NMR assignments and structural characterization of new thiourea and urea kynurenamine derivatives nitric oxide synthase inhibitors

Full text of DOI:10.1002/mrc.4295

Letter ‐ spectral assignments
Received: 11 May 2015
Revised: 12 June 2015
Accepted: 18 June 2015
Published online in Wiley Online Library: 11 September 2015
(wileyonlinelibrary.com) DOI 10.1002/mrc.4295
NMR assignments and structural
characterization of new thiourea and urea
kynurenamine derivatives nitric oxide
synthase inhibitors
Mariem Chayah,a M. Dora Carrión,a Miguel A. Gallo,a
Duane Choquesillo-Lazarteb and M. Encarnación Camachoa*
spectrometers at 298 K. The following parameters were used in
DEPT experiments: pulse width (PW) (135°), 9.0 ms; recycle time,
Introduction
The bioactive molecule of nitric oxide (NO) is an important sec-
ond messenger that participates in regulation of cardiovascular,
nervous and immune systems.[1] NO synthases (NOS) catalyze
the oxidation of the amino acid L-arginine to L-citrulline and
NO with NADPH and molecular oxygen as cosubstrates.[2] In
mammalians, three isoforms of NOS have been identified: neuro-
nal (nNOS), endothelial and inducible NOS.[3] These isoenzymes
are involved in important biological processes and are impli-
cated in many pathological diseases. Thus, overproduction of
NO by nNOS has been associated with neurodegenerative disor-
ders in Alzheimer’s, Parkinson’s or Huntington’s diseases,[4–6] and
the inducible isoform seems to be responsible for the massive
NO production in pathologies such as arthritis, colitis, tissue
damage, cancer or several inflammatory states.[7–9] In this way,
many authors have focused their interest in the development
of NOS selective inhibitors.
1 s; 0.5J (CH)= 4 ms; 65536 data points acquired and transformed
from 1024 scans; spectral width, 15 KHz; and line broadening,
1.3 Hz. Chemical shifts (δ) are quoted in parts per million (ppm)
and are referenced to the residual solvent peak: CDCl3,
δ = 7.26 ppm (1H), δ = 77.4 ppm (13C); (CD3)2CO, δ = 2.05 ppm (1H),
δ = 29.84ppm (13C); (CD3)2SO, δ = 2.50 ppm (1H), δ = 39.52 ppm
(13C); and CD3OD, δ = 3.31 ppm (1H), δ = 49.00 ppm (13C). Spin multi-
plicities are given as s (singlet), bs (broad singlet), d (doublet), dd
(doublet doublet), ddd (doublet, doublet doublet), t (triplet), q (qua-
druplet) and m (multiplet). Coupling constant (J) are given in hertz.
The HMBC spectra were measured with a pulse sequence
gc2hmbc (standard sequence, Agilent Vnmrj_3.2A software)
optimized for 8 Hz (inter-pulse delay for the evolution of long-range
couplings: 62.5 ms). The HSQC spectra were measured with a pulse
sequence gc2hsqcse (standard sequence, Agilent Vnmrj_3.2A
software).
In a recent paper, the synthesis and biological evaluation of a
novel family of kynurenamine derivatives bearing a thiourea or urea
fragment 31–45, which were designed and evaluated as NOS
inhibitor agents, have been described.[10]
Although the structures of these derivatives have been deter-
mined by means of standard spectroscopic techniques (1H and
13C NMR and MS), a detailed NMR study has been performed in
some of them, in order to unequivocally corroborate their
structures.
Nuclear Overhauser spectra were recorded on an Agilent Varian
Direct Drive spectrometer, operating at 600 MHz, with a spectral
widths of 4.96KHz in both F2 and F1 domains; 744 × 200 data
points were acquired with 16 scans per increment and relaxation
delays of 1.0s. The mixing time in NOESY experiments was 0.5s.
Data processing was performed on a 1 × 1 K data matrix.
Molecular dynamics simulations
The present study reports the unambiguous assignment of each
signal in the 1H and 13C NMR spectra in compounds 31–45, using
one-dimensional and two-dimensional resonance techniques. The
spectra of nitro derivatives 1–30, the precursors in the synthetic
pathway, are also included.
