1. X-ray and high-resolution 1
H and 13
C NMR of
smooth muscle relaxant sesquiterpene lactones
Marie-Rose Van Calsteren, Christopher K. Jankowski, Ricardo Reyes-Chilpa,
Manuel Jiménez-Estrada, Maria G. Campos, Abraham Zarazua-Lozada,
Martha Oropeza, and Denis Lesage
Abstract: The structure and stereochemistry of four sesquiterpene lactones, budlein A (1), zaluzanin A (2), and
glaucolides D (3a) and E (3b), isolated from Mexican Asteraceae species, for which only partial NMR data could be
found in the literature, were determined. A combination of 1D and 2D high-resolution NMR experiments, such as
DEPT, COSY, NOESY, ROESY, HMQC, HSQC, and HMBC, were used to completely assign the 1
H and 13
C spectra.
The crystal structures of zaluzanin A (2) and glaucolide E (3b) were also determined. Glaucolides D and E have been
previously reported to relax KCl-induced contraction in rat uterine smooth muscle; therefore, the effects of zaluzanin A
and budlein A were examined in the same model. It was found that both compounds can relax contraction induced by
KCl, but only zaluzanin A induced relaxation when contraction was induced with oxytocin. The preliminary biological
test results according to these profiles are reported in this paper.
Key words: NMR data, X-ray data, sesquiterpene lactones, Mexican Asteraceae, smooth muscle relaxant.
Résumé : On a déterminé la structure et la stéréochimie de quatre lactones sesquiterpéniques, la budléine A (1), la
zaluzanine A (2), et les glaucolides D (3a) et E (3b), isolées d’espèces mexicaines d’Asteraceae, pour lesquelles
seulement des données de RMN partielles ont pu être trouvées dans la littérature. On a utilisé une combinaison
d’expériences de RMN à haute résolution 1D et 2D, telles que DEPT, COSY, NOESY, ROESY, HMQC, HSQC et
HMBC, pour assigner complètement les spectres de 1
H et 13
C. On a aussi déterminé la structure cristalline de la zalu-
zanine A (2) et du glaucolide E (3b). Précédemment, on avait rapporté que les glaucolides D et E relaxent la contrac-
tion induite par le KCl dans le muscle utérin lisse de rate; en conséquence, on a examiné les effets de la zaluzanine A
et de la budléine A dans le même modèle. On a trouvé que les deux composés peuvent relaxer la contraction induite
par le KCl, mais que seule la zaluzanine A induit la relaxation quand la contraction est induite avec l’ocytocine. On
présente les résultats préliminaires des tests biologiques appropriés dans cet article.
Mots-clés : données de RMN, données de rayons-X, lactones sesquiterpéniques, Asteraceae du Mexique, relaxant du
muscle lisse.
Van Calsteren et al. 1084
Introduction
Asteraceae species contain sesquiterpene lactones with in-
teresting biological properties (1); however, complete struc-
tural studies and stereochemistry are needed to understand
how they act. In this study, we isolated four sesquiterpenes,
budlein A (1), zaluzanin A (2), as well as the glaucolides D
(3a) and E (3b), for which only partial NMR data could be
found in the literature (Chart 1). The studied compounds
were isolated from Asteraceae species from Mexico. Con-
cerning the biological properties of these compounds, it has
been described that budlein A is cytotoxic against human tu-
mor cells L-929 and Hep-2, and it also shows antimicrobial
(2) and mild fungistatic properties (3). Budlein A and
zaluzanin A reduced in vitro human sperm motility by 60%
and 16%, respectively, 15 min after incubation, but did not
affect sperm viability. These two compounds also inacti-
vated the thiol groups on the outer membrane of sperm cells
Can. J. Chem. 86: 1077–1084 (2008) doi:10.1139/V08-158 © 2008 NRC Canada
1077
Received 5 March 2008. Accepted 14 June 2008. Published on the NRC Research Press Web site at canjchem.nrc.ca on
7 November 2008.
M.-R. Van Calsteren.1
Centre de recherche et de développement sur les aliments, Agriculture et Agroalimentaire Canada, Saint-
Hyacinthe, QC J2S 8E3, Canada.
