1H, 13C and 15N NMR spectral analysis of substituted 1,2,3,4-tetrahydro-pyrido[1,2-a]pyrimidines

Article abstract of DOI:10.1002/mrc.4005

The NMR spectroscopic data of a series of thirty-four 3-acylpyrido[1,2-a] pyrimidinium salts are analyzed, which were prepared as either perchlorates or chlorides. Methyl group substituted 3-aroyltetrahydropyrido[1,2-a]pyrimidines with the methyl substituent in positions 6, 8 and 9 as well as both in positions 6 and 8 were investigated bearing various aroyl substituents. Unequivocal assignment of all resonances was achieved via two-dimensional ¹H,¹H-COSY measurements, ¹H,¹³C and ¹H,¹⁵N HSQC as well as HMBC experiments, and important diagnostic CH and NH couplings in the heteroaromatic ring system are evaluated. The influence of the methyl substituents was analyzed on the proton, carbon and nitrogen shifts. A significant effect of the counter ion on some chemical shifts of the nuclei under discussion of the pyridopyrimidines is found, allowing the indirect detection of the anion, which is confirmed by direct measurement of the ³⁵Cl nucleus of the perchlorates. Copyright 2013 John Wiley & Sons, Ltd. The ¹H, ¹³C and ¹⁵N NMR spectroscopic data of a series of thirty-four 3-acylpyrido[1,2-a]pyrimidinium salts are analyzed, which were prepared as either perchlorates or chlorides They contain methyl substituent(s) in positions 6, 8 and 9 as well as both in positions 6 and 8. Important CH and NH long range couplings and significant differences of the chemical shifts depending on either the position of the methyl group or the counter ion were evaluated. Copyright

Full text of DOI:10.1002/mrc.4005

Research article
Received: 5 July 2013
Revised: 5 August 2013
Accepted: 9 August 2013
Published online in Wiley Online Library: 2 September 2013
(wileyonlinelibrary.com) DOI 10.1002/mrc.4005
1
13
15
H, C and N NMR spectral analysis of
substituted 1,2,3,4-tetrahydro-pyrido
1,2-a]pyrimidines
[
Ulrich Girreser,* Ullvi Bluhm, Bernd Clement and Dieter Heber
The NMR spectroscopic data of a series of thirty-four 3-acylpyrido[1,2-a]pyrimidinium salts are analyzed, which were prepared
as either perchlorates or chlorides. Methyl group substituted 3-aroyltetrahydropyrido[1,2-a]pyrimidines with the methyl
substituent in positions 6, 8 and 9 as well as both in positions 6 and 8 were investigated bearing various aroyl substituents.
1
1
1
13
1
15
Unequivocal assignment of all resonances was achieved via two-dimensional H, H-COSY measurements, H, C and H, N
HSQC as well as HMBC experiments, and important diagnostic CH and NH couplings in the heteroaromatic ring system are
evaluated. The influence of the methyl substituents was analyzed on the proton, carbon and nitrogen shifts. A significant ef-
fect of the counter ion on some chemical shifts of the nuclei under discussion of the pyridopyrimidines is found, allowing the
3
5
indirect detection of the anion, which is confirmed by direct measurement of the Cl nucleus of the perchlorates. Copyright ©
013 John Wiley & Sons, Ltd.
2
Supporting information may be found in the online version of this article.
1
13
15
35
Keywords: structure analysis; pyrido[1,2-a]pyrimidines; NMR spectroscopy; H NMR; C NMR; N NMR; Cl NMR; counter ion effects
achieve precipitation of the pyridopyrimidines 4–8 during the
synthetic protocol. In total, 34 compounds (Fig. 1) were analyzed,
Introduction
Some years ago, we presented a simple synthetic approach to
and the variability for this data set is generated by (i) varying
aroyl substituents in position 3, (ii) the position of the methyl
group(s), (iii) the different counter ions perchlorate or chloride,
(iv) the varying concentrations of the samples (a factor of 2),
and (v) the different lipophilicity of the structures in the range
enone Mannich bases 2 via condensation of aromatic ketones 1
[
1]
with paraformaldehyde (Scheme 1). These enone Mannich bases
[
2]
could be condensed with a number of bifunctional nucleophiles
[3]
[4]
like amidines or aminopyridines in order to access the corre-
sponding heterocyclic derivatives such as tetrahydropyrimidines
or pyrido[1,2-a]pyrimidines, some structures showing promising
[
6]
of 1.4 to 2.8. Only the unsubstituted 1,2,3,4-tetrahydro-pyrido
1
13
[1,2-a]pyrimidinium bromide has been reported with H and
NMR shifts without assignment.
C
[
5]
[7]
pharmacological properties. Consequently, the approach to
pyrido[1,2-a]pyrimidines was exploited for the search of new NO
[6]
synthase inhibitors. A great number of structures were made
available by using methyl-substituted aminopyridines as precursors
to afford pyridopyrimidines with varying aroyl substituents in posi-
tion 3 and methyl groups in positions 6, 8 and 9 (structures 5–8)
and 6,8-position (pyrimidines 8); additionally, pyridopyrimidines 4
without methyl group were synthesized (Scheme 1). The products
were isolated as either hydroperchlorate or hydrochloride salts.
Experimental
The synthesis and the description of other physical and spectroscopic
properties of the 1,2,3,4-tetrahydro-pyrido[1,2-a]pyrimidines are
described elsewhere.
gated: 3-benzoyl-1,2,3,4-tetrahydro-2H-pyrido[1,2-a]pyrimidine
hydroperchlorate (4a), 1,2,3,4-tetrahydro-3-(4-methylbenzoyl)-2H-
pyrido[1,2-a]pyrimidine hydroperchlorate (4b), 3-(4-bromobenzoyl)-
[
3,6]
The following compounds were investi-
1
They were characterized by their H NMR spectra, the conventional
electron impact mass spectra and elemental analysis, or high
resolution mass spectroscopy. Furthermore, the lipophilicity of
1
3
,2,3,4-tetrahydro-2H-pyrido[1,2-a]pyrimidine hydrochloride (4c),
-(3,4-dichlorobenzoyl)-1,2,3,4-tetrahydro-2H-pyrido[1,2-a]pyrimidine
[
6]
these structures was determined. Some members show a remark-
able NOS inhibition; however, the selectivity for the three isoforms
hydrochloride (4d), 1,2,3,4-tetrahydro-3-(3,4-dimethoxybenzoyl)-2H-
pyrido[1,2-a]pyrimidine hydroperchlorate (4e), 3-(4-fluorobenzoyl)-
(
nNOS, iNOS and eNOS) is not in a range to propose further
[
6]
structural development.