Unrestricted molecular dynamics (MD) simulations were carried out
with the AMBER 12 software suite[11] in explicit solvent using the
AMBER force field leaprc ff12SB. Geometry of compound 39 was
optimized using RHF/6-31G* as implemented in Gaussian 09
(Gaussian, Inc., Wallingford, CT). Charges were then assigned to
Experimental
*
Correspondence to: M. Encarnación Camacho. Departamento de Química
Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, c/
Campus de Cartuja, s/n 18071 Granada, Spain. E-mail: ecamacho@ugr.es
NMR spectra
Nuclear magnetic resonance spectra were recorded on 300-MHz 1H
NMR and 75-MHz 13C NMR Agilent Varian Direct Drive, 400-MHz 1H
NMR and 100-MHz 13C NMR Agilent Varian Direct Drive and 600-
a Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia,
Universidad de Granada, Granada, Spain
1
MHz H NMR and 150-MHz 13C NMR Agilent Varian Direct Drive
b Laboratorio de Estudios Cristalográficos, IACT-CSIC, Armilla, Granada, Spain
Magn. Reson. Chem. 2015, 53, 1071–1079
Copyright © 2015 John Wiley & Sons, Ltd.

M. Chayah et al.
individual atoms by fitting the quantum mechanically calculated
(RHF/6-31G*//RHF/3-21G*) molecular electrostatic potential to a
point charge model. Compound 39 was immersed in a cubic box
of CHCl3 molecules (CL3 model) with a minimum distance of 12 Å
from any atom to the edge. The particle mesh Ewald method for
long-range electrostatic interactions together with standard
periodic boundary conditions was used. SHAKE algorithm was
applied to reach an integration step of 2.0 fs. The followed MD
simulation protocol was the same as previously described.[10] Dis-
tances and conformational clustering were performed using the
program cpptraj implemented in Amber Tools 14.
Table 1. Structural data of the intermediate (1–30) and final (31–45)
compounds
Compound
R1
R2
X
Compound
R1
R2
Et
X
1, 16, 31
2, 17, 32
3, 18, 33
4, 19, 34
5, 20, 35
6, 21, 36
7, 22, 37
8, 23, 38
9, 24, 39
H
H
Me
Et
S
10, 25, 40
11, 26, 41
12, 27, 42
13, 28, 43
14, 29, 44
15, 30, 45
H
H
O
S
S
S
S
S
S
S
S
Pr
Et
Pr
Et
Pr
O
O
O
O
O
H
Pr
OMe
OMe
Cl
OMe
OMe
OMe
Cl
Me
Et
Pr
Cl
Me
Et
Cl
Cl
Pr
X-ray crystallography
A yellow crystal of compound 39 was mounted on a MiTeGen
Micromounts, and this sample was used for data collection. Data
were collected with a Bruker D8 Venture diffractometer. Data
were processed with APEX[12] and corrected for absorption using
SADABS.[13] The structures were solved by direct methods,[14]
which revealed the position of all non-hydrogen atoms. These
atoms were refined on F2 by a full-matrix least-squares proce-
dure using anisotropic displacement parameters.[14] All hydrogen
atoms were located in different Fourier maps and included as
fixed contributions riding on attached atoms with isotropic ther-
mal displacement parameters that are 1.2 times those of the re-
spective atom. Crystallographic data for the structural analysis
have been deposited with the Cambridge Crystallographic Data
Centre, CCDC 1063097.
and (iv) NOESY experiments to determinate the preferred confor-
mation in solution.
Tables 2–4 show the 1H NMR signals of each proton for
compounds 1–45, whereas Tables 5–7 show the corresponding
13C NMR chemical shifts for the same molecules. NMR spectra of
all compounds were carried out in CDCl3 solution, except for
compound 19, which was registered in (CD3)2CO, 20 and 37 in
(CD3)2SO and 26 and 33 in CD3OD. For this reason, some significant
variations are observed in the chemical shifts according to the
solvent.
The 1H NMR and 13C NMR signals of the aromatic ring (H-1″–H-6″,
C-1″–C-6″) and C-3′ are similar in the three families of compounds:
1-(2-(2-(5-substituted-2-nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-
alkylthioureas and alkylureas 1–15, 1-(3-(5-substituted-2-nitrophe-
nyl)-3-oxopropyl)-3-alkylthioureas and alkylureas 16–30, and 1-(3-
(2-aminophenyl-5-substituted)-3-oxopropyl)-3-alkylthioureas and
alkylureas 31–45. However, signals corresponding to the linear
chain (H-1′, H-2′, C-2, C-1′ and C-2′) are influenced by the thiourea
or urea residue of each family of compounds. Finally, H-1 and H-3
signals, linked to nitrogen atoms, do not follow the same pattern
and sometimes are not visible.
