Ent-Kaurene Glycosides from Ageratina cylindrica

Article abstract of DOI:10.1021/acs.jnatprod.5b00488

The aqueous extract of the leaves of Ageratina cylindrica afforded six new ent-kaurenoic acid glycosides together with the known diterpenoid paniculoside V, the flavonoid astragalin, chlorogenic acid, and l-chiro-inositol. The structures were elucidated mainly by NMR and MS methods, and the absolute configuration was established by vibrational circular dichroism spectroscopy. The new compounds showed moderate antiprotozoal activity against Entamoeba histolytica and Giardia lamblia trophozoites.

Full text of DOI:10.1021/acs.jnatprod.5b00488

Article
pubs.acs.org/jnp
ent-Kaurene Glycosides from Ageratina cylindrica
Celia Bustos-Brito,† Mariano Sanchez-Castellanos,† Baldomero Esquivel,† Jose S. Calderon,†
́
́
́
Fernando Calzada,‡ Lilian Yepez-Mulia,§ Pedro Joseph-Nathan,⊥ Gabriel Cuevas,†
́
and Leovigildo Quijano*,†
†Instituto de Química, Universidad Nacional Auton
Mexico
‡Unidad de Investigacion
Siglo XXI, IMSS, Avenida Cuauhtem
§Unidad de Investigacion
Medica en Enfermedades Infecciosas y Parasitarias, UMAE Hospital de Pediatría, Centro Med
Siglo XXI, IMSS, Avenida Cuauhtemoc 330, Col. Doctores, Mexico, D.F. 06725, Mexico
⊥Departamento de Química, Centro de Investigacion
y de Estudios Avanzados del Instituto Politec
Mexico, D.F. 07000, Mexico
́
oma de Mex
́
ico, Circuito Exterior, Ciudad Universitaria, Mex
́
ico, D.F. 04510,
́
́
Med
́
ica en Farmacología, 2° Piso CORCE, UMAE Hospital de Especialidades, Centro Med
́
ico Nacional
́
ico Nacional
́
oc 330, Col. Doctores, Mexico, D.F. 06725, Mexico
́
́
́
́
́
́
́
́
́
nico Nacional, Apartado 14-740,
́
́
S
* Supporting Information
ABSTRACT: The aqueous extract of the leaves of Ageratina
cylindrica afforded six new ent-kaurenoic acid glycosides
together with the known diterpenoid paniculoside V, the
flavonoid astragalin, chlorogenic acid, and L-chiro-inositol. The
structures were elucidated mainly by NMR and MS methods,
and the absolute configuration was established by vibrational
circular dichroism spectroscopy. The new compounds showed
moderate antiprotozoal activity against Entamoeba histolytica
and Giardia lamblia trophozoites.
s a continuation of our search for antiparasitic
RESULTS AND DISCUSSION
■
1
Acompounds, the chemical investigation of the aqueous
Repeated chromatography of the aqueous extracts of the leaves
of A. cylindrica on Sephadex LH-20 and octadecyl-function-
alized silica gel yielded six new compounds (1−6) together
with the known ent-kaurene paniculoside V (7),6 astragalin,7
chlorogenic acid,8 and L-chiro-inositol.9 The compounds were
fully characterized by their physical and spectroscopic proper-
extract of Ageratina cylindrica (McVaugh) R. M. King & H. Rob
(Asteraceae) yielded six new ent-kaurenoic acid glycosides. This
type of compound is predominantly found in species belonging
to several families, but best represented in the Asteraceae,
although they are rarely found in the genera Eupatorium and
Ageratina. Only three kaurenes have been isolated from
Ageratina vacciniaefolia.2 Kaurene glycosides and related
compounds have attracted significant attention due to their
therapeutic potential and economic value.3 In this regard,
steviol glycosides, isolated from the leaves of Stevia rebaudiana,
are natural sugar substitutes sweeter than sucrose, in addition to
their pharmacological activities as hypoglycemic, antitumor and
anticancer, antihypertensive, and antidiarrheal agents.4
1
ties, including H and 13C NMR data (Tables 1 and 2). The
resonances were assigned using COSY, HSQC, ROESY, and
HMBC correlations.
