1H, 13C and 15N NMR assignments of phenazopyridine derivatives

Article abstract of DOI:10.1002/mrc.1542

Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were studied by one- and two-dimensional ¹H, ¹³C and ¹⁵N NMR methodology. The assignments, combined with DFT calculations, re

Full text of DOI:10.1002/mrc.1542

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2005; 43: 256–260
Published online 29 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1542
Note
1H, 13C and 15N NMR assignments of phenazopyridine
derivatives
1
2
2
´
˜
´
Eleuterio Burgueno-Tapia, Yolanda Mora-Perez, Martha S. Morales-Rıos and
Pedro Joseph-Nathan2∗
1
´
´
´
Departamento de Quımica, Unidad Profesional Interdisciplinaria de Biotecnologıa, Instituto Politecnico Nacional, Av. Acueducto s/n, La Laguna
Ticoma´ n, Me´ xico D.F., 07340 Mexico
2
´
´
´
´
Departamento de Quımica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Apartado 14-740, Mexico D.F., 07000
Mexico
Received 7 September 2004; Revised 26 October 2004; Accepted 27 October 2004
Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were
studied by one- and two-dimensional 1H, 13C and 15N NMR methodology. The assignments, combined with
DFT calculations, reveal that the preferred protonation site of the drug is the pyridine ring nitrogen atom.
The chemoselective acetylation of phenazopyridine (2) and its influence on the polarization of the azo
nitrogen atoms were evidenced by the 15N NMR spectra. Molecular calculations of the phenazopyridines
2–4 show that the pyridine and phenyl groups are oriented in an antiperiplanar conformation with
intramolecular hydrogen bonding between the N-b atom and the C-2 amino group preserving the E-azo
stereochemistry. Copyright  2004 John Wiley & Sons, Ltd.
KEYWORDS: NMR; 1H NMR; 13C NMR; 15N NMR; 2D NMR; phenazopyridine derivatives
INTRODUCTION
A large group of compounds formed by azo coupling
of benzenediazonium salts with aromatic amines and
phenols are described as dyestuffs.1 The structures of these
compounds can differ considerably; some are stable as the
azo tautomer, whereas others prefer the hydrazo form or
1
appear as mixtures of both tautomers.2–4 Detailed H, 13C
and 15N interpretations have been used as an important tool
for the study of azo–hydrazo tautomerism processes.1,5,6
Some dyes have been used in medicine, a typical example
being phenazopyridine hydrochloride (1), which has been in
use for many decades as a drug to reduce pain, burning
and discomfort associated with urinary tract infections
or irritation. It is also frequently used in combination
with sulfonamides to treat infections of the urinary tract.7
NMR studies have so far been reported. Consequently, we
Phenazopyridine hydrochloride (1), when ingested orally,
became interested in studying this molecule and some of its
goes via the bloodstream to the kidneys, whence it is
derivatives, and in this paper we report the assignments
eliminated through urine, to which it confers a marked
of the 1H, 13C and 15N NMR signals at the natural
abundance level of these molecules, which, combined with
DFT calculations, were used to confirm the protonation
preference of the drug.
spectacular orange–red color.
The first synthesis of 1, by coupling diazotized aniline
with ˛,˛-diaminopyridine, was reported in 1914,8 and
surprisingly, despite its wide use, no 1H, 13C or 15N
ŁCorrespondence to: Pedro Joseph-Nathan, Departamento de
RESULTS AND DISCUSSION
´
Quımica, Centro de Investigacio´n y de Estudios Avanzados del
The 1H NMR spectrum (Table 1) of phenazopyridine
hydrochloride (1), obtained in DMSO-d6 from a very pure
sample provided by the pharmaceutical industry, showed
Instituto Polite´cnico Nacional, Apartado 14-740, Me´xico D.F., 07000
Mexico. E-mail: pjoseph@nathan.cinvestav.mx
Contract/grant sponsor: CONACYT; Contract/grant number:
G-32631-N.
Copyright  2004 John Wiley & Sons, Ltd.

