11
studies is the saturation transfer difference (STD).
In a 100 mL round bottom flask were added a mixture of
phenanthrenequinone (0.208 g, 1.0 mmol), aminoguanidine
hydrochloride (0.132 g, 1.2 mmol), 20 mL ethanol 95% and
Several compounds have been described in the
literature with impropriate chemical shifts and wrong
structures, sometimes leading to their incorrect interaction
with biological targets. Therefore, the complete structure
determination and chemical shift assignment of new potential
pharmacological agents is very important for the study for
their biological activity and interaction with targets.
In this article are described the synthesis, the NMR
methods used to determine the complete and definitive
three-dimensional, conformational and chemical shift
assignment of pharmacological agents, and the confirmation
by molecular modeling. The docking study is also important
to determine if these compounds can interact with some
biological targets, especially with some specific enzymes.
This process is very important for the study of interaction
from these agents with biological targets.
-1
a few drops of HCl 6 mol L . The reaction was refluxed for
4 h. The solid product obtained after the solvent elimination
by vacuum was washed with acetone and filtered to afford
the pure solid. Orange solid; 93% yield; m.p.: 245-246 °C;
-1
IR (KBr) nmax / cm 3033, 1681, 1634, 1568, 1509, 1447,
1
1278, 1167, 1020, 757; H NMR (600 MHz, DMSO-d )
6
d 14.28 (s, 1H, NH, H12), 8.89 (br s, 2H, NH , H14), 8.64
2
(br s, 2H, NH , H15), 8.48 (d, 1H, J 8.0 Hz, CH, H8),
2
8.27 (d, 1H, J 7.5 Hz, CH, H4), 8.18 (d, 1H, J 7.5 Hz,
CH, H5), 8.13 (d, 1H, J 8.0 Hz, CH, H1), 7.88 (t, 1H,
J 7.5 Hz, CH, H3), 7.61 (t, 1H, J 7.5 Hz, CH, H2), 7.60
(t, 1H, J 8.0 Hz, CH, H6), 7.50 (t, 1H, J 8.0 Hz, CH, H7);
13
C NMR (150 MHz, DMSO-d ) d 181.9 (C, C10), 156.4
6
(C, C13), 139.3 (C, C4a), 136.3 (CH, C3), 134.9 (C, C9),
30.8 (CH, C6), 129.8 (C, C8a), 129.5 (C, C10a), 129.4 (C,
C4b), 129.4 (CH, C7), 128.9 (CH, C2), 128.6 (CH, C1),
26.0 (CH, C8), 124.1 (CH, C4), 124.0 (CH, C6).
1
Experimental
1
General experimental procedures
Synthesis and spectra of phenanthro[9,10-e][1,2,4]triazin-
3-amine (4)
In a 100 mL round bottom flask were added a mixture of
phenanthrenequinone guanylhydrazone (0.301 g, 1 mmol),
Solvents and reagents were obtained from commercial
companies (Sigma-Aldrich, Synth and Merck). Melting
points were determined on a Fisher-Johns apparatus. The
infrared spectra (IR) were measured using a Shimadzu
25 mL of distilled water and 15 drops of NH OH. The
4
2
1 spectrometer, with samples prepared in tablets of
reaction was refluxed for 2 h. The solid obtained was filtered
and washed with distilled water to afford pure solid product.
Yellow solid; 95% yield; m.p.: 260-262 °C; IR (KBr)
anhydrous potassium bromide (KBr). The thin layer
chromatography analyses were conducted using Merck
silica gel 60 F254 aluminum sheets. The NMR spectra
were obtained using a Varian 600 NMR spectrometer,
-1
nmax / cm 3469, 3282, 3120, 3072, 1633, 1521, 1444, 1384,
1
1026, 765, 723; H NMR (600 MHz, DMSO-d ) d 9.07 (dd,
6
with 5 mm tubes and using the DMSO-d as solvent at
1H, J 7.1, 1.9 Hz, CH), 8.97 (d, 1H, J 7.8 Hz, CH), 8.73 (d,
1H, J 7.8 Hz, CH), 8.69 (dd, 1H, J 7.2, 1.5 Hz, CH), 7.89
(t, 1H, J 7.8 Hz, CH), 7.75 (t, 1H, J 7.8 Hz, CH), 7.74 (t,
1H, J 7.2 Hz, CH), 7.73 (t, 1H, J 7.1 Hz, CH), 7.59 (s, 2H,
6
2
5 °C. The solutions of these samples on the NMR tubes
were used with 20 mg at 600 mL. The NMR spectra were
1
13
H, C, attached proton test (APT), heteronuclear single
13
quantum coherence (gHSQC), heteronuclear multiple
bond coherence (gHMBC), correlation spectroscopy
NH ); C NMR (150 MHz, DMSO-d ) d 162.2 (C), 143.1
2 6
(C), 138.4 (C), 133.3 (C), 131.9 (CH), 128.4 (CH), 128.4
(CH), 128.2 (C), 127.9 (C), 127.7 (CH), 127.5 (C), 125.3
(CH), 123.5 (CH), 123.3 (CH), 120.0 (CH).