C.K. Jankowski. Département de chimie et biochimie, Université de Moncton, Moncton, NB EIA 3E9, Canada.
R. Reyes-Chilpa and M. Jiménez-Estrada. Instituto de Química, Universidad Nacional Autónoma de México, Ciudad
Universitaria, México D.F. 04510, México.
M.G. Campos, A. Zarazua-Lozada, and M. Oropeza. Unidad de Investigación en Farmacología, Centro Médico Nacional Siglo
XXI, Instituto Mexicano del Seguro Social, Avenida Cuauhtemoc 330, México D.F. 06725, México.
D. Lesage. Synthèse, Structure et Fonction de Molécules Bioactives, groupe de spectrométrie de masse, UMR 7613, Université
Pierre et Marie Curie–Paris 6, CNRS, 75005 Paris, France.
1
Corresponding author (e-mail: vancalsteren@agr.gc.ca).

2. and have been proposed as potential male contraceptives (4).
Glaucolides D and E are able to relax rat smooth muscle; the
latter compound proved to be more potent than the former
relaxing KCl- or noradrenaline-induced contraction in aorta
and KCl-induced contraction in uterus (5).
The furanoheliangolide budlein A (1) was obtained from
Viguiera buddleiiformis (DC.) Hemsl. This species is a shrub
1–3 m in height with yellow flowers, purplish stems, and
whitened leaves beneath. It grows on rocky canyon slopes,
grassy openings among shrubs and small trees in tropical de-
ciduous, and oak forests or xerophytic vegetation at an alti-
tude of 1800–2500 m in the Sierra Volcánica Transversal. It
is known as “cerote” in the State of Hidalgo (6–8).
The guainolide zaluzanin A (2) was isolated from Zalu-
zania augusta (Lag.) Sch. Bip., a perennial shrub up to 2–
5 m in height with yellow flowers. It grows in the southern
part of the Central Plateau of Mexico. It is locally known as
“Limpia tunas” and “Caxtidán” in the State of Querétaro and
“Cenicilla” in the State of México (6–8). The leaves and
roots water infusion is used as an abortive by peasants of the
State of Querétaro, Mexico (9). There is some ambiguity in
the stereochemistry of 2 as determined by NMR (10); for
this reason, a detailed analysis of 1
H–1
H was performed, and
other evidence was provided by the HMBC experiment. The
structure of 2 was corroborated by means of its X-ray dif-
fraction pattern.
The glaucolides D (3a) and E (3b) were isolated from
Vernonia liatroides DC., a perennial shrub up to 3 m in
height with purple or white flowers. It grows with abun-
dance in openings in oak and pine forest at altitudes of
1000–2300 m in the northern and central parts of Mexico. It
is locally known as “Tlamalacatlacotli” and “Vara de San
Miguel”. In the State of Michoacan, it is a medicinal plant.
A water infusion of leaves and branches is prepared as a
remedy against dysentery. The mashed leaves are applied to
regenerate hair. It produces abundant pollen and nectar;
© 2008 NRC Canada
1078 Can. J. Chem. Vol. 86, 2008
Chart 1. Chemical structures.