1H, 13C and 15N NMR assignments of phenazopyridines 257
Table 1. 1H NMR data and HMBC correlations for phenazopyridine derivatives 1–4a
1
2
3
4
H
υ (J, Hz)
HMBC υ (J, Hz)
HMBC υ (J, Hz)
7.98 (d, 8.5)
HMBC
υ (J, Hz)
HMBC
4
5
o
8.29 (d, 8.4)b
6.39 (d, 8.4)
7.96 (brd, 8.1)
2, 6
7.72 (d, 8.7)
2, 3, 5, 6 8.10 (d, 8.8)
3
i, o, m
2, 6
3
i, p
3, 6
6.03 (d, 8.7)
3, 6
7.51 (d, 8.5)
8.04 (d, 8.8)
7.90 (br d, 8.2)
i, o, m
7.69 (brd, 8.0)
i, o, p
7.82 (brd, 8.0)
m
p
7.50 (brdd, 8.1, 7.4) i, o, m
7.38 (brt, 7.4)
7.42 (brdd, 8.0, 7.4) i, m
7.26 (brt, 7.4) o, m
7.47 (brdd, 8.0, 7.2) i, o, m
7.38 (brt, 7.2)
7.60 (brdd, 8.2, 7.3) i, o
7.56 (br t, 7.3)
o
o
o
a 300 MHz, DMSO-d6, TMS as internal standard. 1: NH2-2 (8.8, brs), NH2-6 (3.7, brs), py-H (8.7, brs). 2: NH2-6 (6.74, brs). 3: NHAc-6
(2.08 s, 10.32 s). 4: NHAc-2 (2.26 s, 10.31 s), NHAc-6 (2.17 s, 10.74 s).
b J4,5 obtained from the spectrum with a trace amount of HCl.
Table 2. 13C NMR data for phenazopyridine derivatives 1–4a
the benzene ring signals at υ 7.96 (brd, 8.1 Hz), 7.50 (brdd,
8.1, 7.4 Hz) and 7.38 (brt, 7.4 Hz), easily assigned from their
relative intensities and multiplicities to H-o, H-m and H-p,
respectively. The pyridine ring signals H-4 and H-5 were
observed as fairly broad signals at υ 8.29 (brs, W1/2 D 25 Hz)
and 6.39 (brd, W1/2 D 7 Hz, J D 8.4 Hz), respectively. Three
signals of protons directly bonded to nitrogen atoms also
appear as very broad signals at υ 8.8 (brs, 2H, W1/2 D 87 Hz),
8.7 (brs, 1H, W1/2 D 25 Hz) and 3.7 (brs, 2H, W1/2 D 270 Hz),
suggesting that neither amino group is protonated due to
the formation the hydrochloride. The ambient probe tem-
perature proton decoupled 13C NMR spectrum of 1, also
obtained in DMSO-d6, showed only eight signals at υ 156.2,
149.5, 135.6, 129.2, 128.4, 122.2, 120.5 and 99.6, of which only
two were sharp peaks. The W1/2 values, obtained from the
full linewidths at half-height, were 179.7, 196.7, 134.3, 2.4,
123.2, 2.8, 84.6 and 280.0 Hz, respectively, providing T2Ł val-
ues of 0.011, 0.010, 0.015, 0.833, 0.016, 0.714, 0.024 and 0.007 s,
respectively, from the relationship9 W1/2 D 2/T2Ł. In order to
find the expected ninth signal, the 13C NMR spectrum was
C
1b
2
3
4
2
3
4
5
6
i
o
m
p
151.6
122.1
136.0
101.7
156.2
149.7
120.6
128.9
128.4
153.6
124.0
138.1
100.0
160.9
152.7
121.0
129.0
127.7
152.1
127.8
137.0
102.6
153.3
152.3
121.8
129.1
129.7
148.2
134.7
127.6
110.1
153.0
152.1
122.6
129.3
131.2
a 75.4 MHz, DMSO-d6, TMS as internal standard. 3: NHAc-6
(24.2, 169.5). 4: NHAc-2 (24.0, 169.3), NHAc-6 (24.2, 169.6).
b
°
Measured at 60 C.
of 2 occurs at N-6. In contrast, using DFT calculations, we
estimated the relative stabilities of the three protonated
species 1, 1a and 1b and compared the values with that
calculated for phenazopyridine (2). The EDFT values for 1,
1a, 1b and 2 are ꢀ439 205.9, ꢀ439 197.1, ꢀ439 189.3 and
ꢀ438 965.9 kcal molꢀ1, respectively (1 kcal D 4.184 kJ). Since
the protonation energy, to estimate the protonation of
phenazopyridine (2) at different sites, is obtained from the
differences between the total energy of 2 and the total energy
of 1, 1a and 1b (EprotC is defined as the negative proton
°
obtained at 60 C, whereby the broad signal at υ 149.5 was
shifted and split into two broad signals at υ 151.6 and 149.7.