1
1
,2,3,4-tetrahydro-2H-pyrido[1,2-a]pyrimidine hydroperchlorate (4f),
,2,3,4-tetrahydro-3-(3,4,5-trimethoxybenzoyl)-2H-pyrido[1,2-a]
Continuing our investigations, we considered heteronuclear
chemical shifts as an important tool for the analysis of the
tetrahydro-pyrido[1,2-a]pyrimidines 4–8, so all structures were
investigated using standard NMR pulse techniques to assign,
evaluate and compare the homo- and heteronuclear spectra of
these compounds. Of special interest was the effect of substitu-
* Correspondence to: Ulrich Girreser, Pharmaceutical Institute, Department of
Pharmaceutical Chemistry, Christian-Albrechts-University of Kiel, Gutenbergstraße
76, D-24118 Kiel, Germany. E-mail: girreser@pharmazie.uni-kiel.de
1
5
tion on the N NMR shifts and the influence of the counter ion,
Pharmaceutical Institute, Department of Pharmaceutical Chemistry, Christian-
Albrechts-University of Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany
as either hydrochloric acid or perchloric acid was necessary to
Magn. Reson. Chem. 2013, 51, 714–721
Copyright © 2013 John Wiley & Sons, Ltd.

1
13
15
H, C and N NMR spectral analysis of substituted 1,2,3,4-tetrahydro-pyrido[1,2-a]pyrimidines
3-(4-cyanobenzoyl)-1,2,3,4-tetrahydro-8-methyl-2H-pyrido[1,2-a]py-
rimidine hydroperchlorate (6m), 3-[4-(4-cyanobenzyloxy)benzoyl]-
1,2,3,4-tetrahydro-8-methyl-2H-pyrido[1,2-a]pyrimidine
hydroper
chlorate (6n), 3-[4-(4-chlorobenzyloxy)benzoyl]-1,2,3,4-tetrahydro-8-
methyl-2H-pyrido[1,2-a]pyrimidine hydroperchlorate (6o), 3-[4-(4-
Bromobenzyloxy)benzoyl]-1,2,3,4-tetrahydro-8-methyl-2H-pyrido [1,2-a]
pyrimidine hydrochloride (6p), 3-benzoyl-1,2,3,4-tetrahydro-9-
methyl-2H-pyrido[1,2-a]pyrimidine hydroperchlorate (7a), 1,2,3,4-
tetrahydro-9-methyl-3-(4-methylbenzoyl)-2H-pyrido[1,2-a]pyrimidine
hydroperchlorate (7b), 1,2,3,4-tetrahydro-3-(3,4-dimethoxybenzoyl)-
Scheme 1. Synthesis of substituted 1,2,3,4-tetrahydro-pyrido[1,2-a]
pyrimidines 4–8.
9-methyl-2H-pyrido[1,2-a]pyrimidine hydroperchlorate (7c), 1,2,3,4-
tetrahydro-6,8-dimethyl-3-(4-phenylbenzoyl)-2H-pyrido[1,2-a]
pyrimidine hydroperchlorate (8a), 3-[4-(4-bromophenyl)benzoyl]-
1,2,3,4-tetrahydro-6,8-dimethyl-2H-pyrido[1,2-a]pyrimidine hydroper
chlorate (8b), 1,2,3,4-tetrahydro-6,8-dimethyl-3-[2-(6-methylnaph
thoyl)]-2H-pyrido[1,2-a]pyrimidine hydroperchlorate (8c) and
1
,2,3,4-tetrahydro-6,8-dimethyl-3-[2-(6-methylnaphthoyl)]-2H-
pyrido[1,2-a]pyrimidine hydrochloride (8d).
NMR measurements – general
All spectra were obtained on a Bruker Avance III 300 spectrometer
(
Bruker, Rheinstetten, Germany) with a variable temperature unit
[8]
at 300 K calibrated with methanol-d4 with a multinuclear probe
head using the manufacturer’s pulse programs described later on.
All spectra were recorded in DMSO-d as solvent. The compounds
6
are not sufficiently soluble in other solvents like CDCl or D O.
3
2
Between 8 and 25 mg of the pyridopyrimidinium salt was used in
0
.5 ml of solvent. Furthermore, 2μl of nitromethane were added as
15
a 20% solution in DMSO-d as internal standard for the N NMR
measurements (30.4MHz) with N NMR δ = 0.0ppm, affording an
additional signal at 4.46 ppm ( H) and 63.5 ppm ( C). For the more
6
15
1
13
lipophilic structures, heating of the samples for complete dissolu-
1
13
tion was necessary. H (300 MHz) and C (75.4 MHz) spectra were
1
Figure 1. Structures of pyridopyrimidines 4–8 investigated.
referenced to internal DMSO-d ( H NMR δ 2.50 ppm) and internal
5
13
19
DMSO-d
ments, external CFCl
the second largest peak); for Cl NMR measurements (29.4 MHz),
6
( C NMR δ 39.5ppm). For F NMR (282 MHz) measure-
19
pyrimidine hydrochloride (4g), 1,2,3,4-tetrahydro-6-methyl-3-(4-
methylbenzoyl)-2H-pyrido[1,2-a]pyrimidine hydroperchlorate (5a),
3 6
in DMSO-d was used ( F δ 0.0ppm using
35
35
1,2,3,4-tetrahydro-3-(3,4-dimethoxybenzoyl)-6-methyl-2H-pyrido[1,2-
external NaCl (0.1M in D
2
O, Cl NMR, δ 0.0ppm) was used. All
a]pyrimidine hydroperchlorate (5b), 3-(4-fluorobenzoyl)-1,2,3,4-
tetrahydro-6-methyl-2H-pyrido[1,2-a]pyrimidine hydroperchlorate
coupling constants (J values) are quoted in Hz.