Coupling constants (Hz)
Experimental Calculated Dihedral angles (°)
Proton δ Multiplicitya
from MM+ from MM+
H2 5.682 s
H5 6.222 dt 3
J5,6 4.2 5.0 –121
4
J5,15 –1.7 –1.1 –68, 172b
H6 5.380 m 3
J6,7 4.4 5.1 –126
H7 3.759 m 3
J7,8 2.2 1.9 –103
4
J7,13a –2.6 –1.8 –123c
H8 5.269 m 3
J8,9α 3.5 2.0 59
H9α 2.318 dd 2
J9α,9β –15.2
H9β 2.546 dd 3
J8,9β 5.6 4.3 –57
H13a 6.369 d 2
J13a,13b <1
H13b 5.707 d 4
J7,13b –2.4 –1.8 –123c
H14 (3H) 1.494 s
H15 (2H) 4.409 t 5
J6,15 1.9
H3 ′ 6.118 qq 3
J3′,3′-Me 7.3 5.9 –151, –31, 88
H2 ′-Me (3H) 1.799 quintet 4
J3′,2′-Me –1.5 –1.0 –89, 30, 150d
H3 ′-Me (3H) 1.936 dq 5
J2′-Me,3′-Me 1.5
a
Multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad.
b
H15–C15–C4–C3.
c
H7–C7–C11–C12.
d
H2 ′-Me–C2 ′-Me–C2 ′–C1 ′.
Table 1. 1
H NMR data (CDCl3) for budlein A 1.

3. therefore, it is important for local apiculture. The leaves can
be consumed by cattle (11). The structure of 3b was corrob-
orated by means of its X-ray diffraction pattern.
We have previously reported that glaucolides D and E,
sesquiterpene γ-lactones lacking an exocyclic methylene
double bond, can relax rat uterine smooth muscle contrac-
tion induced by KCl; this property was attributed to the exis-
tence of alternative alkylating sites such as C10 provided by
the germacra-1(10),4-diene-4-epoxide skeleton (5). In this
work, the effects of sesquiterpene lactones bearing an
exocyclic methylene double bond in the γ-lactone (budlein
A) and δ-lactone (zaluzanin A) moieties were examined us-
ing the same model.
Results and discussion
Budlein A
The 1
H NMR spectrum (Table 1) of 1 showed, among
others, the following signals: a pair of low-field doublets
at δ 6.37 and 5.71, characteristic of a γ-lactone conjugated
with an exocyclic methylene group; a doublet of triplets cen-
tered at δ 6.22 for the vinyl hydrogen H5; a quartet of
quartets at δ 6.12, a chemical shift typical of the vinyl proton
of angelic acid; two complex signals centered at δ 5.38
and 5.27 (H6 and H8); a triplet at δ 4.41 (2H) assigned to the
–CH2–O– protons of an allylic primary alcohol; a signal cor-
responding to H7 (δ 3.76); signals for the vinyl methyl
groups (δ 1.94 and 1.80) of the angelate ester moiety; a sin-
glet at δ 1.49 for the C14 methyl group. The stereochemistry
of 1 at C6 and C8 was established from the dihedral angles
(Table 1) calculated from the coupling constants. The calcu-
lated dihedral angle of –122° at the C6–C7 bond indicated a
β-configuration for H6 or a trans-lactone closure. The
configuration of the oxygen function at C8 was found to be
β-oriented, since the coupling constants between the pairs
H7–H8, H8–H9α, and H8–H9β have small values. This is in
agreement with the required dihedral angles (Table 1). The
ROESY spectrum displayed through-space correlations be-
© 2008 NRC Canada
Van Calsteren et al. 1079
1 2 3a 3b
Carbon δ Multiplicitya
δ Multiplicitya
δ Multiplicitya
δ Multiplicitya
C1 205.01 s 42.51 d 127.45 d 127.14 d
C2 104.92 d 37.02 t 68.22 d 68.29 d
C3 182.32 s 72.61 d 42.38 t 42.46 t
C4 135.66b
s 41.09 d 59.56 s 59.54 s
C5 134.64 d 92.94 s 66.23 d 66.36 d
C6 75.25c
d 66.38 d 82.02 d 82.18 d
C7 48.36 d 42.02 d 162.44 s 164.13 s
C8 74.02c
d 24.35 d 70.16 d 69.54 d
C9 42.15 t 22.58 t 45.84 br t 45.73 br t
C10 87.85 s 14.65 s 133.52 s 134.01 s
C11 138.50b
s 140.67 s 129.30 s 128.42 s
C12 168.76 s 168d
s 170.17 s 170.14e
s
C13 123.86 t 123.93 t 56.31 t 56.16 t
C14 21.30 f
q 23.35 q 17.90 br q 17.92 br q
C15 62.61 t 6.72 q 17.58 q 17.62 q
Angg
Emacg
Macg
C1′ 165.74 s 169.94 s 165.98 s
C2′ 126.28 s 53.39 s 135.07 s
C3′ 141.63 d 53.04 t 127.42 t
C2′-Me 20.06 f
q 17.11 q 18.11 q
C3′-Me 15.82 q
2-Ac
CO 170.25 s 170.29e
s
CH3 21.02 q 21.04 q
13-Ac
CO 170.35 s 170.41e
s
CH3 20.80 q 20.75 q
a
Multiplicity (determined by the DEPT experiment): s, singlet (quaternary); d, doublet (tertiary); t, triplet (secondary); q, quartet (primary); br, broad.