The W1/2 values now were 36.8, 64.8, 31.2, 34.2, 1.9, 11.2,
Ł
1.9, 13.1 and 68.3 Hz, respectively, providing T2 values of
0.054, 0.031, 0.064, 0.058, 1.053, 0.179, 1.053, 0.153 and 0.029 s.
Other 13C NMR signals, in particular C-5, also underwent
°
some shifts, in the spectrum measured at 60 C, to provide
the values given in Table 2. Addition of a trace amount of
HCl to the NMR solution permitted the room temperature
observation of the 13C NMR signals as sharp peaks at υ 157.8,
153.8, 148.6, 135.4, 129.7, 128.8, 122.4, 120.6 and 104.4. The line
broadening might be due to slow equilibria between species,
which become fast upon addition of a trace amount of acid.
The 13C NMR data assignment was achieved using one- and
two-dimensional NMR experiments, including gHSQC, and
gHMBC measurements.
Results of ab initio calculations and the differences
in relative stabilities between protonated species and the
corresponding free amino group have been used to predict
protonation sites of aminopyridines, since net electronic
charge values are not adequate to predict different basicities
of nitrogen atoms.10,11 The net electronic charge values
obtained by density functional theory (DFT)12 for N-1, N-
2, N-6, N-a and N-b in 2 are ꢀ0.573, ꢀ0.795, ꢀ0.818, ꢀ0.198
and ꢀ0.293 e, respectively, which suggest that protonation
affinity),13 it follows that Eprotꢀ2 ꢀ 1ꢁ D ꢀ240.0 kcal molꢀ1
,
Eprotꢀ2 ꢀ 1aꢁ D ꢀ231.2 kcal molꢀ1 and Eprotꢀ2 ꢀ 1bꢁ D
ꢀ223.4 kcal molꢀ1. These data allow us to conclude that
the structure of phenazopyridine hydrochloride is 1, a
conclusion which is in agreement with the 1H NMR
spectrum, wherein three signals of hydrogen atoms bonded
to nitrogen are observed (Table 1). As can be seen in the
molecular structure obtained from DFT calculations (Fig. 1),
the amino group at C-2 is involved in stabilization of a
coplanar arrangement of the pyridine and benzene rings due
to formation of an intramolecular hydrogen bond with the
N-b atom. The calculated distance between NH-2 and N-b is
˚
1.88 A. The equation for the plane defined by all carbon and
nitrogen atoms, obtained by using the Parst program from
the WinGX system,14 is 0.315x ꢀ 0.662y ꢀ 0.680z D 0.011. The
proton involved in the hydrogen bonding deviates by only
˚
0.001 A from this plane, which is meaningless.
Copyright  2004 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: 256–260

258
E. Burguen˜o-Tapia et al.
99% in favor of the NH/N-1-syn, NH/Me-syn conformer,
which is represented by 3 in Fig. 1. This is in concordance
with the W-type coupling between the amido proton and the
C-4 signals observed in the gHMBC experiment. As in the
case of 2, no 1H NMR signal was observed for NH2-2.
On the other hand, in the case of 4, the unmistakable
assignment of the two acetamido groups at C-2 and C-
6 could be achieved with the aid of gHMBC experiment.
Thus, the observed correlations between the amido signal at
υ 10.74 and the C-5 signal at υ 110.1, and correlations between
the amido signal at υ 10.31 and the C-2 and C-3 signals,
allowed assignment of the amido groups. Assuming the
calculated conformation of the acetamido group at C-6 as in
3, and the hydrogen bonding between the C-2-NH and N-b,
two minimum energy conformations were calculated having
the carbonyl group coplanar to the phenazopyridine atoms.
The DFT total energy values for these conformers were
ꢀ630 554.0 (C-2-NH/Me-anti) and ꢀ630 552.4 (C-2-NH/Me-
syn) kcal molꢀ1. From these values, as above, was deduced
the equilibrium is shifted 95% in favor of the C-2-NH/Me-
anti conformer, represented by 4 in Fig. 1. Complete 1H and
13C NMR data assignments (Tables 1 and 2, respectively)
were achieved with the aid of COSY, NOESY, gHSQC and
gHMBC experiments.