(
5c), 1,2,3,4-tetrahydro-6-methyl-3-(4-phenylbenzoyl)-2H-pyrido[1,2-
1
13
H NMR and C NMR measurements
a]pyrimidine hydrochloride (5d), 1,2,3,4-tetrahydro-6-methyl-3-[2-
(
(
6-methylnaphthoyl)]-2H-pyrido[1,2-a]pyrimidine hydroperchlorate
5e), 1,2,3,4-tetrahydro-8-methyl-3-benzoyl-2H-pyrido[1,2-a]pyrimi-
Conventional one-dimensional proton and proton-decoupled
carbon spectra using a 30° flip angle were measured. A gradient-
1
1
dine hydroperchlorate (6a), 3-(4-chlorobenzoyl)-1,2,3,4-tetrahydro-
8
1
a]pyrimidine hydroperchlorate (6c), 1,2,3,4-tetrahydro-[2-(6-
methoxynaphthoyl)]-8-methyl-3-2H-pyrido[1,2-a]pyrimidine
enhanced double-quantum filtered nonphase sensitive H,
COSY spectrum with 512 t increments was recorded in order to
evaluate the coupling proton and unresolved diagnostic H,H long
H
-methyl-2H-pyrido[1,2-a]pyrimidine
hydroperchlorate
(6b),
1
,2,3,4-tetrahydro-8-methyl-3-[2-(6-methylnaphthoyl)]-2H-pyrido[1,2-
1
13
range couplings. For the recording of H, C correlations, gradi-
ent-enhanced HSQC spectra were used with carbon decoupling
hydroperchlorate
tetrahydro-8-methyl-2H-pyrido[1,2-a]pyrimidine hydroperchlorate
6e), 3-(4-chloro-3-methylbenzoyl)-1,2,3,4-tetrahydro-8-methyl-2H-
(6d),
3-[4-(4-bromophenyl)benzoyl]-1,2,3,4-
with 512 t
tions per t
1
increments, an interpulse delay of 1.5s and two repeti-
increment. For the recording of gradient-enhanced
1
1
13
13
(
H, C HMBC spectra without C decoupling, the pulse program
pyrido[1,2-a]pyrimidine hydroperchlorate (6f), 3-(3-fluoro-4-
methoxybenzoyl)-1,2,3,4-tetrahydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine hydroperchlorate (6g), 3-[4-(4-cyanophenyl)benzoyl]-
with two low-pass filters was employed with 256 t increments,
1
an interpulse delay of 1.5 s and eight repetitions per t increment.
1
Delays were optimized for a coupling constant of 8 Hz. The determi-
nation of CH long range coupling constants was achieved via the
EXSIDE pulse program with selective excitation using a shaped
pulse with a length of 50ms providing sufficient selectivity in most
cases; in a few cases, a length of 120 ms was necessary. 1024 t1
increments, an interpulse delay of 2 s, four or eight repetitions per
t1 increment and a J factor of 30 were used.
1
,2,3,4-tetrahydro-8-methyl-2H-pyrido[1,2-a]pyrimidine
hydrochloride (6h), 1,2,3,4-tetrahydro-3-[4-(4-methoxyphenyl)ben-
zoyl]-8-methyl-2H-pyrido[1,2-a]pyrimidine hydroperchlorate (6i), 3-(4-
cyclohexylbenzoyl)-1,2,3,4-tetrahydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine hydroperchlorate (6k), 3-(4-carboxylatobenzoyl)-1,2,3,4-
tetrahydro-8-methyl-2H-pyrido[1,2-a]pyrimidine hydrochloride (6l),
Magn. Reson. Chem. 2013, 51, 714–721
Copyright © 2013 John Wiley & Sons, Ltd.
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U. Girreser et al.
1
5
N NMR measurements
heterocyclic skeleton is delocalized on positions 5, 6, 8 and 9a, lead-
ing to a high-frequency shift of the corresponding protons H-6 of
about 1.2 ppm and H-8 of about 1 ppm, compared with H-7 and
H-9, which resonate at 6.9ppm (Table 1). We have shown before
that the exchangeable proton is bound to N-1; there is no evidence
15
N data are obtained via indirect detection of proton reso-
1
15
nances. So, for the recording of H, N gradient-enhanced HSQC
spectra, a sweep width of 550 ppm, 1024 t1 increments, an
acquisition time of 0.24 s, an interpulse delay of 1.5 s and two
[4]
for tautomeric isomers. The signal of this NH proton is usually
repetitions per t increment were employed. Resolution enhance-
1
rather broad with line widths of several Hz up to 50 Hz (the broad-
ening originating presumably on impurities like traces of excess of
acid or water or exchange processes); in some cases, a triplet struc-
ture can be anticipated, in those cases the corresponding couplings
ment was achieved via linear prediction. The calibration of this
1
15
H, N experiment was performed with the signal of the internal
1
15
standard nitromethane in the subsequently measured H, N
HMBC: For the recording of this gradient-enhanced HMBC
spectra without decoupling with 512 t
tion time of 0.12 s, an interpulse delay of 1.5 s, and 16 or 24
repetitions per t
1
1
1
13
1
15
of this NH proton can be observed in the H, H, H, C and H, N
correlations as well. The aliphatic part of the heterocycle is forming
a partially overlapping ABMNX spin system, which was not investi-
gated in detail; the chemical shifts of the corresponding protons
could be sometimes only unraveled using the results of the
1
increments, an acquisi-
15
1
increment were necessary. Here, also a
N
sweep rate of 550 ppm and resolution improvement via linear
prediction were employed.
1
13
H, C HSQC experiment.
The aromatic ring protons of the heterocycle show extensive
coupling, typical coupling constants, which are found in the
1
9
F NMR measurements
1
9
1
one-dimensional proton spectra of 4 in the range of 6.2–6.9 Hz
Conventional one-dimensional F spectra with and without H
decoupling were measured with a sweep rate of 300 ppm and
the acquisition of 128 k data points. Sixty-four repetitions were
accumulated with an interpulse delay of 1.5 s.
3
3
3
[
J(H-6,H-7)], 6.8–7.0 Hz [ J(H-7,H-8)] and 8.4–8.9 Hz [ J(H-8,H-9)].