b
Assignments reversed in ref. (12).
c
Assignments reversed in ref. (12).
d
From HMBC.
e
Assignments are interchangeable.
f
Assignments reversed in ref. (13).
g
Ang, angelate; Emac, epoxymethacrylate; Mac, methacrylate.
Table 2. 13
C NMR data (CDCl3) for budlein A 1, zaluzanin A 2, and glaucolides D 3a and E 3b.

4. tween the following noncoupled nuclei: H6–H9β, H7–H9α,
H8–H13b, H9α–H14, and H9β–H14. The 13
C NMR spectrum
(Table 2) was essentially identical to that reported previ-
ously (12, 13), with some re-assignments based on direct
and long-range 1
H–13
C correlations. The NMR data are in
agreement with the stereochemistry deduced for goya-
zensolide from X-ray studies (14), this compound is the C8
α-methacrylate epimer of budlein A.
Zaluzanin A
The 1
H NMR spectrum of 2 exhibited the following sig-
nals (Table 3): two low-field signals at δ 6.15 and 5.54, cor-
responding to geminal olefinic protons; two broad signals, a
multiplet centered at δ 4.20 and a triplet at δ 3.94, attributed
to protons attached to carbons bearing hydroxyl groups; a
broad doublet at δ 3.15 assigned to an allylic proton; a sin-
glet at δ 1.06 and a doublet centered at δ 0.99 ascribed to a
tertiary and a secondary methyl group, respectively; three
protons, which exhibit signals to lower frequency, two dou-
blets of doublets at δ 0.86 and 0.63 and a doublet of doublets
of triplets at δ 0.57, indicative of a cyclopropane ring. Im-
portant differences can be noted between the 1
H (Table 3)
and 13
C (Table 2) NMR data compared with reported values
(10), such as reversed assignments for H8 and H9β as well as
C4 and C7. Biogenetic considerations suggest that the C7
side chain possesses a β-configuration. Therefore, O5 in-
volved in the six-membered lactone is also β-oriented, be-
cause it accepts a hydrogen bond from 3-OH, as indicated
by the large coupling constant between H3 and 3-OH, com-
pared with J6,6-OH, which shows an averaged value. The long-
range couplings exhibited by H6 to both H2α and H8 indicate
that it possesses the β-configuration, the hydroxyl group in po-
sition 6 being α-oriented. In the HMBC spectrum, H6 shows
a long-range coupling to C8 but not to C11, a further indica-
tion of its β-orientation. The NMR data are in agreement
with the stereochemistry deduced from X-ray studies of
this compound (Fig. 1). Previous correlations have been
done using X-ray stereodiagrams of zaluzanin B and a
diacetylated dibromated derivative of zaluzanin A (10).