Compound 1 gave the molecular formula C32H50O12, as
determined by HRESIMS and 13C NMR data. The IR spectrum
showed absorption bands for OH and COOR groups at 3370
and 1732 cm−1, respectively. The 1H and 13C NMR spectra of 1
1
were similar to those of 7. The H NMR spectrum (Table 1)
showed the presence of two methyl singlets at δ 1.28 and 1.30
and two olefinic singlets at δ 5.08 and 5.88 of an exocyclic
methylene group, which showed cross-peaks with the
methylene carbon signal at δ 106.5 in the HSQC experiment.
The above data suggested an ent-kaurenoic acid derivative. Two
doublets at δ 4.80 (J = 8.0 Hz) and δ 6.29 (J = 8.0 Hz), coupled
with the carbon resonances at δ 107.3 and 96.2, respectively,
were assigned to anomeric hydrogens, suggesting the presence
of two sugar units. Their β-orientation follows from the
Entamoeba histolytica and Giardia lamblia are protozoa
associated with parasitic infections for which metronidazole
has proven to be effective. However, due to its adverse side
effects, efforts for improving the therapy for giardiasis and
amoebic dysentery are needed. In this sense, vegetal extracts
have been used for many years in the treatment and prevention
of several diseases, including parasitic infections,5 since
medicinal plants could be a source of new drugs with high
activity and low toxicity. Herein, the isolation, structural
characterization, and antiprotozoal activity of six new ent-
kaurenoic glycosides from A. cylindrica are discussed.
Received: June 9, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00488
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. NMR Data (1H 400 MHz, 13C 100 MHz, Pyridine-d5) of 1 and 2
1
2
position
1a
δH (J in Hz)
1.82, d (13.2)
δC, type
HMBC
δH (J in Hz)
1.75, m
δC
41.2, CH2
2, 3, 9, 10, 20
2, 3, 10
41.0, CH2
20.0, CH2
38.9, CH2
1b
0.83, td (13.2, 3.6)
2.23, m
0.75, td (13.2, 3.6)
2.18, m
a
a
a
2a
20.0, CH2
38.8, CH2
5, 9, 20
a
2b
1.40, m
1, 3, 9
1.38, m
3a
2.39, m
2, 18, 19
2.35, dd (13.6, 2.8)
0.85, td (13.6, 4.4)
3b
1.00, m
2, 4, 5, 19
4
44.6, C
44.5, C
5
1.25, dd (12.0, 1.6)
2.48, d (13.6)
2.13, dd (13.6, 1.6)
2.35, m
57.1, CH
22.5, CH2
6, 10, 18, 19, 20
1.12, dd (12.0, 1.2)
2.48, m
56.8, CH
22.4, CH2
6a
4, 7, 8, 10