(
COSY), total correlation spectroscopy (TOCSY) and gated
1
3
decoupling C. The NMR frequency was 600 MHz for
1
13
the H and 150 MHz for the C. The molecular modeling
calculations were executed with the Spartan’10 and using
density functional theory (DFT) with the B3LYP and the
M06 methods by 6-311G* basis set. These methods were
used to determine the natural atomic charges (QNPA) for
Synthesis and spectra of 2-[(4-chlorophenylamino)
methylene]malononitrile (5)
12
In a 100 mL round-bottomed flask were added 0.500 g
(3.94 mmol) 4-chloroaniline, followed by addition of
ethoxymethylenemalononitrile 0.250 g (2.04 mmol)
dissolved in 30 mL of ethanol 95% and was heated under
reflux for 3 h. The precipitated product was filtered and
washed with distilled water.Yellow solid; 76% yield; m.p.
-1
2
each atom, and the values of energy (kJ mol ), PSA (Å ),
element volume (Å ) and the dipole moment (Debye), and
2
the calculated chemical shift of hydrogens and carbons
to confirm with the NMR results. The docking study was
performed with AutoDock Vina.
-1
252-254 °C; IR (KBr) nmax / cm 3449, 3294, 3225, 3024,

Vol. 28, No. 6, 2017
Azeredo et al.
977
2
368, 2214, 1659, 1589, 1489, 1335, 1180, 1095, 995, 818,
long-range heteronuclear coupling constants from spin-
state selective multiplets. This method is very efficient,
1
16
740, 548; H NMR (600 MHz, DMSO-d ) d 11.18 (s,1H,
6
NH), 8.51 (s, 1H, CH), 7.45 (d, 2H, J 9.1 Hz, 2 CH), 7.44
but sometimes requires several conditions for analysis.
Other method, which was developed by our research
group is the selective heteronuclear simultaneous short
13
(
d, 2H, J 9.1 Hz, 2 CH); C NMR (150 MHz, DMSO-d )
6
d 155.9 (CH), 138.2 (C), 129.3 (CH), 129.2 (C), 119.8
CH), 116.3 (C), 114.0 (C), 52.4 (C).
17
(
and long-range correlations (SHESSLOC), which is also
appropriate. However, one of the simplest methods is the
13
Results and Discussion
gated decoupling of C NMR, which can also be applied
15
19
to other atoms, as N and F, being an easy application
and affording very good results for the small molecules.
However, this method is usually not used by all specialists
on NMR. In general terms, the gated decoupling of
The assignments of new potential pharmacological
agents is normally executed with different one
1
13
19
15
dimensional NMR spectra (1D), as H, C, F and N, and
with some dimensional (2D) spectra, as COSY, TOCSY,
HSQC and HMBC. In general, the 1D spectra afford the
ppm chemical shift values of all atoms, but to confirm the
correct assignment of all atoms is fundamental the use of
the 2D spectra. The COSY affords the coupling between
all hydrogen atoms, but sometimes this method affords
13
C NMR is very important to determine the chemical shift
of all quaternary carbons, a process that have been used to
confirm the structure and chemical shift of pharmacological
18
agents.
In order to determine the three dimensional structure of
molecules, despite the complete chemical shift assignment,
it is also necessary the application of NOE. The NOE
methods allow the calculation of dipolar coupling, a process
that determines which hydrogens are close to other ones
and the correlation distance without the participation of
bonds. This methodology is very efficient to determine
the diverse position of several hydrogens, allowing the
confirmation of the three dimensional structure of these
compounds. Another important procedure to confirm the
three-dimensional structure of molecules is the application
of molecular modeling. These NMR methodologies are
described and applied for the non questionable assignment
of molecules in this article.