© 2008 NRC Canada
1080 Can. J. Chem. Vol. 86, 2008
Coupling constants (Hz)
Experimental Calculated Dihedral angles (°)
Proton δ Multiplicitya
from MM+ from X-ray from MM+ from X-ray
H1 2.168 dd 3
J1,2α 9.0 5.6 6.1 –45 –42
3
J1,2β 13.3 11.8 11.4 –166 –162
H2α 2.670 dddd 3
J2α,3 7.8 9.7 9.6 11 14
5
J2α,6 0.9
H2β 1.828 td 2
J2α,2β –13.6
H3 4.195 m 3
J2β,3 4.2 5.0 5.6 132 135
H4 1.970 quintet d 3
J3,4 7.3 5.9 6.6 26 20
H6 3.945 br t 3
J6,7 2.8 2.8 2.0 64 71
4
J6,8 1.0 3.0 2.8 –171, 167b
–166, 164b
H7 3.147 br d 3
J7,8 1.0 1.9 1.3 –67 –73
H8 0.574 ddt 3
J8,9α 6.2 7.8 9.4 139 148
H9α 0.855 dd 2
J9α,9β –4.5
H9β 0.633 dd 3
J8,9β 9.7 10.3 10.4 –3 3
H13a 6.153 d 2
J13a,13b 1.1
H13b 5.544 dd 4
J7,13b –0.5 –0.3 –0.2 –161c
–165c
H14 (3H) 1.061 s
H15 (3H) 0.993 d 3
J4,15 7.3 6.5 6.4 –61, 58, 178 –62, 58, 178
3-OH 1.98 br d 3
J3,3-OH 10.7
6-OH 1.944 d 3
J6,6-OH 3.5
a
Multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad.
b
H6–C6–C7–C8 and H8–C8–C7–C6.
c
H7–C7–C11–C12.
Table 3. 1
H NMR data (CDCl3) for zaluzanin A 2.
Fig. 1. Crystal structure of zaluzanin A 2.

5. Glaucolide D
The 1
H NMR spectrum (Table 4) of 3a displayed the fol-
lowing features: a triplet of doublets at δ 5.57 (H2); a broad
doublet at δ 5.17 re-assigned from H8 (15) to H1 based on a
cross-peak to H14; a doublet of doublets at δ 5.04 re-assigned
from H2 (15) to H8 based on its couplings to both protons on
C9 and on a cross-peak to H6; two doublets of doublets at
δ 5.03 and 4.87 corresponding to the vinyl methylene group
at C13 substituted with an acetoxyl function; a doublet at δ
4.76 (H6); the 13-acetate methyl singlet at δ 2.12; two sig-
nals overlapped at δ 2.03 (H14 and 2-Ac CH3); two sharp
three-proton singlets at δ 1.55 and 1.37 for two tertiary
methyl groups, H2′-Me and H15, respectively. Protons belong-
ing to the epoxymethacrylate substituent were distinguished
based on chemical shifts and two- and four-bond coupling
constants (16). The following correlations were observed on
the NOESY and ROESY spectra: H1–H5, H1–H9α, H2–H15, H3α–
H5, H6–H15, H8–H14, H8–H15. Table 2 reports the 13
C NMR
data of this compound, which could not be found in the liter-
ature. Peaks corresponding to C9 and C14 were broadened,
likely due to a conformational exchange on the 13
C chemical-
shift timescale. The stereochemistry of 3a is not ambiguous,
since it has been determined by X-ray crystallography (17).