Additionally, long range couplings in the order of 1.0–1.5 Hz
4
4
[
J(H-6,H-8)] and 0.5–1.2 Hz [ J(H-7,H-9)] are found. An
5
unresolved J coupling of H-6 with H-9 is observed in the H,
H-COSY spectrum of 4, too. For the methyl-substituted
pyridopyrimidines 5, 6 and 7, the J coupling constants were
3
5
Cl NMR measurements
3
35
Conventional one-dimensional Cl spectra were measured with
a sweep rate of 1500 ppm, acquisition of 8800 data points and
an acquisition time of 100 ms. One thousand repetitions were
accumulated with an interpulse delay of 100 ms.
found to be in the same range. For structures 5, 7 and 8, the
4
4
J coupling constants were unresolved; for 6, this J coupling
constant value was about 1.5–1.8 Hz.
The signal broadening of the aromatic protons of 5 to 8 is
originated by a long range quartet splitting due to the methyl
protons in the ring. For one derivative of 6, this coupling constant
could be determined to be 0.8 Hz. The corresponding H,H-COSY
spectra do of course show all these long range couplings for
the tetrahydropyridopyrimidines 5 to 8. Also, crosspeaks for an
Mass spectrometry
Electrospray ionization mass spectra were recorded with a Bruker
Esquire ~LC in the positive ion mode. Samples were applied via
chromatographic separation on an RP 8 column with a 0.1%
aqueous acetic acid/methanol gradient. The molecular ion was
subjected to a second fragmentation step in the ion trap,
delivering the fragment ions given in the supplementary
experimental part.
7
interesting J coupling of the methyl protons in position 8 of 6
and 8 with the aliphatic protons in position 4 can be found.
The effect of the methyl group on the chemical shifts of the
aromatic ring protons H-6 to H-9 is summarized in Table 3, where
the average shifts of the hydroperchlorates of structures 4 to
8
are compared. The additivity of the methyl substitution effect
in positions 6 and 8 (pyrimidines 5 and 6) with the dimethyl de-
rivatives 8 is also clearly visible (Table 3).
Results and Discussion
The results of the NMR experiments, especially the chemical
shifts, are presented in tables for the different heteroaromatic
1
3
C NMR data
1
13 15
35
nuclei ( H, C, N and Cl; Tables 1 and 2); the complete listing
of all chemical shifts are given in the supporting information
section for each compound, together with the results of the mass
spectroscopic analysis. The basic statistical evaluation of the
chemical shifts is also given in the supporting information section
with the results of the Shapiro–Wilk test for normal distribution, F
or Levene’s test for homogeneous variances, and student’s t test,
Welch’s t test or the Mann–Whitney rank sum test for significant
differences, depending on the results for normality and
homoscedasticity testing.
The focus of this discussion is on the aromatic carbon chemical shifts
of the heterocyclic part of the substituted pyridopyrimidines 4–8.
The following pattern is recognizable for these carbon atoms, and
the chemical shifts are summarized in Table 2. C-7 and C-9 resonate
at low frequency of about 110–114 ppm and the partially positively
charged carbon centers C-6 and C-8 at about 140 ppm; the
annulated carbon atom C-9a is observed at 150 ppm. Introducing
a methyl group in positions 6, 8 or 9 leads to a general high-
frequency shift of about 10 ppm of the connected carbon center,
[
9]
a typical substituent shift for this group. The additivity of the
1
methyl substitution effect in positions 6 and 8 (pyrimidines 5 and
H NMR data
13
6) on the C chemical shifts compared with the dimethyl deriva-
The chemical shifts of the protons of the aroyl substituent are also
given in the supporting information section and are not discussed
any further; the substituent in position 3 of the pyridopyrimidinium
moiety has no detectable influence on the chemical shifts of the
aromatic protons of the heterocycle. The positive charge in the
tives 8 is again clearly visible (Table 4).
For the unequivocal assignment, HSQC and HMBC spectra
were recorded; for the pyridopyrimidines, a large number of CH
long range couplings are observable, which are too numerous
to be summarized in a single graphical representation. In general,
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Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2013, 51, 714–721

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H, C and N NMR spectral analysis of substituted 1,2,3,4-tetrahydro-pyrido[1,2-a]pyrimidines
1
Table 1. Selected H NMR shifts for pyridopyrimidines 4–8
Comp.
Anion
H-6
6-Me
H-7
H-8
8-Me
H-9
9-Me
NH
4À
4
4
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
7
7
7
8
8
8
8
a
b
e
f
ClO
8.03
8.01
8.01
8.02
8.00
8.01
8.06
6.87
6.86
6.86
6.85
6.86
6.84
6.84
6.81
6.81
6.80
6.83
6.81
6.72
6.73
6.76
6.75
6.74
6.73
6.73
6.74
6.73
6.73
6.73
6.73
6.71
6.72
6.70
6.86
6.85
6.85
6.71
6.71
6.71
6.80
7.83
7.78
7.79
7.80
7.81
7.79
7.78
7.70
7.70
7.69
7.72
7.70
6.99
6.99
7.00
7.05
6.99
7.16
7.19
6.87
6.87
6.86
6.90
6.97
6.82
6.78
6.79
6.78
6.77
6.80
6.76
6.77
6.75
6.81
6.76
6.76
6.95
6.91
6.90
9.05
9.35
9.40
9.61
9.35
10.00
10.18
9.29
9.30
9.28
9.36
9.66
9.44
9.28
9.22
9.20
9.28
9.35
9.14
9.13
9.16
9.38
9.19
9.17
10.00
9.88
9.83
8.54
8.55
8.56
9.21
9.13
9.14
9.71
4À
4À
4À
ClO
ClO
ClO
À
c
Cl
À
d
g
a
b
c
Cl
À
Cl
4À
4À
4À
4À
ClO
ClO
ClO
ClO
2.51
2.52
2.51
2.53
2.53
e
d
a
b
c
À
Cl
4À
4À
4À
4À
4À
4À
4À
4À
4À
4À
4À
4À
ClO
ClO
ClO
ClO
ClO
ClO
ClO
ClO
ClO
ClO
ClO
ClO
7.92
7.91
7.93
7.93
7.92
7.91
7.90
7.93
7.91
7.91
7.90
7.91
7.98
7.94
7.92
7.97
7.95
7.95
2.30
2.31
2.33
2.33
2.32
2.31
2.32
2.32
2.32
2.31
2.32
2.32
2.30
2.30
2.30
d
e
f
g
i
k
m
n
o
h
l
À
Cl
À
Cl
À
p
a
b
c
Cl
4À
4À
4À
4À
4À
4À
ClO
ClO
ClO
ClO
ClO
ClO
7.73
7.72
7.73
2.17
2.17
2.18
a
b
c
2.50
2.49
2.48
2.48
2.28
2.28
2.28
2.26
6.68
6.66
6.67
6.69
À
d
Cl
the aliphatic protons of the heterocycle show crosspeaks with
the other aliphatic carbon atoms and the carbon atom of the
carbonyl group, which resonates at around 196 ppm. H-2
couples with C-9a and H-4 with C-6. For the pyridopyrimidines
pyridine are reported to be À0.9 Hz for H-5 with C-2 and
[10c]
À1.7 Hz for H-6 with C-3.