1
difficult results, especially when some H signals are with
13
superposition or with very similar chemical shift. For this
reason it is important the use of the TOCSY method, which
indicates the hydrogen groups that are part of different
14
coupling systems.
The HSQC affords one single bond correlation between
1
13
19
15
H with C, F, N and other atoms. This methodology
confirms which hydrogens are directly bounded to the
other atoms, being this information important to partial
determination of the chemical shifts. The other important
spectrum is the HMBC, which displays the correlation
1
of H with the other atoms with two, three and four
bond correlations, being very important to determine the
chemical shift and position of all atoms, especially the
quaternary carbons. This method is very important for the
obtention of the complete chemical shift of all atoms from
different compounds. Therefore, these 2D spectra are very
The phenanthro[9,10-e][1,2,4]triazin-3-amine (4)
was prepared with two reactions. Initially was selected
phenanthrenequinone (1) to react with aminoguanidine
hydrochloride (2), to form the phenanthrenequinone
guanylhydrazone (3), which was selected as an agent for
decreasing the cancer radiotherapy and for treatment of
Alzheimer’s disease. Finally, the product 3 was transformed
to phenanthro[9,10-e][1,2,4]triazin-3-amine (4) by
cyclization, as shown in Figure 1, which was also selected
as intercalate to DNA.
15
important to determine the molecular position of all atoms.
Interestingly, sometimes the application of these
methods does not allow the complete assignment of the
analyzed agents, being necessary the application of other
methods to determine the complete coupling constants,
a process that usually confirms the complete and non
questionable chemical shift assignment. There are some
methods than can be applied for this type of information,
especially IPAP-HSQMBC, which is measurement for
The compound 3 was previously tested as intercalate
of DNA and with condition to decrease 50% of the cancer
radiotherapy. For this reason compound 4 was designed as
DNA intercalate with the possibility to decrease the dose
Figure 1. Synthesis of phenanthro[9,10-e][1,2,4]triazin-3-amine (4).

9
78
Synthesis, Three-Dimensional Structure, Conformation and Correct Chemical Shift Assignment Determination
J. Braz. Chem. Soc.
Figure 2. Mechanism of synthesis of phenanthro[9,10-e][1,2,4]triazin-3-amine (4).
1
Table 1. H, COSY and TOCSY of phenanthro[9,10-e][1,2,4]triazin-3-amine (4)
a
dH
Integration
1H
Mult.
JHH / Hz
7.1, 1.9
7.8
COSY
TOCSY
9
8
8
8
7
7
7
7
.07 (H12)
dd
d
d
dd
t
7.73, 7.74
7.75, 7.89
8.69, 7.89
8.73, 7.74
8.73, 7.75
8.97, 7.89
8.69, 7.73
9.07, 7.74
-
9.07-8.69-7.74-7.75
8.97-8.73-7.89-7.75
8.73-8.97-7.89-7.75
8.69-9.07-7.74-7.75
7.89-8.97-8.73-7.75
7.75-8.97-8.73-7.89
7.74-9.07-8.69-7.73
7.73-9.07-8.69-7.74
-
.97 (H5)
.73 (H8)
.69 (H9)
.89 (H13)
.75 (H6)
.74 (H10)
.73 (H11)
.59 (H13)
1H
1H
7.8
1H
7.2, 1.5
7.8
1H
1H
t
7.8
1H
t
7.2
1H
t
7.1
7
2H
s
-
a
The COSY signals with bold are the most effectives. COSY: correlation spectroscopy; TOCSY: total correlation spectroscopy.
1
3
of cancer radiotherapy. Therefore, the complete chemical
shift assignment of this compound is fundamental to study
its interaction with DNA.
The mechanism of cyclization of phenathrenequinone
guanylhydrazone (3) to prepare phenanthro[9,10-e][1,2,4]
triazin-3-amine (4) is shown in Figure 2.
Table 2. C, HSQC and HMBC of phenanthro[9,10-e][1,2,4]triazin-
-amine (4)
3
a
dC
gHSQC
-
gHMBC
1
1
1
62.2 (C3)
-
43.1 (C4a)
-
8.97, 8.73
9.07
38.4 (C12b)
-
The initial NMR methods to obtain information for the
chemical shift assignment of phenanthro[9,10-e][1,2,4]
133.3 (C8a)
131.9 (C7)
-
8.97, 8.73, 8.69, 7.89, 7.75
8.97, 8.73, 7.75
9.07, 8.69
1
triazin-3-amine (4) were H, COSY and TOCSY, which
7.89
7.74
7.73
-
13
results are shown in Table 1. The C, gHSQC and gHMBC
results are shown in Table 2.