Heteronuclear single-quantum correlation and HMBC exper-
iments were performed on some compounds of each series,
and the results of these experiments have been extrapolated
to the other compounds. Table 8 shows the HSQC correla-
tions for some representative compounds, whereas Fig. 1
shows the more important connectivities found in the HMBC
spectra.
Results and discussion
Scheme 1 represents the previously reported synthetic pathway
followed in the preparation of final compounds 31–45,[10] and
Table 1 shows the structural data of all the intermediate and final
compounds synthesized.
Structural elucidation of these compounds has been made by
1
routine H and 13C techniques. Nevertheless, a definitive assign-
ment of all signals needs the use of several NMR techniques as
follows: (i) DEPT experiments to determine the number of
protons attached to each carbon atom; (ii) HSQC spectra to
determine the 13C resonances of the tertiary, secondary and pri-
mary carbons; (iii) HMBC sequences to assign the signals of
quaternary carbons via two-bond and three-bond interactions;
Heteronuclear single-quantum correlation experiments per-
formed on compounds 1, 6, 9 and 11 allow the assignment of
Scheme 1. General synthetic pathway followed in the preparation of final compounds 31–45. (a) Phthalimide, NaOMe, DMSO; room temperature (RT), 2 h;
(b) (CH2OH)2, p-TsOH; toluene, reflux, 10 h; (c) NH2NH2, dry EtOH, reflux, 4.5 h; (d) XCNR2, anhyd CH2Cl2, RT, overnight; (e) HCl, CH2Cl2, RT, 4 h; (f) Fe/FeSO4, H2O,
95 °C, 3 h.
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Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 1071–1079

NMR assignments of new thiourea and urea kynurenamine derivatives
Magn. Reson. Chem. 2015, 53, 1071–1079
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

M. Chayah et al.
wileyonlinelibrary.com/journal/mrc
Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 1071–1079

NMR assignments of new thiourea and urea kynurenamine derivatives
Magn. Reson. Chem. 2015, 53, 1071–1079
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

M. Chayah et al.
Table 5. 13C NMR chemical shifts of compounds 1–15
Compound
C-2
C-1′
C-2′
C-3′
C-1″
C-2″
C-3″
C-4″
C-5″
C-6″
2xCdiox
OCH3
1
2
180.82
180.92
180.39
182.19
181.98
181.34
181.66
180.68
180.79
158.50
158.54
158.57
158.40
158.77
158.78
40.04
40.09
39.96
40.35
40.31
40.28
40.06
39.98
40.07
35.66
35.59
35.59
35.57
35.43
35.25
38.06
38.18
37.98
37.97
37.95
37.94
38.12
38.09
38.10
39.37
39.04
39.01
39.01
42.39
42.39
109.28
109.46
109.23
109.54
109.43
109.54
109.06
109.06
109.07
109.51
109.24
109.45
109.46
108.93
108.89
134.66
134.79
134.56
137.65
137.42
137.61
137.21
137.18
137.18
135.15
135.07
137.62
137.79
137.20
137.26
149.51
149.64
149.46
143.01
143.14
143.05
147.85
147.86
147.88
149.66
149.46
144.92
142.97
147.69
147.61
131.61
131.70
131.47
126.24
126.22
126.19
125.18
125.16
125.16
131.47
131.29
125.74
125.76
124.70
124.66
129.82
129.93
129.72
114.28
114.29
114.27
130.00
129.99
130.00
129.68
129.52
114.08
114.00
129.43
129.48
123.48
123.59
123.37
161.94
161.72
161.92
137.91
137.90
137.89
123.38
123.20
161.42
161.58
137.30
137.35
128.38
128.48
128.29
113.38
113.33
113.38
128.59
128.59
128.59
128.63
128.42
113.21
113.26
128.41
128.44
65.04
65.14
64.93
65.14
65.12
65.13
65.33
65.32
65.33
65.07
64.88
64.83
64.91
64.87
64.97
—
—
3
4
56.17
56.14
56.16
—
5
6
7
8
—
9
—
10
11
12
13
14
15
—
—
55.62
55.94
—
—
δ (in ppm) relative to CDCl3. 13C signals for the R2 substituent: 1, CH3: 32.81; 30.29; 2, CH2CH3: 38.18, 14.28; 3, CH2CH2CH3: 45.60, 22.16, 11.44; 4, CH3: 30.50;
5, CH2CH3: 42.25, 22.65; 6, CH2CH2CH3: 45.78, 22.39, 11.63; 7, CH3: 30.47; 8, CH2CH3:38.80, 14.26; 9, CH2CH2CH3: 45.80, 22.37, 11.66; 10, CH2CH3: 35.66,
15.64; 11, CH2CH2CH3: 42.56, 23.26, 11.36; 12, CH2CH3: 41.79, 15.47; 13, CH2CH2CH3: 42.49, 23.50, 11.44; 14 CH2CH3: 35.35, 15.31; 15, CH2CH2CH3:
39.02, 23.34, 11.31.