In order to substantiate the discus-
sion on diagnostic CH long range couplings, the following values
[
11]
were determined for 4a using the EXSIDE pulse program,
a
4
–8, which have a suitably resolved triplet signal for the NH
two-dimensional technique, starting with a selective pulse on
one proton and determining the coupling constant by the split-
ting of the carbon resonances in the indirect dimension. This
technique uses indirect detection via a double INEPT transfer
proton in position 1, CH long range couplings to C-9 and C-9a
are found.
For the pyridopyrimidinium salts 4, the following couplings are
observed for the aromatic moiety part of the heterocycle: H-6
couples with C-4, C-7, C-8, C-9 (w, weak) and C-9a; H-7 couples
with C-6, C-9 and C-9a (w); H-8 couples with C-6, C-9 (w) and
C-9a; and finally, H-9 couples with C-4 (w), C-6 (w), C-7, C-8 (w)
and C-9a. The assignment of a crosspeak as a weak signal is
based on the investigation of the one-dimensional projection of
the column of the respective proton in the two-dimensional
spectrum; here, signals with less than about 10% of the intensity
of the major peaks are denominated weak. Reference values for
the unsubstituted pyridinium ion as model compound are
[
11]
with gradient enhancement
samples already analyzed. Recently, the usage of a proton and
and allows the usage of the
[
12]
carbon selective version (Selexside) was published. Thus, the
following values were obtained for 4a: H-6 couples with C-4
(4.1Hz), C-7 (3.2 Hz), C-9 (ca. 1 Hz) and C-9a (7.2Hz); H-7 couples
with C-6 (6.3Hz), C-8 (<2 Hz), C-9 (7.9 Hz) and C-9a (<2 Hz); and
H-8 couples with C-6 (9.4Hz), C-7 (<2 Hz), C-9 (0.9Hz) and C-9a
(9.1Hz). Finally, H-9 shows CH long range couplings to C-4
(<2 Hz), C-6 (<2 Hz), C-7 (7.4Hz), C-8 (<2 Hz) and C-9a (3.1 Hz).
In the 6-methyl-substituted derivatives 5, crosspeaks of the
protons of the methyl group with C-4 (w), C-6 and C-7 are
observed. H-7 couples with C-6, 6-Me and C-9. H-8 couples also
with C-6, C-7 (w), C-9 and C-9a. H-9 shows coupling to C-4 (w),
C-6 (w), C-7, C-8 (w) and C-9a.
[
10]
reported by Kalinowski et al. ; they give the following coupling
constants: 3.3 Hz for J(H-3,C-2) and 5.1 Hz for J(H-2,C-3).
Couplings over three bonds are in the range of 6.1 to
2
2
[10a]
[10b]
7
.2 Hz.
Couplings over four bonds in the unprotonated
Magn. Reson. Chem. 2013, 51, 714–721
Copyright © 2013 John Wiley & Sons, Ltd.
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U. Girreser et al.
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Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2013, 51, 714–721

1
13
15
H, C and N NMR spectral analysis of substituted 1,2,3,4-tetrahydro-pyrido[1,2-a]pyrimidines
1
Table 3. Summary of average H NMR chemical shifts for the
different substitution pattern in 4 to 8
Eventually, the CH long range couplings of the pyridopyrimidines
bearing two methyl groups are analyzed. The protons of the
8
methyl group in position 6 couple with C-4, C-6, C-7 and C-9 (w),
and H-7 couples with C-6, 6-Me, 8-Me and C-9; the protons of the
methyl group in position 8 exhibit couplings to C-7, C-8 and C-9,
and H-9 couples to C-7, 8-Me and C-9a. For one example, 8c,
the following coupling constants were determined: The protons
of the methyl group in position 6 couple to C-4 (<2 Hz), C-6
(6.8 Hz), C-7 (4.7 Hz), C-8 (<2Hz) and C-9 (<2 Hz), and H-7
couples to C-6 (5.1 Hz), 6-Me (3.7 Hz), C-8 (<2 Hz), 8-Me (4.4 Hz),
C-9 (5.0 Hz) and C-9a (<2 Hz); the protons of the methyl group
in position 8 couple to C-7 (6.6Hz), C-8 (4.6 Hz) and C-9 (5.7 Hz),
and H-9 couples to C-6 (<2 Hz), C-7 (6.9 Hz), C-8 (<2 Hz) and
C-9a (3.5 Hz).
Comp.
H-6/6-Me
H-7
H-8/8-Me H-9/9-Me
NH
4
5
Δ
6
Δ
7
Δ
8
Δ
Δ
(n = 4)
8.02
2.52
6.86
6.81
7.80
7.70
7.01
6.88
9.35
9.31
(n = 4)
(n = 12)
(n = 3)
(n = 3)
À0.05
6.74
À0.10
2.32
À0.13
6.78
À0.04
9.24
7.92
À0.10
7.96
À0.12
6.85
À0.23
2.17
À0.11
8.55
7.73
À0.07
2.28
À0.03
2.49
À0.01
6.72
À0.80
9.16
6.68
À0.33
À0.36
À0.14
À0.17
À0.19
À0.15
calculated
So, the heterocyclic framework shows many diagnostic CH
long range couplings, besides the usually large coupling
constants over three bonds; the large coupling constants over
two bonds in the vicinity of the positively charged heteroatom
have to be considered for this class of compounds as well.
Only the perchlorates were compared; Δ is the difference in
chemical shift to the unsubstituted pyrimidine 4; and Δcalculated
is the calculated difference for substituted pyrimidines 8 using
the incremental shift values of 5 and 6.
