SHA ET AL.
643
window function was exponential multiplication (EM),
sizes of real spectrum were 64 K and 32k, respectively,
and line broadenings were 0.30 and 1 Hz, respectively.
For two-dimensional experiments, NOESY data were
collected with 1,024 × 128 data points, HSQC and HMBC
data were achieved with 2,048 × 256 and 2,048 × 128 data
points, respectively. The other experiment conditions are
as follows: for NOESY, the pulse program was
Using the σref data, theoretical 13C chemical shifts (δcal
)
of other structural models (tautomer II-2, tautomer I-1,
and tautomer I-2) were worked out according to the fol-
lowing Equation (1):
δ
cal = σref −σcal
:
ð1Þ
1
noesyesgpph,[8] center of the H spectrum was 5.32 ppm,
1
spectral width in the H dimension was 14.92 ppm, and
duration of mixing time was 0.7 s; for HSQC, the pulse
program was hsqcetgpsisp2.2,[9] center of the 1H
spectrum was 5.50 ppm, center of the 13C spectrum
3 | RESULTS AND DISCUSSION
3.1 | NMR spectral studies
1
was 89.96 ppm, spectral width in the H dimension was
14.92 ppm, and spectral width in the 13C dimension
was 205.95 ppm; for HMBC, the pulse program was
hmbcgpndqf,[10] center of the 1H spectrum was 5.52 ppm,
center of the 13C spectrum was 89.96 ppm, spectral width
in the 1H dimension was 13.69 ppm, spectral width in the
13C dimension was 205.90 ppm, and interpulse evolution
period of long-range proton–carbon coupling constants
was 62.5 ms. The NOESY, HSQC, and HMBC processing
parameters are as follows: sizes of real spectrum in F2
and F1 were 2 K, window functions in F2 and F1 were
QSINE, and sine bell shift in F2 and F1 was 2. All NMR
data were analyzed using TopSpin4.0.5 (Bruker NMR
Software).
Firstly, H-2, H-5 of benzene-ring and H-13, H-14 of
heterocyclic-ring could be straightforwardly assigned in
the 1H NMR spectrum of fenobam (Figure 1). One singlet
peak at 7.86 ppm is attributed to H-2, and a doublet of
doublets appearing as a triplet at 7.25 ppm is belonged to
H-5; whereas, the singlet peak at 4.01 ppm is assigned to
methylene group (H-13), and another singlet peak at
2.99 ppm is identified as N-methyl group (H-14). Using
the integration of H-2 (the integral area of H-2 as 1) as
the reference, those of the methylene peak (H-13) and
the methyl peak (H-14) are 2.03 and 2.99, respectively,
which firmly proving the assignation of H-13 and H-14.
In addition, two singlet peaks at high frequencies at 9.54
and 10.91 ppm belong to two amino groups, and two dou-
blet peaks at 7.48 and 6.96 ppm are assigned to protons at
positions 4 and 6 of benzene-ring, respectively.
2.5 | Theoretical calculation
However, it is difficult to distinguish these two dou-
blet peaks at 7.48 and 6.96 ppm due to their similar
chemical environments. As can be seen from 1H–1H
NOESY spectrum of fenobam (Figure 2), one amino pro-
Molecule in tautomer II configuration was extracted
directly from the reported crystal structure of anhydrous
form IV (tautomer II-1) and form III (tautomer II-2),
respectively.[7] Tautomer I was obtained by twisting the
dihedral angle τ(C10–N–C8–N) and moving the proton
from position 11 to position 9. Structural optimization
and single-point energy calculations were performed
using Gaussian (09) software[11] at B3LYP/6-31G* level
with (DMSO) and without solvent effect. Self-consistent
reaction field (SCRF) method coupled with the integral
equation formalism polarizable continuum model
(IEFPCM) was used in simulated DMSO solution,
describing the solvent implicitly.[12] Each structure opti-
mized was verified to be stable by subsequent vibrational
analysis. Theoretical 13C isotropic chemical shielding ten-
sors were calculated at B3LYP/6-311+G(2d,p) level with
solvent (DMSO) effect using same strategy.[12] One of the
tautomer II models (tautomer II-1, vide infra) was
selected as a reference compound. The σref parameters
for each carbon atom can be obtained by adding the cal-
culated 13C shielding values (σcal) of tautomer II-1 and
the corresponding experimental chemical shifts (δexp).[13]
1
ton (9.54 ppm) shows intramolecular H–H1 NOE inter-
action with the two protons on the benzene ring at 7.86
and 7.48 ppm, respectively. Because the position of H-2
(7.86 ppm) has been assigned, it can be considered that
the doublet peak at 7.48 ppm is ascribed to H-6, and the
other doublet peak at 6.96 ppm is directly attributed to
H-4. Reasonably, the two singlet peaks at 9.54 and
10.91 ppm are identified as H-7 and H-9/H-11, respec-
tively. Further observation from the NOESY spectrum
reveals that H-2 and H-6 have similar correlation peaks
with H-7, which is believed to be caused by the flexible
rotation of the C1–N7 bond in solution. Therefore, the
NOESY spectrum only gave the average results and could
not tell which distance is longer.
The further assignment of the tautomeric form could
be accomplished from the NOESY spectroscopy
(Figure 2). If fenobam tautomeric I exists as the main
form in solution, we should observe an intramolecular
NOE cross peak between H-9 and H-14 in the NOESY