1
1
1
1
1
1
1
1
28.4 (C10)
28.4 (C11)
28.2 (C8b)
27.9 (C12a)
27.7 (C6)
27.5 (C4b)
25.3 (C5)
23.5 (C8)
9.07, 8.69
1
The H NMR spectrum affords the chemical shift
9.07, 7.74, 8.73, 8.69
9.07, 8.69, 7.73
8.97, 7.89, 8.73
8.97, 8.73, 7.75
7.89, 8.73, 7.75
7.89, 7.75
1
and multiplicity of all H signals and the integration
-
procedure allows the determination of the number of
hydrogen atoms on each signal. These results, as shown
on columns 1-4 of Table 1, indicate that the hydrogen
signals of 8.69 to 9.07 ppm are hydrogens basically with
7.75
-
8.97
8.73
9.69
9.07
only one other hydrogen at 3 bond distance, a process that
3
allows J constants from 7.1 to 7.8 Hz. Interestingly,
123.3 (C9)
7.74, 7.73
HH
only two of these hydrogens also display coupling with
122.0 (C12)
7.74, 7.73
4
other hydrogens at 4 bond distances with JHH of 1.5 and
a
The signals with bold are the strong correlations, in normal font are
average and in italic are the lowest ones. HSQC: heteronuclear single
quantum coherence; HMBC: heteronuclear multiple bond coherence.
1
.9 Hz. This result indicates that the condition of the two
benzyl rings containing hydrogens (A and B) are different,
especially because the ligation condition of the aromatic
ring containing the four nitrogen atoms with these two
benzyl rings are different.
The COSY spectrum affords certain difficulty to
determine the coupling of some hydrogen which display
chemical shift very similar to other ones. In order to use
better the COSY experiment is necessary to perform
the spectrum with lower expansion and selecting all the
coupling hydrogens. However, the best methodology is the
use of TOCSY spectrum, which indicates that the H signals
from 9.07, 8.69, 7.74 and 7.75 ppm are the hydrogens from
one of the two aromatic rings and 8.97, 8.73, 7.89 and
7.75 ppm are from the other benzyl ring. This information
1

Vol. 28, No. 6, 2017
Azeredo et al.
979
is important to confirm the area of all hydrogen signals,
especially because some of these signals are almost as
similar superposition, for example, 7.74 and 7.73 ppm.
Despite being discovered which hydrogens are from
each benzyl ring it is also important to determine which
ring is A and B, as shown on Figure 1. For this reason, it
is necessary the obtention of the C spectrum to determine
the chemical shift of all carbons. The next process is the
gHSQC spectrum to determine which hydrogens are
directly bounded to the CH carbons. Finally, the gHMBC
spectrum is used to determine the long range coupling of
hydrogens with carbons. In this case, the H-C coupling is
the strongest at three bonds, being less with coupling at two
bonds and very low or zero at four bonds. In general, when
the coupling bounds are at 5 or more bonds, the coupling
is normally zero.
The gHMBC data, from column 2 of Table 2, confirm
which carbons are directly with one bond to the respective
hydrogens.
The gHMBC results, from column 3 of Table 2, are
shown with different forms. The bold signals display
1
13
strong H- C interaction, usually being from three bonds.
For example, 8.97 ppm from line 5 of Table 2, indicates
that this hydrogen (8.97 ppm) is at three bonds from the
carbon of 133.3 ppm. All the other bold signals in Table 2
are also with this same condition. The hydrogen signals
with normal size not marked in bold, from column 3
on Table 2, usually indicate a three bond correlation
with their corresponding carbon, but sometimes could
also correspond to a two bond correlation with their
corresponding carbon. The hydrogen signals with the
13
1
13
same size, but without being are low H- C interaction
results, but sometimes could be from three or two
bond correlations, being more possible for the 3 bond
correlation. The low results of these hydrogen signals,
which are shown blue in structure, could be due to low
Because the coupling between atoms depends from
the electrons involved in the coupling bonds, these certain
conditions lead to modification of the coupling constant.