In the same way, the crosspeaks of pyridopyrimidines 6 in the
15
N NMR data
HMBC spectra are given: H-6 couples with C-4, C-7, C-8 and C-9a;
H-7 couples with C-6, 8-Me, C-9 and C-9a (w); the protons of the
methyl group in position 8 couple with C-7, C-8 and C-9; and
finally, H-9 shows couplings with C-4 (w), C-7, 8-Me and C-9a.
Exemplarily for 6o, the following values for the coupling
constants were determined: H-6 with C-4 (4.1 Hz), C-7 (2.8 Hz),
C-8 (ca. 8 Hz), C-9 (<2 Hz) and C-9a (7.3 Hz); H-7 with C-6
1
5
N NMR spectroscopy has developed to a routinely employed
[
13]
technique in structure elucidation and analysis,
in natural products, and in the investigation of drugs and drug
decomposition products
for example
[
14]
[
15]
despite its inherent low sensitivity
that is overcome by indirect proton detection. So, all 34
compounds were investigated by proton-detected HSQC and
HMBC experiments; the chemical shifts were evaluated by the
resolution-enhanced two-dimensional data sets, whereby
nitromethane was employed as internal standard (Fig. 2). For
nearly all structures, two signals are observed in the range of
À220 to À228 ppm and À290 to À294 ppm, corresponding to
the charged pyridinium nitrogen atom and the amino function
(Tables 2 and 4). Here, also the comparison with literature data
is helpful. Städeli et al. reported chemical shifts of the protonated
2-aminopyridine of À229.5 and À292.6 ppm in fluorosulfonic
acid and À226.0/-305.6 ppm in trifluoroacetic acid relative to
(
5.7 Hz), C-8 (<2 Hz), 8-Me (3.5 Hz), C-9 (6.8 Hz) and C-9a (<2 Hz);
and H-9 with C-4 (<2 Hz), C-6 (<2 Hz), C-7 (7.9 Hz), C-8 (<2 Hz),
-Me (4.9 Hz) and C-9a (2.3 Hz). The protons of the methyl group
8
couple with C-7 (4.7 Hz), C-8 (6.5 Hz) and C-9 (approx. 5 Hz).
For the pyridopyrimidinium salts 7, the following CH long
range couplings are observed in general: H-6 couples with C-4,
C-7, C-8, C-9 (w) and C-9a; H-7 with C-6, C-9 and C-9a (w); and
H-8 with C-6, 9-Me and C-9a. The protons of the methyl group
couple with C-6 (w), C-7 (w), C-8, C-9 and C-9a. For 7c, the follow-
ing values for the CH long range coupling constants were deter-
mined: H-6 with C-4 (3.6 Hz), C-7 (3.1 Hz), C-8 (8.6 Hz), C-9 (0.7 Hz)
and C-9a (7.2 Hz); H-7 with C-6 (5.6 Hz), C-8 (<2 Hz), C-9 (7.7 Hz)
and C-9a (<2 Hz); and H-8 with C-6 (8.0 Hz), C-7 (<2 Hz), C-9
[
16]
neat external nitromethane.
by subtraction of 2 ppm to values relative to nitromethane in
DMSO. More recently, Beltrame et al. investigated the proton-
These values can be referenced
[
17]
(
<2 Hz), 9-Me (5.3 Hz) and C-9a (9.3 Hz). The protons of the
methyl group in position 9 show the following couplings to C-6
<2 Hz), C-8 (5.5 Hz), C-9 (6.7 Hz) and C-9a (4.2 Hz).
ation of monoaminopyridines, and they reported values relative
[
18]
to deuterated N,N-DMF,
which requires re-referencing. For N,
(
N-DMF-d , LeGoff reported a chemical shift of 105 ppm at
7
13
15
Table 4. Summary of average C and N NMR chemical shifts for the different substitution pattern in 4 to 8
N-1
C-2
C-3
C-4
N-5
C-6
6-Me
C-7
C-8
8-Me
C-9
9-Me
C-9a
4
5
Δ
6
Δ
7
Δ
8
Δ
Δ
À291.0
À291.3
À0.3
40.4
40.4
0.0
35.0
35.5
0.5
51.1
46.9
À4.2
50.6
À0.5
51.6
0.5
À225.3
À222.2
+3.1
139.5
148.2
8.7
112.0
113.0
1.0
141.2
140.6
À0.6
153.2
12.0
114.4
112.2
À2.2
112.6
À1.8
123.0
8.6
151.0
152.0
1.0
19.4
À293.7
À2.7
40.3
À0.1
41.4
1.0
35.3
0.3
À229.1
À3.8
138.7
À0.8
137.4
À2.1
147.3
7.8
114.2
2.2
20.9
20.7
150.6
À0.4
150.1
À0.9
151.6
0.6
À292.6
À1.6
35.1
0.1
À225.1
0.2
111.8
À0.2
115.1
3.1
140.1
À1.1
152.4
11.2
16.5
À293.5
À2.5
40.3
À0.1
À0.1
35.9
0.9
46.5
À4.6
À4.7
À225.9
À0.6
19.2
110.6
À3.8
À4.0
calculated
À3.0
0.8
À0.7
7.9
3.2
11.4
0.6
Only the perchlorates were compared; Δ is the difference in chemical shift to the unsubstituted pyrimidine 4; and Δcalculated is the calculated
difference for substituted pyrimidines 8 using the incremental shift values of 5 and 6.
Magn. Reson. Chem. 2013, 51, 714–721
Copyright © 2013 John Wiley & Sons, Ltd.
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U. Girreser et al.
Figure 3. Graphical representation of diagnostic NH long range
couplings in pyridopyrimidines 4–8 (w, weak).
1
15
Figure 2.[ H, N HMBC spectrum of the most potent NOS inhibitor in
6]
6
the series 6e in DMSO-d .
impossible to detect. Only as a perchlorate, the chlorine
resonance can be detected easily even in organic solvents; so,
the measurement of this signal at about +1010 ppm relative to
the chlorine in sodium chloride in water was employed to proof
the identity of the perchlorate anion. This perchlorate signal is
of course not influenced by the type of compound and the
substitution position of the methyl group (Table 2).
[
19]
3
ppm
53 K,
and Kohlemainen gave a shift for N,N-DMF of 103.8
[
20]
(obviously, both values are referenced to the ammonia
scale); these values can be recalculated to values relative to
nitromethane in DMSO
[
17]
affording a chemical shift of DMF
versus nitromethane of À277.2 and À278.4 ppm, respectively.