Although phenazopyridine hydrochloride (1) contains
five nitrogen atoms, its low solubility in DMSO precluded
the determination of its 15N NMR spectrum. In contrast, in the
case of 2 the 15N resonances could be obtained from gHMQC
and gHMBC experiments optimized to detect one-bond
1JꢀN,Hꢁ D 90 Hz and long-range nJꢀN,Hꢁ D 3 Hz coupling
constants. However, the gHMQC experiment reveled the
presence of only one amino group signal at υ ꢀ293.4, which
was assigned to NH2-6 from its correlation with the signal at υ
6.74, and no signal for NH2-2 could be detected. The gHMBC
(1H,15N) measurements allowed detection of the pyridine
ring nitrogen at υ ꢀ156.3 from its correlation to H-5 at υ
6.03, while the N-a and N-b nitrogen atoms were assigned at
υ C102.0 and C48.1 from their respective correlations with
H-4 at υ 7.72 and H-o at υ 7.69. The chemical shift assignments
of all nitrogen atoms of 3 and 4 were straightforward from
the corresponding gHMQC and gHMBC experiments to the
values given in Table 3. The observed 15N chemical shifts of
2–4 are in agreement with the donor- or acceptor-substituted
pyridine moiety. The acetylation of each amino group on
the pyridine ring shifts N-b some 35 ppm to lower field,
evidencing that the electron-withdrawing effect of the N-
acetyl groups reduces or prevents the conjugation of the
acetamido nitrogen electron lone pair with the pyridine
moiety.
1
2
EDFT = -439205.9 kcal/mol
EDFT = -438965.9 kcal/mol
3
4
EDFT = -534762.2 kcal/mol
EDFT = -630554.0 kcal/mol
Figure 1. Density functional theory (B3LYP/6–31GŁ) minimum
energy molecular models of phenazopyridine derivatives 1–4.
Treatment of 1 with base afforded 2, whose 1H NMR sig-
nals, obtained in DMSO-d6, were straightforwardly assigned.
Thus, the pyridine ring signals H-4 and H-5 were assigned at
υ 7.72 (d, 8.7 Hz) and υ 6.03 (d, 8.7 Hz), respectively, whereas
the H-o, H-m and H-p signals were observed at υ 7.69, (brd,
8.0 Hz), 7.42 (brdd, 8.0, 7.4 Hz) and 7.26 (brd, 7.4 Hz), respec-
1
tively. The H NMR spectrum of 2 shows only one amino
group signal at υ 6.74 (brs), which could be assigned after
inspection of the gHMBC contour plot (Table 1) to NH2-6,
since it shows a clear correlation with the C-5 signal at υ
100.0. The failure to detect the NH2-2 signal can be attributed
to the weak and significantly exchange-broadened signal15
of the amino protons. The 13C NMR spectrum of 2 shows
the expected nine signals (Table 2), from which the proto-
nated carbons at υ 138.1, 129.0, 127.7, 121.0 and 100.0 were
unambiguously assigned, using the gHSQC experiment, to
C-4, C-m, C-p, C-o and C-5, respectively. The remaining qua-
ternary carbons were assigned on the basis of their chemical
shifts and with the aid of a gHMBC experiment. Signals at
υ 160.9, 153.6, 152.7 and 124.0 were assigned to C-6, C-2, C-i
and C-3, respectively.
Reaction of 2 with acetic anhydride and pyridine at room
temperature for 3 h yielded 3 (79%) and 4 (12%). When the
reaction was left overnight, exclusively 4 was obtained in
88% yield. The assignment of the acetamido group at C-6,
for 3, was supported by gHMBC correlations between the
amido proton signal at υ 10.32 and the C-5 and C-6 signals at υ
102.6 and 153.3, respectively. In addition, a W-type coupling
between the NH-6 signal at υ 10.32 and C-4 was observed.