When atomic coupling occurs using two bonds, this two-
2
n
bond coupling ( J) is usually negative, while coupling at 3
coupling constants ( JCH), a process that sometimes
3
bonds, named three-bond coupling ( J), is more positive.
depends on the structure conformation, bonds rotation
and the presence of electronegative or electropositive
atoms bounded to the carbons from the involved coupling
pathways. The complete assignment of phenanthro[9,10-e]
[1,2,4]triazin-3-amine (4) is shown in Figure 3. In this case
it is used the possible chemical shifts of the quaternary
carbons 4a and 12b, being selected 4a as the one with
the higher chemical shift (143.1 ppm). Unfortunately,
it could be possible that carbon 12b could be of 143.1
ppm, a process that would exchange the chemical shift of
compound 4 from A to B, as shown in Figure 3.
To confirm the assignment of structureA from Figure 3,
are obtained from the spectra data of gHSQC and gHMBC.
For example, the hydrogen of 9.07 ppm is bounded to the
carbon of 122.0 ppm (gHSQC) and displays long length
coupling with the carbons 138.4, 128.4, 128.4, 128.2 and
127.9, being the most strong with the carbons 128.4 and
2
3
For this reason the J is much lower than J ,especially
CH
CH
because with the two-bond coupling sometimes exists a
mixture of positive and negative coupling, a process that
2
3
sometimes leads to J = zero. For this reason, J is much
CH
CH
2
more effective than J . Another condition that decreases
CH
all coupling constants is the presence of electron attractive
groups bounded to the atoms involved on the ligands for
the coupling. This condition diminishes the density of
electrons, a process that decreases the J . The coupling
CH
constants are also depending from the angles involved
between the bonds with connection of the atoms, therefore,
the conformation of the agents structure also affects the
3
coupling constants. In general term, the J coupling
CH
depends on the angle, being major when the angle is 0°
or 180° and very low when this angle decreases, which
affords coupling constants of zero when the angle is of 90°.
Figure 3. Two different assignment of phenanthro[9,10-e][1,2,4]triazin-3-amine (4) (A and B).

9
80
Synthesis, Three-Dimensional Structure, Conformation and Correct Chemical Shift Assignment Determination
J. Braz. Chem. Soc.
1
1
20.2, indicating that it is at three bonds from carbon
28.4, which is bounded to the hydrogen of 7.74 ppm, and
Table 3. Molecular modeling calculation results of compound (4)
-1
Energy / (kJ mol )
-0.3035
249.35
242.79
50.120
with carbon 128.2 that is not bounded to any hydrogen.
Interestingly, the 9.07 ppm signal to the other quaternary
carbon, 138.44 ppm, is not very strong, but that must be due
to a three bonding distance. The other interactions are with
the CH carbon 128.4, which is bounded to the hydrogen of
2
Area / Å
2
Volume / Å
2
PSA / Å
Dipole moment / Debye 2.53
Electronic charge
7.73 ppm, and with another quaternary carbon, 127.9 ppm,
being at two bonds from the 9.07 hydrogen.
C1
-0.166
-0.186
-0.187
-0.176
-0.042
-0.013
-0.181
-0.171
-0.196
-0.070
-0.072
0.239
The only problem is if the chemical shift of carbons 4a
and 12b that could be exchanges, a process that could also
exchange the position of atoms 1, 2, 3, 4 and 13, leading to
structure B. The aromatic carbons bounded with C-N=N
or C-N=C could be with chemical shift 143.1 and 138.4
on the structure B. However, there is the possibility that the
aromatic carbons bounded to C-N=N and C-N=C could
also be of 143.1 and 138.4 on the structure B.
The possibility to confirm the structure of compound 4
is the study of the NMR spectra and comparison by
molecular modeling, especially to determine which of
the carbons 4a and 12b displays lower electronic density,
a process that could indicate which carbon contains the
highest chemical shift.
C2
C3
C4
C4a
C4b
C5
C6
C7
C8
C8a
C9
C10
0.104
C10a
-0.044
0.572
The structure calculation of compound 4 using density
functional with B3LYP and 6-311G*, affords the electronic
density calculated with natural form, the molecular
C11
PSA: polar surface area.
-1
2
energy (kJ mol ), polar surface area (PSA, Å ), element
2
volume (Å ) and their dipole moment (Debye), as shown
is of 138.4 ppm, indicating that the correct assignment of
in Table 3.
compound 4 is the structure B shown in Figure 3.