We measured a value of À276.6ppm for the chemical shift of
6
undeuterated N,N-DMF versus internal nitromethane in DMSO-d ,
which is in accordance with the above reported values. So, the
shifts of Beltrame reported for a protonated 2-aminopyridine in
Influence of the counter ion
The comparison of chemical shifts of pyrimidinium perchlorates
versus chlorides requires some statistical calculations. All results
are summarized in the supporting information section in
tables 1–10. For these calculations, the rounded numbers in
tables 1 and 2 are used; chemical shift differences that are
CDCl
3
/DMSO-d
6
7/3 are calculated to be À228.1 and À302.5ppm.
Therefore, the description of the pyrido[1,2-a]pyridines as deriva-
tives of alkylated aminopyridinium ions is confirmed also by the
chemical shifts of the N nuclei, as Bertrame et al. proved the
protonation of the pyridine nitrogen in their investigation.
The effect of methyl group substitution on the chemical shift of
the partially positively charged nitrogen atom N-5 can be
summarized as +3, approx. 0 and À4 ppm for the methyl group
in ortho, meta and para positions, of the nitrogen, corresponding
to the pyridopyrimidines 5, 7 and 6, when compared with
structures 4 (Table 4). For uncharged pyridine and quinoline, this
methyl group substituent effect has been reported to be in the
order of +4.9, 0, +8.3 ppm and +4.9, +0.2, +5.3 ppm, respec-
15
1
13
15
below 0.02 ( H), 0.1 ( C) and 0.4 ppm ( N) are considered
15
to be near the experimental error (especially for the
N
NMR results, which are extracted from two-dimensional spectra).
For the pyridopyrimidines 4–8, the relevant and significant differ-
1
13
15
ences in H, C and N NMR shifts that are observed are summa-
rized in Table 5.
A diagnostic difference can be observed in general for NH, H-9,
N-1 and C-8; the difference is smaller and not uniformly observed
for all pyridopyrimidines for N-5 and other proton and carbon
shifts. It can be speculated that the NH function is more involved
in any ion pairing process in solution; this might explain the
strong effects on chemical shifts on this part of the molecule,
especially a Δδ value of about 0.4 ppm for NH and a Δδ of
[21]
tively.
So, the shielding effect of the additional methyl group
in ortho position and the negligible effect in meta position is also
observed for the pyridopyrimidines 4–8; however, the interesting
deshielding of the methyl group para to the nitrogen might re-
quire a more theoretical analysis. The effect of the methyl group
substitution on the chemical shift of the amine function is of
course much smaller; in all cases, methyl group substitution leads
to a more or less pronounced deshielding (À0.3 to À2.7 ppm).
0
.1 ppm for H-9 are observed. The group of Burgess et al.
1
15
investigated H and N NMR chemical shift changes upon salt
formation for simple bases like pyridine and reported greater
[22]
1
chemical shift changes with stronger acids
; however,
For nearly all structures, the J coupling constant for H with N-1
perchloric acid can be considered as the stronger acid, also in
DMSO; so, a greater shift compared with the unprotonated base
was determined from the HMBC spectrum as a rough estimate in
the range of 92 to 95 Hz, proving the strong involvement of the
nitrogen lone pair in mesomeric stabilization of the positive charge.
The observed diagnostic NH long range couplings are
summarized in graphical form; as in the case of CH long range
correlations, only sufficiently narrow signals for the NH proton
led to observable correlations in the H,N HMBC spectra (Fig. 3).
should be observed for the perchlorate. Similarly, Somoshekar
[23]
et al.
reported on the influence of the counter ion (maleate,
mesylate and chloride) for trimipramine hydrochloride in CDCl
and derived a linear correlation between pk value and chemical
shift difference compared with the nonprotonated base for N in
3
a
15
[23]
the range of Δδ = 0.5 ppm per pk
a
unit.
When taking a pk
a
value for À7 for hydrochloric acid and À10 for perchloric acid,
15
3
5
a difference for the N resonance of N-1 in the range of
.5 ppm would be expected, but in this case, the chlorides show
Cl NMR data
1
The chlorine nucleus is usually of no importance in structure
elucidation; because of the quadrupole moment, it exhibits broad
lines, which are of no value in the investigation of organic
structures. Also, as a counterion for organic salts like the
the greater shift difference (calculating a chemical shift by
referencing to nitromethane in DMSO of about À315.6 ppm for
[
18]
nonprotonated aminopyridine ). Recently, Metaxas and Cohrt
reported on interesting counter ion effect in strychnine salts
1
13
pyrimidinium ions in DMSO-d , the surrounding of the nucleus
effecting H and C shifts in CDCl3 and CD OD, but not in
3
6
[
24]
is not symmetric, leading to broad lines, which are difficult or
D2O.
Vinalryay and Pandiarajan discussed solvent-free and
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Magn. Reson. Chem. 2013, 51, 714–721

1
13
15
H, C and N NMR spectral analysis of substituted 1,2,3,4-tetrahydro-pyrido[1,2-a]pyrimidines
Table 5. Relevant and significant chemical shift differences for pyridopyrimidines 4–6 and 8 between perchlorates and chorides, p < 0.05, if not
stated otherwise
Comp.
Atom (chemical shift difference Δδ in ppm)
4
5
6
8
NH (Δδ = 0.44, p = 0.11), H-9 (Δδ = 0.10, p = 0.11), N-1 (Δδ = 1.3, p = 0.14), N-5 (Δδ = 0.6), C-8 (Δδ = 0.2)
NH (Δδ = 0.35), H-9 (Δδ = 0.10), N-1 (Δδ = 1.0), N-5 (Δδ = 0.3), C-8 (Δδ = 0.2)
NH (Δδ = 0.66), N-1 (Δδ = 2.1), N-5 (Δδ = 0.5), C-7 (Δδ = 0.2), C-8 (Δδ = 0.4)
NH (Δδ = 0.55), H-7 (Δδ = 0.09), H-9 (Δδ = 0.02, p = 0.074), N-1 (Δδ = 2.0), C-3 (Δδ = 0.2), C-8 (Δδ = 0.4)
solvent containing ion pairing for picrates, nitrates and chlorides of
to be mentioned. The perchlorate anion could be determined
directly by measurement of the Cl resonance.
[
25]
35
protonated substituted piperidinium ions in different solvents.