Glaucolide E
The 1
H NMR spectrum (Table 5) of compound 3b showed
a number of striking similarities to that obtained for 3a; in-
deed, the differences could be accounted for on the basis of
a methacrylate ester instead of an epoxydized methacrylate
ester at C8: the methacrylate vinyl protons as quintets at
δ 6.12 and 5.68; a triplet of doublets at δ 5.59 (H2); a broad
doublet at δ 5.18 re-assigned from H8 (15) to H1 as for 3a;
doublets of doublets at δ 5.00 and 4.85 for the methylene
protons at C13, indicating the same lactone function as in 3a;
a broad doublet at δ 4.84 (H6); methyl group signals at
δ 2.08 for the 13-acetyl methyl group, at δ 2.04 for the C14
vinyl methyl and at δ 2.03 for the 2-acetyl methyl; a second
vinyl methyl signal at δ 1.93 (H2′-Me); a tertiary methyl
singlet (δ 1.39) corresponding to the C15 methyl group. Es-
sentially, the same NOEs as those found for 3a were found
on the ROESY spectrum, except that H2–H14 was present
and H2–H15 was absent. 13
C NMR data for 3b are reported
here for the first time (Table 2). A broadening of the same
carbon peaks as in 3a was observed. This compound must
be the precursor of 3a and would thus have the same stereo-
chemistry. The NMR data are in agreement with the
stereochemistry deduced from X-ray studies for this com-
pound (Fig. 2).
Effect of budlein A and zaluzanin A on smooth muscle
contraction
Both budlein A (1) and zaluzanin A (2) relaxed the oxytocin-
induced (20 nmol/L) contractile response in rat uterine
smooth muscle; however, KCl-induced contraction was re-
laxed only by zaluzanin A. These data indicate that zaluzanin
A, but not budlein A, might exert its relaxant effect through
a mechanism related with calcium influx through voltage-
operated calcium channels (18), and that both sesquiterpene
lactones might have a positive effect on the phosphatidyl-
inositol signaling pathway of the smooth muscle cell (19–21).
Experimental section
Budlein A 1 (MW = 374, C20H22O7) was isolated from
Viguiera buddleiaeformis as previously described (22). The
leaves and stems (10 kg) were extracted twice with ethanol.
After evaporating the solvent, an oily residue was obtained,
© 2008 NRC Canada
Van Calsteren et al. 1081
Coupling constants (Hz)
Proton δ Multiplicitya
Experimental Calculated from X-ray Dihedral angles (°) from X-ray
H1 5.173 br d 3
J1,2 10.3 11.6 –177
H2 5.574 td 3
J2,3α 10.9 10.5 166
H3α 1.271 br dd 2
J3α,3β –12.4
H3β 2.581 dd 3
J2,3β 6.7 3.5 64
H5 2.460 d 3
J5,6 8.9 8.8 174
H6 4.758 d 5
J6,13 0.9
H8 5.036 dd 3
J8,9α 10.6 9.2 –145
H9α 2.993 br dd 2
J9α,9β –12.5
H9β 2.568 br d 3
J8,9β 1.4 1.8 110
H13 4.872 dd 2
J13,13 –13.0
H13 5.033 dd 5
J6,13 1.0
H14 (3H) 2.026 s
H15 (3H) 1.369 s
H3′ pro-R 3.109 dd 4
J3 ′R,2 ′-Me <1
H3′ pro-S 2.803 d 2
J3 ′R,3 ′S 6.0
H2′-Me (3H) 1.554 s
2-Ac CH3 (3H) 2.026 s
13-Ac CH3 (3H) 2.120 s
a
Multiplicity: s, singlet; d, doublet; t, triplet; br, broad.
Table 4. 1
H NMR data (CDCl3) for glaucolide D 3a.

6. which was dissolved in hot benzene and subjected to column
chromatography (alumina). Fractions eluted with CHCl3–
acetone (9:1) were concentrated, obtaining an amorphous
residue. Recrystallization from acetone – isopropylic ether
yielded 1 (3.65 g, mp 106–108 °C).