Table 6. 13C NMR chemical shifts of compounds 16–30
Compound
C-2
C-1′
C-2′
C-3′
C-1″
C-2″
C-3″
C-4″
C-5″
C-6″
OCH3
16
17
18
19a
20b
21
22
23
24
25
26c
27
28
29
30
181.50
181.54
180.70
184.29
182.37
181.83
181.76
181.43
181.60
158.53
160.00
158.73
160.01
159.95
158.99
39.50
39.71
39.51
39.44
39.41
39.86
39.70
39.64
39.66
35.64
34.82
35.54
34.94
36.65
35.58
42.50
42.51
42.30
42.36
42.63
42.73
42.64
42.64
42.65
43.43
41.66
43.47
43.09
44.52
43.35
202.54
202.64
202.38
201.28
201.95
202.74
201.09
201.15
201.16
202.55
202.03
202.55
202.23
202.02
200.91
137.20
137.23
137.01
138.56
138.43
138.26
138.87
138.86
138.87
137.58
137.25
138.18
138.31
140.24
139.03
145.76
145.91
145.73
140.82
140.64
140.44
143.95
143.96
143.96
145.92
146.22
140.59
140.68
145.07
143.92
124.50
124.81
124.60
127.21
127.86
127.50
126.35
126.34
126.32
124.74
124.29
127.43
126.99
127.42
126.30
131.09
131.19
130.99
115.60
116.31
115.49
131.07
131.07
131.06
130.99
131.01
115.43
115.04
132.11
131.00
134.50
134.61
134.39
164.71
164.70
164.75
141.70
141.68
141.66
134.53
134.21
164.72
164.82
142.80
141.69
127.30
127.45
127.27
112.51
113.20
112.06
127.51
127.53
127.54
127.50
127.64
112.04
112.31
128.75
127.58
—
—
—
56.36
57.31
56.63
—
—
—
—
—
56.61
55.85
—
—
aSolvent used (CD3)2CO.
bSolvent used (CD3)2SO.
cSolvent used CD3OD.
δ (in ppm) relative to CDCl3. 13C signals for the R2 substituent: : 16, CH3: 32.80; 17, CH2CH3: 38.75, 14.22; 18, CH2CH2CH3: 45.62, 22.13, 11.43; 19, CH3:
30.20; 20, CH2CH3: 38.93, 15.09; 21, CH2CH2CH3: 45.68, 22.32, 11.64; 22, CH3: 29.93; 23, CH2CH3:38.73, 14.21; 24, CH2CH2CH3: 45.75, 22.33, 11.65; 25,
CH2CH3: 35.35, 15.61; 26, CH2CH2CH3: 34.86, 23.27, 10.46; 27, CH2CH3: 35.88, 15.35; 28, CH2CH2CH3: 41.68, 23.27, 10.46; 29 CH2CH3: 37.07, 16.52; 30,
CH2CH2CH3: 42.92, 23.27, 11.53.
the secondary carbon atoms C-1′ and C-2′ chemical shifts, and
the tertiary carbon atoms C-3″, C-4″, C-5″ and C-6″ chemical shifts
in the dioxolane derivatives 1–15. These atoms show signals in
ranges of 35.25–40.35 (C-1′), 37.94–42.39 (C-2′), 124.66-131.70
(C-3″), 114.00–130.00 (C-4″), 123.20–161.94 (C-5″) and 113.21–
128.63 (C-6″).