DFT calculations for 3 showed four minimum energy
conformations having the carbonyl group coplanar to the
phenazopyridine atoms, which were originated by rotations
at the C-6—NH and HN—CO bonds. The DFT total
energy values for these conformers were ꢀ534 762.2 (NH/N-
1-syn, NH/Me-syn), ꢀ534 758.0 (NH/N-1-syn, NH/Me-
anti), ꢀ534 755.1 (NH/N-1-anti, NH/Me-syn) and ꢀ534 759.5
(NH/N-1-anti, NH/Me-anti) kcal molꢀ1. From these values
EXPERIMENTAL
General
UV–visible and IR spectra were determined on Perkin-
Elmer UV/vis Lambda 12 and Perkin-Elmer 16F PC FT-IR
spectrophotometers, respectively. Low-resolution electron
ionization mass spectrometry (EIMS) was performed on
a Varian Saturn 2000 spectrometer at 70 eV. Melting-
points were determined on a Fisher-Johns apparatus and
°
and G D ꢀRT ln K it follows that the equilibrium is shifted
Copyright  2004 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: 256–260

1H, 13C and 15N NMR assignments of phenazopyridines 259
Table 3. 15N NMR data and HMBC (1H,15N) correlations for 2–4.a
2
3
4
N
υ
HMBC
υ
HMBC
υ
HMBC
1
2
6
a
b
ꢀ156.3
5, NH2(6)
ꢀ140.2
ꢀ298.4
ꢀ233.8
C111.3
C82.8
5, MeCONH-6
ꢀ113.4
ꢀ245.4
ꢀ243.2
C114.3
C116.3
5
b
—
4
4, MeCONH-2
4, 5, MeCONH-6
ꢀ293.4
C102.0
C48.1
4, 5
4
o
5, MeCONH-6
4
o
4
o
a From external neat MeNO2, υ15N D 0.
b Not observed.
1368 cmꢀ1. EIMS: m/z (relative intensity, %) 213 [M]C (100),
136 (21), 108 (29), 81 (19), 54 (15). 1H and 13C NMR: see
Tables 1 and 2, respectively.
are uncorrected. Molecular models were generated using
the MMX force-field,16 as implemented in the PCMODEL
program. The structures generated from the PCMODEL
program were optimized by DFT (B3LYP/6–31GŁ)11 using
the PC Spartan ’04 program from Wavefunction (Irvine, CA,
USA). Acetylations of 2 were performed in the usual manner
with acetic anhydride in pyridine.
6-Acetamido-2-amino-3-phenylazopyridine (3)
°
Pale-orange amorphous solid, m.p. 169–170 C. UV (EtOH):
ꢂmax (log ε) 248 (3.9), 270 (4.0), 306 (3.9), 404 (4.2). IR (CHCl3):
ꢃmax 3480, 3416, 3290, 1752, 1704, 1606, 1578, 1508 cmꢀ1. EIMS:
m/z (relative intensity, %) 255 [M]C (100), 213 (17), 150 (11),
136 (13), 108 (47), 81 (22), 43 (14). 1H and 13C NMR: see
Tables 1 and 2, respectively.
NMR spectra
Natural abundance 1H, 13C and 15N NMR spectra were
recorded on a Varian Mercury 300 instrument at 300, 75.4
and 30.4 MHz, respectively. Spectra were referenced to
TMS for 1H and 13C and to neat MeNO2, υ D 0, for 15N.
2,6-Diacetamido-3-phenylazopyridine (4)
Pale-yellow amorphous solid, m.p. 212–213 C. UV (EtOH):
1
The H,13C correlation experiments were performed using
°
standard manufacturer-supplied gHSQC17 and gHMBC18
pulse programs. Typically the proton spectral widths in the
gHSQC experiment were 12 820.5 Hz for F1 and 3601.0 Hz
for F2, the experiment being optimized for a 1J(C,H)
coupling of 140 Hz, whereas in the gHMBC experiment the
spectral widths for F1 and F2 were 18 115.9 and 3601.0 Hz,
ꢂmax (log ε) 244 (3.3), 262 (3.4), 298 (3.2), 378 (3.5). IR (CHCl3):
ꢃmax 3408, 1706, 1590, 1544, 1516, 1484 cmꢀ1. EIMS: m/z
(relative intensity, %) 297 [M]C (19), 213 (17), 254 (100), 212
1
(13), 150 (34), 108 (20), 77 (14), 43 (5). H and 13C NMR: see
Tables 1 and 2, respectively.
Acknowledgements
Financial support from CONACYT (G-32631-N) is acknowledged.
We thank Jorge Ebrard (Laboratorios Mixim SA de CV) and Aditivos
respectively, and the J(C,H) value set to 8 Hz. The H,15N
correlation experiments were performed using standard
gHMQC and gHMBC pulse sequences.18b,c,19 The HMQC
experiment was acquired with 1024 data points and spectral
widths of 2362.4 Hz for F2 and 54 945.1 Hz for F1, and was
n
1
´
y Farmoquımicos, SA de CV, Mexico City, for the generous supply
of compound 1.
1
optimized for a J(N,H) coupling of 90 Hz. The multibond
REFERENCES
correlation experiment was optimized for a long-range
coupling of 3 Hz. Data were acquired with 1024 points and
512 increments for time evolution, the number of scans per
increment was 64 and the delay between transients was
always 1 s.