Zaluzanin A 2 (MW = 264, C15H20O4) was isolated from
Zaluzania augusta as previously described (23). The aerial
parts (1 kg) were extracted twice with ethanol (6 L, 20 h)
under reflux. The volume was reduced to 2 L and treated
with lead acetate aqueous solution (20 g/2 L) for 2 h at room
temperature. The extract was filtrated through Celite and
then partitioned with chloroform. The organic layer was
concentrated, dissolved in benzene, and subjected to column
chromatography (alumina). Fractions eluted with chloroform
and ethyl acetate yielded a solid, which was recrystallized
with acetone – diethyl ether and then with methanol,
obtaining 2 as white crystals (1.2 g, mp 265 °C). X-ray:
orthorhombic, space group P212121, a = 6.7277(5) Å, b =
10.0202(7) Å, c = 19.5724(13) Å, V = 1319.45(16) Å3
, T =
291(2) K, Z = 4, Dcalcd. = 1.331 g/cm3
, F(000) = 568, λ =
0.71073 Å, µ = 0.095 mm–1
, crystal size 0.34 mm ×
0.23 mm × 0.16 mm. A total of 10770 reflections were
collected for 2.08° < θ < 25.00° and –8 ≤ h ≤ 7, –11 ≤ k ≤ 11,
–23 ≤ l ≤ 23. 2312 reflections with I > 2σ(I) were used in the
refinement. The final R indices were R1 = 0.0357 and wR2 =
0.0886 and R indices (all data): R1 = 0.0401, wR2 = 0.0906.
Glaucolide D 3a (MW = 464, C23H28O10) and glaucolide
E 3b (MW = 448, C23H28O9) were obtained as previously
described from the aerial parts of Vernonia liatroides (24).
The dried plant material (3 kg) was extracted with hot meth-
anol; while concentrating the extract, 3a precipitated as a
white amorphous solid (mp 186–188 °C). The methanol
extract was then partitioned with hexane and CHCl3. This
last fraction was subjected to column chromatography (silica
gel). Fractions eluted with hexane–EtOAc (6:4) afforded 3b
as white crystals (mp 149–150 °C) from acetone–hexane. X-
ray: monoclinic, space group P21, a = 10.6585(8) Å, b =
8.2373(7) Å, c = 13.2507(11) Å, β = 90.334(2)°, V =
1163.36(16) Å3
, T = 291(2) K, Z = 2, Dcalcd. = 1.280 g/cm3
,
F(000) = 476, λ = 0.710 73 Å, µ = 0.099 mm–1
, crystal size
0.46 mm × 0.18 mm × 0.15 mm. A total of 9537 reflections
were collected for 1.91° < θ < 25.00° and –12 ≤ h ≤ 12, –9 ≤
k ≤ 9, –15 ≤ l ≤ 15. 4070 reflections with I > 2σ(I) were used
in the refinement. The final R indices were R1 = 0.0430 and
© 2008 NRC Canada
1082 Can. J. Chem. Vol. 86, 2008
Coupling constants (Hz)
Proton δ Multiplicitya
Experimental Calculated from X-ray Dihedral angles (°) from X-ray
H1 5.185 br d 3
J1,2 10.5 11.1 –167
H2 5.588 td 3
J2,3α 10.8 10.4 165
H3α 1.282 br dd 2
J3α,3β –12.4
H3β 2.591 dd 3
J2,3β 6.7 6.1 47
H5 2.477 d 3
J5,6 8.8 8.9 170
H6 4.840 br d 5
J6,13 0.8
H8 5.019 dd 3
J8,9α 10.5 10.7 –157
H9α 3.011 br dd 2
J9α,9β –12.4
H9β 2.677 br d 3
J8,9β 1.0 1.4 85
H13 4.854 dd 2
J13,13 –12.9
H13 5.003 dd 5
J6,13 0.9
H14 (3H) 2.036 d 4
J1,14 –1.1 –1.1 –57, 63, 173b
H15 (3H) 1.390 s
H3 ′a 6.125 quintet 4
J3 ′a,2 ′-Me –1.2 –1.1 –69, 51, 171c
H3 ′b 5.685 quintet 2
J3 ′a,3 ′b 1.3
H2 ′-Me (3H) 1.931 t 4
J3 ′b,2 ′-Me –1.4 –1.1 –69, 51, 171c
2-Ac CH3 (3H) 2.031 s
13-Ac CH3 (3H) 2.078 s
a
Multiplicity: s, singlet; d, doublet; t, triplet; br, broad.
b
H14–C14–C10–C9.
c
H2 ′-Me–C2 ′-Me–C2 ′–C1′ .