Similar HSQC experiments performed on compounds 18, 21, 23,
27 and 30 indicate that the 13C NMR signals for the analogue sec-
ondary and tertiary carbon atoms in the intermediate nitrophenyl
derivatives 16–30 are in the following ranges: 34.82–39.86 (C-1′),
41.66–44.52 (C-2′), 124.29–127.86 (C-3″), 115.04–132.11 (C-4″),
134.21–164.82 (C-5″) and 112.04–128.75 (C-6″).
In the same way, HSQC experiments performed on compounds
32, 35, 39, 41, 43 and 45 confirm that the peaks corresponding
to the analogue secondary and tertiary carbon atoms in the final
aminophenyl derivatives 31–45 appear around 35.54–41.11 (C-1′),
38.47–39.93 (C-2′), 117.38–120.05 (C-3″), 123.54–135.68 (C-4″),
116.13–151.54 (C-5″) and 113.23–132.57 (C-6″).
To confirm the signals corresponding to quaternary carbons,
HMBC spectra on the intermediate (1, 6, 11, 18, 21 and 30) and final
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NMR assignments of new thiourea and urea kynurenamine derivatives
Table 7. 13C NMR chemical shifts of compounds 31–45
Compound
C-2
C-1′
C-2′
C-3′
C-1″
C-2″
C-3″
C-4″
C-5″
C-6″
OCH3
31
32
33b
34
35
36
37a
38
39
40
41
42
43
44
45
182.21
181.34
182.86
182.41
181.36
181.56
181.43
181.40
181.52
158.82
158.85
158.57
158.49
158.62
158.88
40.06
40.19
41.11
40.31
40.18
40.29
39.99
40.01
40.00
35.54
35.88
35.64
35.73
35.80
35.86
38.47
38.70
39.76
38.87
38.93
38.89
38.82
38.78
38.85
39.41
39.71
39.93
39.81
39.59
39.56
201.92
202.14
202.76
201.61
201.69
201.67
200.61
201.23
201.24
201.96
202.16
201.91
201.76
201.15
201.06
117.62
117.77
118.77
117.51
117.89
117.51
117.76
118.59
119.03
117.91
118.48
119.33
117.90
118.59
118.63
150.38
150.60
152.83
145.44
144.74
145.45
150.52
148.55
147.84
150.16
149.87
142.75
145.29
148.82
148.70
117.47
117.65
118.48
119.13
119.39
119.13
119.67
119.31
119.62
117.38
117.94
120.05
119.04
119.19
119.28
134.97
135.16
135.68
124.60
124.44
124.61
134.73
135.13
135.11
134.61
134.88
123.54
124.13
134.98
134.99
116.26
116.42
116.42
150.51
150.74
150.52
121.65
121.13
121.65
116.13
116.79
151.54
150.48
120.83
120.93
131.22
131.43
132.57
113.24
113.30
113.23
130.84
130.50
130.52
131.20
131.43
113.83
113.47
130.49
130.45
—
—
56.30
56.29
56.30
—
—
—
—
—
56.24
56.25
—
—
aSolvent used (CD3)2SO.
bSolvent used CD3OD.
δ (in ppm) relative to CDCl3. 13C signals for the R2 substituent: 31, CH3: 30.25; 32, CH2CH3: 38.70, 14.23; 33, CH2CH2CH3: 39.76, 23.55, 11.66; 34, CH3:
30.42; 35, CH2CH3: 38.93, 14.23; 36, CH2CH2CH3: 45.74, 22.32, 11.66; 37, CH3: 31.12; 38, CH2CH3:38.78, 14.22; 39, CH2CH2CH3: 45.82, 22.35, 11.67; 40,
CH2CH3: 35.35, 15.38; 41, CH2CH2CH3: 42.62, 23.56, 11.59; 42, CH2CH3: 35.55, 15.63; 43, CH2CH2CH3: 42.63, 23.61, 11.58; 44 CH2CH3: 35.82, 15.49; 45,
CH2CH2CH3: 42.80, 23.42, 11.57.