1. Lycˇka A. Annu. Rep. NMR Spectrosc. 1993; 26: 247.
´
2. Beka´rek V, Doba´sˇ J, Socha J, Vetesˇnık P, Vecˇerˇa M. Collect. Czech.
Chem. Commun. 1970; 35: 1406.
3. Berrie AH, Hamnpson P, Longworth SW, Mathias A. J. Chem.
Soc. B 1970; 1308.
4. Beka´rek V, Rothschein K, Vetesnic P, Vecˇerˇa M. Tetrahedron Lett.
1968; 3711.
5. Lycˇka A, Snobl D, Macha´cˇek V, Vecˇerˇa M. Org. Magn. Reson.
Characterization data of phenazopyridine
derivatives
ˇ
1981; 15: 390.
2,6-Diamino-3-phenylazopyridine hydrochloride (1)
6. Lycˇka A, Jirman J, Schneider B, Strada J. Magn. Reson. Chem.
1988; 26: 507.
°
Dark-red microcrystals, m.p. 232–234 C. UV (EtOH): ꢂmax
7. Medline Plus. Online database produced by the National Library
of Medicine. Phenazopyridine. http://www.nlm.nih.gov/med-
line/druginformation.html and search Phenazopyridine
(Systemic), 2001.
8. Chichibabin AE, Zeide OA. J. Russ. Phys. Chem. 1914; 46: 1216.
9. Gu¨nther H. NMR Spectroscopy. Wiley: New York, 1980; 233.
10. Catala´n J, Mo´ O, Pe´rez P, Ya´n˜ez M. J. Am. Chem. Soc. 1979; 101:
6520.
11. Fossey J, Loupy A, Strzelecka H. Tetrahedron 1980; 37:
1935.
12. (a) Perdew JP. Phys. Rev. B 1986; 33: 8822; (b) Becke AD. Phys.
Rev. A 1988; 38: 3098.
(log ε) 242 (3.98), 277 (4.10), 306 (3.76), 413 (4.39). IR (KBr):
ꢃmax 3465, 3288, 3066, 2912, 1636, 1602, 1563, 1452, 1418, 1368,
1265 cmꢀ1. EIMS: m/z (relative intensity, %) 213 [M ꢀ HCl]C
1
(100), 184 (5), 136 (21), 108 (36), 81 (29), 54 (20). H and 13C
NMR: see Tables 1 and 2, respectively.
2,6-Diamino-3-phenylazopyridine (2)
Pale-orange amorphous solid, m.p. 136–137 C. UV (EtOH):
°
ꢂmax (log ε) 244 (3.95), 277 (4.08), 304 (3.75), 416 (4.39).
IR (CHCl3): ꢃmax 3518, 3474, 3414, 1612, 1584, 1538, 1486,
Copyright  2004 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: 256–260

260
E. Burguen˜o-Tapia et al.
13. Aue DH, Webb HM, Bowers MT. J. Am. Chem. Soc. 1976; 98: 311.
14. (a) Nardelli M. Comput. Chem. 1983; 7: 95; (b) Nardelli MJ. Appl.
Crystallogr. 1995; 28: 659; (c) Farrugia LJ. J. Appl. Crystallogr. 1999;
32: 837.
15. Anderberg PI, Luck IJ, Harding MM. Magn. Reson. Chem. 2002;
40: 313.
16. Burkert U, Allinger NL. Molecular Mechanics. ACS Monograph
177. American Chemical Society: Washington, DC, 1982.
17. (a) Kay LE, Keifer P, Saarinen T. J. Am. Chem. Soc. 1992; 114:
10 663; (b) Palmer III AG, Cavanagh J, Wright PE, Rance M.
J. Magn. Reson. 1991; 93: 151; (c) Kontaxis G, Stonehouse J,
Laur ED, Keeler J. J. Magn. Reson. A 1991; 111: 70.
18. (a) Bax A, Summers MF. J. Am. Chem. Soc. 1986; 108: 2093;
(b) Willker W, Leibfritz D, Kerssebaum R, Bermel W. Magn.
Reson. Chem. 1993; 31: 287; (c) Ruiz-Cabello J, Vuister GW,
Moonen CTW, van Gelderen P, Cohen JS, van Zijl PCM. J. Magn.
Reson. 1992; 100: 282.
19. Hurd RE, John BK. J. Magn. Reson. 1991; 91: 648.
Copyright  2004 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: 256–260