When the calculated electronic density of one atom is
a positive value, this indicates that the number of electrons
on this atom is low, leading to higher chemical shift. On the
other hand, if the electronic density is negative, the number
of electrons is higher and affords low chemical shift. When
these numbers are positive, if they contain grate value, their
chemical shift is higher. If the numbers are negative, when
the negative number is major, the chemical shift is very
low. For this reason the electronic density values indicate
the possible chemical shifts of the carbons. These results
display that the electronic density of the carbon bounded
with two nitrogen atoms (C-N=N-) is 0.104, and for the
carbon bounded with only one nitrogen (C-N=C-) is
In some cases the complete assignment of certain
n
molecules requires the determination of the J in order
CH
to determine the position and chemical shift of some
n
quaternary carbons. To determine the values of J several
CH
16
procedures can be used, such as IPAP-HSQMBC, and
SHESSLOC, but the simple and very easy to apply
this method is the gated decoupling of C NMR. One
1
7
13
18
13
example of gated decoupling of C NMR application is on
compound 5, which was synthesized as shown in Figure 4.
The assignment of compound 5, which was selected
for treatment of leishmaniasis, is generally simple, but the
only problem is the definition of the chemical shift and
position of carbons C10 and C11, which are very similar.
The complete NMR results of compound 5 are shown in
Tables 4 and 5. This compound was selected as inhibitor of
0
.239. The major electronic density (0.239) is for a higher
chemical shift, confirming that 4a is 143.1 ppm and 12b
Figure 4. Synthesis of 2-[(4-chlorophenylamino)methylene]malononitrile (5).

Vol. 28, No. 6, 2017
Azeredo et al.
981
the enzyme nucleoside hydrolases (NH) from Leishmania
donovani and Leishnamia chagasi, because we already
discovered some of these agents.
with H-8 (8.51 ppm) as shown by gHMBC, it confirms
that these carbons are from the C≡N groups. On the other
19
13
n
hand, the gated decoupling of C NMR affords all the J
CH
3
coupling constants, especially indicating that the J of the
CH
1
Table 4. H, gHSQC and gHMBC of 2-[(4-chlorophenylamino)
methylene]malononitrile (5)
116.3 and 114.1 ppm are 4.7 and 10.4 Hz, respectively.
These results confirm that the carbon at the trans position
about H-8 is the 114.1 ppm and the carbon at the cis
position is the 116.3 ppm. Therefore, it is confirmed that
a
dH (Int., mult., J Hz)
gHSQC
-
gHMBC
-
1
8
7
1.18 (s, 1H, H7)
13
the application of gated decoupling of C NMR is very
.51 (s, 1H, H8)
155.9
119.8
129.3
138.2, 116.3, 114.1, 52.4
138.2, 129.3, 129.2
138.2, 129.2, 119.8
important to obtain the complete and non questionable
chemical shift of quaternary carbons. Since hydrogens 8.51
and 7.44 display strong interaction with the quaternary
carbon of 138.2 ppm by gHMBC, this condition indicates
that the hydrogen 7.44 is close to this carbon and far from
the 8.51 ppm.As the 7.54 hydrogen interacts strongly with
the quaternary carbon of 129.3 ppm, which is bounded
to the Cl, this hydrogen is close to 8.51. The complete
assignment of compound 4 is shown in Figure 6.
.45 (d, 2H, 9.1, H2 and H6)
.44 (d, 2H, 9.1, H3 and H5)
7
a
The signals with bold are the strong correlations, in normal font are
average. HSQC: heteronuclear single quantum coherence; HMBC:
heteronuclear multiple bond coherence.
13
13
Table5. C,APTandgateddecouplingof Cofof2-[(4-chlorophenylamino)
methylene]malononitrile (5)
Gated
dC
APT
n
Mult.
d
JCH / Hz
1
1
1
1
1
1
1
5
55.9 (C8)
CH
C
176.9
38.2 (C1)
dt
6.9 and 4.6
167.9 and 4.6
8.7
29.3 (C3 and C5)
29.2 (C4)
CH
C
dd
tm
dd
d
19.8 (C2 and C6)
16.3 (C10)
CH
C
165.4 and 5.8
4.7
Figure 6. Complete assignment of 2-[(4-chlorophenylamino)methylene]
malononitrile (5).