Table 8. HSQC correlations found for compounds 1, 6, 9, 11, 18, 21, 23, 27, 30, 33, 35, 39, 41, 43 and 45
1H/13
C
Compound
1
6
9
11
3.37
18
4.03
21
4.05
23
27
30
3.64
32
3.96
35
3.96
39
3.99
41
3.56
43
3.57
45
3.58
H-1′
C-1′
H-2′
C-2′
H-3″
3.67
40.04
2.48
38.06
7.51
3.68
40.28
2.52
37.94
7.48
3.70
4.04
3.65
35.54
2.99
40.07
2.48
35.50
2.37
39.51
3.19
39.86
3.10
39.64
3.18
35.58
3.04
40.19
3.30
40.18
3.25
40.00
3.26
35.88
3.15
35.73
3.14
35.86
3.14
38.10
7.40
39.04
7.49
42.30
8.07
42.72
8.13
42.64
8.07
43.47
8.13
43.35
8.06
38.70
6.62
38.93
6.63
38.85
6.69
39.71
6.64
39.81
6.60
39.56
6.62
C-3″ 131.61
H-4″ 7.43
C-4″ 129.82
H-5″ 7.43
C-5″ 123.48
H-6″ 7.79
C-6″ 128.38
126.19 125.16 131.29 124.60 127.50 126.34 127.43 126.30 117.65 119.39 119.62 117.44 119.04 119.28
6.85 7.40 7.40 7.59 6.98 7.56 6.98 7.54 7.25 6.95 7.23 7.24 6.94 7.19
114.27 130.00 129.52 130.99 115.49 131.07 115.43 131.00 135.16 124.44 135.11 134.88 124.13 134.99
7.40
123.20 134.39
7.58 7.42
7.71
6.62
116.42
7.67
6.64
116.79
7.67
7.06
7.58
6.74
7.38 6,75
7.35
7.11
7.67
7.14
7.62
113.38 128.59 128.42 127.27 112.06 127.53 112.04 127.58 131.43 113.30 130.52 131.43 113.47 130.45
Figure 1. Important connectivities found in the HMBC spectra of compounds (a) 1–15, (b) 16–30 and (c) 31–45.
(35, 39, 41 and 45) compounds were recorded (Fig. 1). Correlations
in compound 1 between H-6″ (δ 7.59 ppm) and the 13C signal at
149.51 ppm, H-3″ (δ 7.51 ppm) and the 13C signal at 134.66 ppm
and H-2″ (δ 2.48ppm) and the 13C signal at 109.28 ppm allow the
unequivocal assignment of C-2″, C-1″ and C-3′, respectively. In
addition, compound 6 HMBC analysis indicates a correlation
between OCH3 (δ 3.85) and the 13C signal at 161.92 ppm, which
can be attributed to C-5″. Also, correlation between H-1′ (δ 3.37)
in compound 11 and the peak at 158.54 ppm shows that this signal
corresponds to C-2.
Heteronuclear multiple-bond correlation experiment performed
on compound 18 indicates that the signal at δ 8.07 (H-3″) is corre-
lated with the 13C signal at 137.01 ppm, which can be assigned to
C-1″, whereas H-6″ signal (δ 7.42) is correlated with the 13C signal
Magn. Reson. Chem. 2015, 53, 1071–1079
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M. Chayah et al.
at 145.73 ppm; accordingly, this signal corresponds to C-2″. Further-
more, a correlation between H-1′ signal (δ 4.03) and 13C signal at
180.70 ppm has been observed, and this signal can be designated
as C-2 of thiourea. Finally, H-2′ (δ 3.19) is correlated with a signal
at δ 202.38 that can be assigned to C-3′. In addition, similar
experiment of compound 21 indicates a correlation between the
OCH3 (δ 3.90) and the 13C signal at 164.75ppm that is identified
as C-5″. Lastly, HMBC experiment on the urea 30 demonstrates that
H-6″ signal (δ 7.35) is correlated with the 13C signal at 141.69 ppm,
and this signal corresponds to C-5″, whereas correlation between
H-1′ (δ 3,64) and a signal at 158.99 ppm indicates that this signal
corresponds to C-2 of urea.