On the other hand, Barczynski reported small counter ion effects
1
13
of H and C nuclei within experimental errors of complexes of var-
[
26]
ious acids with 1-methylquinolinium-3-carboxylate, a zwitter ion.
References
So, to date, there is no general predictability of the influence of the
counter ion in different solvents and depending on the acid
strength of the conjugate anion. However, the chemical shift
differences of the pyridopyrimidines 4–8 investigated here, at
least for some nuclei, are sufficiently large to allow the indirect
detection of the type of counter ion, namely either the chloride
or perchlorate anion.
[
[
1] U. Girreser, D. Heber, M. Schütt. Synthesis 1998, 30, 715–717.
2] U. Girreser, D. Heber. J. Prakt. Chem. 2000, 342, 230–234.
[3] U. Girreser, D. Heber, M. Schütt. Synthesis 1999, 31, 1637–1641.
[
[
4] U. Girreser, D. Heber, M. Schütt. Synlett 1998, 9, 263–264.
5] U. Bluhm, J.-L. Boucher, B. Clement, U. Girreser, D. Heber,
B. Ramassamy, U. Wolschendorf. J. Het. Chem. in press.
6] U. Bluhm, J.-L. Boucher, U. Buss, B. Clement, F. Friedrich, U. Girreser,
D. Heber, T. Lam, M. Lepoivre, M. Rostaie-Gerylow, U. Wolschendorf.
Eur. J. Med. Chem. 2009, 44, 2877–2887.
[
[
[
7] R. Paira, A. Maity, S. Mondal, S. Naskar, K. B. Sahu, P. Saha, A. Hazra,
E. Padmanaban, S. Bannerjee, N. B. Mondal. Tetrahedron Lett. 2011,
Mass spectrometry
5
2, 1653–1657.
8] M. Findeisen, T. Brand, S. Berger. Magn. Reson. Chem. 2007, 45,
75–178.
For the assignment and proof of structure, mass spectra after
electrospray ionization in the positive ionization mode were
measured, and the base peak, which was the protonated
molecular ion, was fragmented in a second step (MS/MS) in the
ion trap. This way, the information about the number of methyl
groups of the pyrido[1,2-a]pyrimidine skeleton was confirmed,
but the position of the methyl group could not be determined.
For the smaller pyridopyrimidines, the defragmentation of the
protonated pyrimidine can be explained via a simple retro
Diels–Alder reaction, delivering the uncharged enone together
with an o-chinoid heterocyclic structure bearing none, one or
two methyl substituents.
1
[9] E. Pretsch, P. Bühlmann, C. Affolter, M. Badertscher, Spektroskopische
Daten zur Strukturaufklärung organischer Verbindungen, 4th edn,
Springer, Berlin, 2001.
1
3
[
[
10] H.-O. Kalinowski, S. Berger, S. Braun,
C-NMR-Spektroskopie,
Thieme, Stuttgart, 1984; a) p. 472, b) p. 489, c) p. 491.
11] V. V. Krishnamurthy. J. Magn. Res. Ser. A 1996, 121, 33–41.
[12] C. P. Butts, B. Heise, G. Tatolo. Org. Lett. 2012, 14, 3256–3259.
13] R. Marek, A. Lyčka. Curr. Org. Chem. 2002, 6, 35–66.
[
[
15
14] G. E. Martin, M. SoIntseva, A. J. Williams. N NMR spectroscopy,
in Modern Alkaloids, (Ed: E. Fattorusso, O. Taglialatele-Scafati),
Wiley-VCH, Weinheim, 2008, pp. 409–471.
[15] A. Czyrski, U. Girreser, T. Hermann. J. Mol. Struct. 2013, 1036,
11–114.
16] W. Städeli, W. von Philipsborn, A. Wick, I. Kompiš. Helv. Chim. Acta
980, 63, 504–522.
17] R. M. Claramunt, D. Sanz, C. Lopéz, J. A. Jiménez, M. L. Jimeno,
J. Elguero, A. Fruchier. Magn. Res. Chem. 1997, 35, 35–75.
18] P. Beltrame, E. Cadoni, C. Floris, G. Gelli, A. Lai. Spectrochim. Acta A
1
[
[
[
[
[
1
Conclusion
A series of 1,2,3,4-tetrahydro-pyrido[1,2-a]pyrimidinium per-
chlorates and chlorides, with and without methyl substituents
in the unsaturated part of the heterocycle, were analyzed,
2
002, 25, 2639–2697.
19] G. Le Goff, M. T. Martin, C. Servy, S. Cortial, P. Lopes, A. Bialecki,
J. Smadja, J. Ouazzani. J. Nat. Prod. 2012, 75, 915–919.
20] E. Kolehmainen, Novel Applications of Dynamic NMR in Organic
Chemistry, in Annual Reports on NMR Spectroscopy, vol 49,
1
13 15
35
affording a robust data set of H, C, N and Cl chemical shifts
1
13
1
15
6
in DMSO-d . H, C and H, N long range couplings were inves-
tigated, which are an important tool in structure elucidation.
Exemplarily, for a few compounds, the CH long range coupling
constants were determined using the exside pulse program.
(
Ed: G. Webb), Academic Press, U.K., 2003, pp. 2–41.
[
21] L. Stefaniak, J. D. Roberts, M. Witanowski, G. A. Webb. Org. Magn. Res.
1984, 22, 201–208.
1
5
Significant differences for the N chemical shifts of N-5 were
found, depending on the position of the methyl substituent.
Large and significant shift differences were found for all the
nuclei under investigation when comparing perchlorates with
chlorides; here, especially at the NH signal, N-1, C-8 and H-9 have
[22] H. Kim, J. Gao, D. J. Burgess. Int. J. Pharm. 2009, 377, 105–11.
23] B. S. Somashekar, G. A. Nagana Gowda, A. R. Ramesha, C. L. Khetrapal.
Magn. Res. Chem. 2005, 43, 166–170.
24] A. E. Metaxas, J. R. Cort. Magn. Res. Chem. 2013, 51, 292–298.
25] V. Vimalraj, K. Pandiarajan. Magn. Res. Chem. 2011, 49, 682–687.
[26] P. Barczynski, M. Szafran. Polish J. Chem 2009, 83, 1061–1074.
[
[
[
Magn. Reson. Chem. 2013, 51, 714–721
Copyright © 2013 John Wiley & Sons, Ltd.
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