7
6b
2.10, m
a
a
7a
39.0, CH2
20, 11,
6
2.33, m
37.0, CH2
7b
1.46, m
6, 5
1.47, m
8
46.8, C
46.7, C
9
1.90, m
46.7, CH
40.1, C
8, 10, 12, 14
1.83, m
46.7, CH
40.1, C
10
11a
11b
12a
12b
13
1.60, m
18.9, CH2
8, 10, 12
1.91, m
18.8, CH2
1.42, m
10
1.83, m
1.61, m
34.4, CH2
11
1.60, m
34.3, CH2
a
1.46, m
11, 14, 16, 17
1.44, m
2.58, br s
2.25, m
41.1, CH
37.2, CH2
8, 11, 12, 15, 16
9, 12, 13
2.58, br s
2.21, m
41.1, CH
38.6, CH2
14a
14b
15
1.02, m
9, 12, 13, 15, 16
9, 16, 17
1.03, dd (12.0, 4.4)
4.10, t (2.4)
4.11, t (2.0)
91.1, CH
157.3, C
90.1, CH
157.2, C
16
a
17a
17b
18
5.88, br s
5.08, br s
1.28, s
106.5, CH2
12, 13, 15, 16
5.91, br s
5.09, dd (1.2)
1.23, s
106.4, CH2
a
12, 13, 15, 16
29.1, CH3
177.4, C
3, 4, 5, 19
28.9, CH3
177.4, C
19
20
1.30, s
16.6, CH3
107.3, CH
72.9, CH
76.0, CH
73.1, CH
71.8, CH
17.8, CH3
96.2, CH
79.6, CH
74.5, CH
71.5, CH
79.8, CH
62.6, CH2
1, 5, 9, 10
1.28, s
16.5, CH3
107.0, CH
73.0, CH
73.6, CH
74.6, CH
70.1, CH
17.4, CH3
96.2, CH
79.6, CH
74.5, CH
71.5, CH
79.8, CH
62.5, CH2
1′
2′
3′
4′
5′
6′
1″
2″
4.80, d (8.0)
15
4.87, d (8.0)
4.41, t (8.8)
1′
4.36, dd (9.2)
4.28, dd (9.6, 4.0)
5.64, dd (3.6, 1.2)
3.90, qd (6.4, 0.8)
1.37, d (6.4)
4.12, dd (9.2, 3.6)
4.06, dd (3.6, 0.8)
3.79, qd (6.4,0.8)
1.59, d (6.4)
1′, 5′
3′
1′, 2′, 6′
a
4′, 5′
6.29, d (8.0)
3″, 5″, 19
6.28, d (8.0)
a
a
4.23, t (8.8)
1″, 5″, 6″
4.25, t (8.8)
3″
4″
5″
4.28, t (8.8)
2″, 4″
2″, 3″, 6″
4.28, t (8.8)
4.36, t (9.2)
4.37, dd (9.6, 9.2)
a
4.04, ddd (9.2, 4.4, 2.4)
4.48, dd (12.0, 2.4)
4.42, dd (12.0, 4.0)
1″, 2″, 3″, 4″
4.04, ddd (9.6, 4.4, 2.4)
4.47, dd (12.0, 2.4)
4.40, dd (12.0, 4.4)
1.80, s
a
6″a
6″b
OCOCH3
OCOCH3
1″, 5″
5″
21.1, CH3
171.4, C
a
4J interaction.
magnitude (8 Hz) of the coupling constants. An additional
methyl doublet at δ 1.59 (J = 6.4, 3H) suggested one of the
sugar residues to be a methyl pentose moiety.
correlations showed two spin systems, from anomeric H-1″ [δH
6.29 (δC 96.2)] to H2-6″ [δH 4.42 and 4.48 (δC 62.6)] and from
anomeric H-1′ [δH 4.80 (δC 107.3)] to H3-6′ [δH 1.59 (δC
17.8)] (Table 1). Carbon chemical shifts and H−H coupling
constants permitted identification of the sugar moieties as β-
glucopyranosyl and β-fucopyranosyl residues, respectively.