Table 5. 1
H NMR data (CDCl3) for glaucolide E 3b.
Fig. 2. Crystal structure of glaucolide E 3b.

7. wR2 = 0.0859 and R indices (all data): R1 = 0.0563, wR2 =
0.0901.
X-ray crystallography2
Data were collected on a Bruker Smart APEX AXS CCD
area-detector diffractometer. No absorption correction was
applied. The structures were solved by direct methods using
SHELXS-97 and refined by full-matrix least squares. All
non–hydrogen atoms were refined with anisotropic displace-
ment parameters, the hydrogen atoms coordinates were esti-
mated in riding mode.
NMR spectroscopy
The compounds were dissolved in CDCl3, and TMS was
used as an internal standard. High-resolution 1
H (300.3 MHz)
and 13
C (75.5 MHz) NMR spectra were acquired at 25 °C on
a Chemagnetics Infinity 300 spectrometer using a Nalorac
z-gradient 5 mm inverse broadband 1
H{15
N–31
P} probe. A
combination of 1D and 2D high-resolution NMR experi-
ments were used to assign the 1
H and 13
C spectra.
Connectivities between protons were obtained using 2D
gradient-selected COSY (25). The 2D phase-sensitive
NOESY (26) and ROESY (27) experiments were performed
with a mixing time of 600 ms. The 1D 13
C NMR spectra
were recorded with WALTZ proton decoupling (28). The
multiplicity of the carbon signals was established using the
1D DEPT experiment (29) with a reading pulse of 135° and
an evolution time of 3.5 ms. The 2D gradient-selected mag-
nitude HMQC (30) and phase-sensitive HSQC (31) spectra
were acquired with 3.5 and 1.7 ms evolution times, respec-
tively, and MPF9 (32) carbon decoupling during acquisition.
The 2D gradient-selected magnitude HMBC (33) was run
without carbon decoupling with one-bond and long-range
evolution times of 3.5 and 80 ms, respectively. Accurate 1
H
chemical shifts and coupling constants were obtained by
spectral simulation using the program gNMR from
IvorySoft. Conformations were estimated from three-bond
vicinal 1
H–1
H coupling constants and substituent electro-
negativities (34), from four-bond long-range 1
H–1
H coupling
constants (35), and from three- and four-bond vinyl–allylic
1
H–1
H coupling constants (36), taking signs into account.
Molecular modeling
Geometry optimization was performed with the program
HyperChem 7.03 Professional from Hypercube using the
force field Hyper MM+, starting from the modified crystal
structure of goyazenzolide A (14) for 1 and from a built
structure for 2.
Uterine smooth muscle assays
Relaxant activity of budlein A and zaluzanin A was evalu-
ated on the contractile response to 60 mmol/L KCl and
oxytocin (20 nmol/L) as previously described (5).
Acknowledgments
Part of this research was financed by the grant DGAPA-
UNAM IN 219305–2. The authors are grateful to Simón
Hernández-Ortega for X-ray recordings.
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© 2008 NRC Canada
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2
Supplementary data for this article are available on the journal Web site (canjchem.nrc.ca) or may be purchased from the Depository of
Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Ottawa, ON K1A 0R6, Canada. DUD 3848. For more
information on obtaining material, refer to cisti-icist.nrc-cnrc.gc.ca/cms/unpub_e.shtml. CCDC 699880 and 699881 contain the crystallo-
graphic data for this manuscript. These data can be obtained, free of charge, via www.ccdc.cam.ac.uk/conts/retrieving.html (Or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax +44 1223 336033; or deposit@ccdc.cam.ac.uk).

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1084 Can. J. Chem. Vol. 86, 2008