14.1 (C11)
C
d
10.4
2.4 (C9)
C
d
4.6
The molecular modeling calculation using density
functional theory (DFT) with the B3LYP and the M06
method with 6-311-G* basis set, indicates the electronic
atomic charges (QNPA) for each carbon, as shown in
Table 6.
APT: attached proton test.
The hydrogen 8.51 ppm, which is a simple signal
because it is not bounded to the benzyl ring and with other
hydrogens, but it is coupled with the carbons 116.3 and
Table 6. Carbons molecular modeling calculation of electronic atomic
charge from compound 5
114.1 ppm. The coupling condition between 8.51 ppm with
1
16.3 and 114.1 ppm, because it is by three bonds, indicate
3
that the respective J values depend on the conformation
CH
of the hydrogen with the two carbons, being at position cis
of trans, as are shown in Figure 5.
Carbon
C1
Electronic charge
0.158
Figure 5. Conformation of three bound coupling of hydrogen with carbon.
C2 and C6
C3 and C5
C4
-0.206 and -0.219
-0.199 and -0.194
-0.034
The forms A and B from Figure 5 are linear cis and
trans, respectively. The trans conformation allows the
greater H-C coupling than the cis conformation. When the
system is planar, the angle between C and H is of 180° in
conformation A and of 0° in conformation B. Because the
C8
0.172
C9
-0.370
C10
0.276
C11
0.285
116.3 and 114.1 ppm carbons are only strongly coupled

9
82
Synthesis, Three-Dimensional Structure, Conformation and Correct Chemical Shift Assignment Determination
J. Braz. Chem. Soc.
The electronic condensation on each atom indicates
indicated that this compound is an appropriate intercalate
to DNA, as shown in Figure 7.
their possible chemical shift. When the electronic
concentration is major or lower, the chemical shift is lower
or major, respectively. The carbons electronic charge from
compound 5 are between -0.219 and +0.285, being the
negative values with major electronic concentration, and
the positive ones with lower concentration. In this case, the
C≡N carbon of major electronic charge display electronic
charges of 0.276 of 116.3 ppm and the other one 0.285 with
This result indicates that compound 4 could be used
as an agent to decrease the dose of cancer radiotherapy,
in order to diminish the negative effects of radiotherapy
to other human cells. This compound interacts better with
the groups deoxydenosine 5’-monophosphate (A) and
deoxythymidine 5’-nonophosphate (T) of DDD.
The docking study of compound 5 as inhibitor of the
enzyme nucleoside hydrolase from Leishmania donovani
(NH) is shown in Figure 8.
1
14.1 ppm. The definitive chemical shift of these carbons
is determined by all NMR methods, being also important
13
the gated decoupling C and molecular modeling.
This result indicates that the CN groups of compound 5
interact with the amino acids Asp14, Th4128, Asn168
The study of the potential capacity of these compounds
to interact with the selected biological targets was executed
by docking with AutoDock Vina. The interaction of
compound 4 was selected to DNA. For this topic it was used
the Drew-Dickerson-Dodecamer (DDD), and the docking
2+
and the Ca of this enzyme. On the other hand, the NH
group interacts with Gly12 and the aromatic ring displays
p-stacking with Phe167. For this reason, this compound
could display activity against leishmaniasis.
Figure 7. Docking of compound 4 with Drew-Dickerson-Dodecamer (DDD).
Figure 8. Docking study of the interaction of compound 5 with nucleoside hydrolase (NH).

Vol. 28, No. 6, 2017
Azeredo et al.
983
The biological texts of these compounds will be
performed on the future, and also with other similar
compounds against cancer and Leishmania.
Acknowledgments
We appreciate the financial support afforded by CAPES,
CNPq and INBEB.
Conclusions
References
The determination of the non questionable and complete
chemical shift assignment of potential pharmacological
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interaction with the biological targets involved on their
biological activity. This information can be used for
inhibition of enzymes and interaction with DNA, being a
process to determine which areas of these pharmacological
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In general terms, the methods to definitely confirm the
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confirm the definitive structure of the obtained compounds
prepared by synthesis. This condition is to motivate the
complete structure and chemical shift assignment of all
new synthesized compounds, in order to confirm their
condition. This procedure is also confirmed by molecular
modeling.
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the active site of this enzyme, and the chlorinated benzyl
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this enzyme. The biological tests of these compounds will
be executed on the future.
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Submitted: April 29, 2016
Published online: August 31, 2016