Heteronuclear multiple-bond correlation spectrum of compound
35 denotes that H-3″ (δ 6.63) and OCH3 (δ 3.73) are correlated with
the 13C signals at 117.89 and 150.74 ppm, which are assigned to C-1″
and C-5″, respectively. In compound 39, the signal of 13C at
147.84 ppm can be assigned to C-2″ because of its correlation with
H-4″ (δ 7.23), whereas H-1′ (δ 3.99) is correlated with the 13C signal
at 182.52 ppm, and consequently, this peak is identified as C-2 of
the thiourea. Also, H-2′ of the urea 41 (δ 3.15) is correlated with a
13C signal at 202.16 that is assigned to C-3′. Finally, in compound
45, the H-6″ (δ 7.62) and H-1′ (δ 3.58) are correlated with the 13C
signals at 120.93 and 158.88 ppm that are designed to C-5″ and C-2
of the urea, respectively.
of the amides and the equivalent C-2 atom of the ureas or
thioureas). The amide C-1 atom shows signals in a range of
167.4–176.4, whereas for the equivalent urea C-2 atom, the signals
appear in a range of 158.5–160.0, and for the thiourea C-2 atom, the
range is 180.7–184.3ppm, according to similar compounds de-
scribed in literature.[16,17]
Nuclear Overhauser spectroscopy experiments performed on
compound 39 (R1 = Cl, R2 = Pr, X = S) (Fig. 2) demonstrate a correla-
tion between H-6″ and H-2′ and vice versa, which proves that they
have close distance in space. These NOE effects are possible if the
molecule adopts a conformation where there is an intramolecular
hydrogen bond between the carbonyl group of the linear chain
and the amino group in 2″-position of the aromatic ring.
To explore the conformational behavior of this molecule in
solution, we performed an unrestricted MD simulation (100ns) of
compound 39 in similar conditions to those where NOESY spectra
were recorded (box of CHCl3 molecules and 300K) (Fig. 3(a)). In
the three calculated conformational clusters of 39 along the MD
simulation, a permanent intramolecular hydrogen bond between
the carbonyl oxygen and the amino group of the phenyl ring is
observed. In this regard, the distance between NH2 and O¼C
remains under 2.8 Å along the MD simulation (Fig. 3(b)). As a conse-
quence, hydrogen atoms H-2′ and H-6″ are close in space (2.0–
2.5 Å), which is in line with the experimental NOEs. Finally, the major
differences between the three groups of conformers are due to the
These compounds have been designed from a series of N-(3-(2-
amino-5-substitutedphenyl)-3-oxopropyl)alkylamide
derivatives
previously synthesized by our research group,[15] by substitution
of the main chain alkylamide for an alkylurea or alkylthiourea ones.
The main differences between the 13C chemical shifts of the previ-
ously kynurenamine derivatives[15] and the new kynurenamine-
ureas and thioureas are the 13C signals of the main chain (C-1 atom
Figure 4. Asymmetric unit in the crystal of compound 39. Only one of the
Figure 2. Selected NOESY correlations for compound 39.
disordered positions of the propyl group is depicted for clarity.
Figure 3. Molecular dynamics (MD) simulation of 39 in a box of CHCl3 molecules. (a) Representative structure of the major conformer of 39 and (b) distance
evolution between carbonyl oxygen and the amino group calculated along the 100-ns MD simulation. Spatial proximity of H-2″ and H-6″ atoms is indicated as
dotted lines.
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Magn. Reson. Chem. 2015, 53, 1071–1079

NMR assignments of new thiourea and urea kynurenamine derivatives
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relative orientation of the thiourea side chain to the ring due to the
rotation of C2–C1 bonds in the chain.
Derivative 39 has been crystallized with the object of confirming
the preferred conformation of these molecules in solid state, and its
3D structure has been determined using X-ray diffraction. This
compound crystallizes in the triclinic system, space group P-1. The
asymmetric unit consists of one molecule (Fig. 4) where the mean
planes of the aromatic ring and the thiourea moiety defines a
dihedral angle of 75.44°. In this conformation, one intramolecular
involving
H-bond is observed
aromatic-NH2 and CO groups (N–
H⋯O: 2.683 A 129.0°). The propyl group is disordered over two
alternative positions, with site-occupancy factors 0.52:0.48. In the
crystal lattice, pairs of adjacent molecules are symmetrically
connected by intermolecular H-bonds that involve aromatic-NH2
and CO groups (N–H⋯O: 3.184 A 154.0°). The aforementioned pairs
of molecules are further associated by additional intermolecular H-
bonding interactions where aromatic Cl and thiourea groups are
involved, building a 3D H-bonded network.
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Acknowledgement
This work was partially supported by the Instituto de Salud Carlos III
through the grant FI11/00432.
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Magn. Reson. Chem. 2015, 53, 1071–1079
Copyright © 2015 John Wiley & Sons, Ltd.
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