The 13C NMR data (Table 1) showed 32 resonances due to
three methyl, 10 methylene, and 14 methine groups, four
quaternary carbon atoms, and one carbonyl carbon evidenced
by DEPT experiments. Eighteen signals at low frequencies,
The location of the sugar moieties was determined by the
HMBC correlations between the carbonyl group at δ 177.4 (C-
19) and the anomeric hydrogen at δ 6.29, indicating that one
sugar moiety must be located at C-19 as an ester, while the
other one must be located at C-15, since its anomeric doublet
at δ 4.80 showed long-range coupling with the carbon signal at
δ 91.1 (δH 4.11) assigned to C-15. Identification of the spin
system of the individual monosaccharide moieties and complete
assignment of the hydrogen resonances were achieved through
COSY and selective 1D-TOCSY experiments. The NMR
́
together with a carbonyl (δ 177.4) and an olefinic methylene (δ
106.5), are assignable to the kaurene skeleton, while 10
methines and one methylene in the δ 62.6−107.3 range,
B
DOI: 10.1021/acs.jnatprod.5b00488
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Journal of Natural Products
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Table 2. NMR Data (1H 400 MHz, 13C 100 MHz, Pyridine-d5) of 3−5
a
3
4
5
position
1a
δH (J in Hz)
1.80, s
δC
δH (J in Hz)
1.81, s
δC
δH (J in Hz)
1.84, m
δC
41.2, CH2
41.4, CH2
41.2, CH2
1b
0.83, td (12.0, 4.0)
2.21, s
0.87, td (13.6, 4.0)
2.25, m
0.81, td (13.5, 4.0)
2.24, m
2a
20.0, CH2
39.0, CH2
20.3, CH2
39.0, CH2
20.3, CH2
38.9, CH2
2b
1.39, m
1.48, m
1.45, m
3a
2.31, m
2.38, td (13.2, 4.0)
1.01, m
2.42, m
3b
1.45, m
0.89, td (13.0, 4.0)
4
44.5, C
44.3, C
44.2, C
5
1.87, m
2.45, m
2.11, m
2.22, m
1.01, m
46.8, CH
22.4, CH2
1.23, m
56.6, CH
22.8, CH2
1.1,1 dd (12.0, 2.0)
2.42, d (12.0)
2.14, m
56.4, CH
22.8, CH2
6a
2.21, m
6b
2.14, m
7a
37.0, CH2
2.05, d (12.0)
1.05, m
37.3, CH2
1.05, dd (11.5, 4.5)
2.04, d (11.5)
37.2, CH2
7b
8
46.8, C
46.8, C
46.7, C
9
1.22, m
57.2, CH
40.1, C
1.21, m
46.7, CH
40.1, C
1.85, d (7.5)
46.7, CH
40.1, C
10
11a
11b
12a
12b
13
1.88, m
18.8, CH2
1.92, m
18.8, CH2
1.91, m
18.8, CH2
1.43, m
1.44, m
1.42, m
1.56, m
34.3, CH2
1.58, m
34.4, CH2
1.58, m
34.4, CH2
1.43, m
1.48, m
1.46, m
2.57, br s
2.37, m
41.1, CH
38.8, CH2
2.61 s
41.1, CH
38.9, CH2
2.62, s
41.1, CH
37.2, CH2
14a
14b
15
2.46, d (12.8)
2.05, d (11.5)
1.06, dd (11.5, 4.5)
4.11, t (2.5)
1.01 m
1.00, dd (13.2, 4.0)
4.13, m
4.10, t (2.4)
91.3, CH
157.0, C
91.0, CH
157.2, C
90.9, CH
157.1, C
16
17a
17b
18
5.84, s
106.6, CH2
5.92, s
106.4, CH2
5.94, s
5.12, s
1.28, s
106.4, CH2
5.07, dd (1.2, 1.2)
1.27, s
5.11, t (1.2)
1.33, s
29.0, CH3
177.4, C
29.7, CH3
180.7, C
29.7, CH3
180.8, C
19
20
1.30, s
16.5, CH3
107.1, CH
69.7, CH
78.5, CH
70.5, CH
71.5, CH
17.6, CH3
96.2, CH
79.6, CH
74.5, CH
71.5, CH
79.8, CH
62.5, CH2
1.21, s
16.7, CH3
107.3, CH
72.9, CH
76.0, CH
73.1, CH
71.8, CH
17.8, CH3
1.19, s
16.6, CH3
107.1, CH
73.0, CH
73.6, CH
74.6, CH
70.1, CH
17.4, CH3
1′
2′
3′
4′
5′
6′
1″
2″
4.87, d (8.0)
4.85, d (8.0)
4.91, d (8.0)
4.39, dd (9.5, 8.0)
4.30, dd (9.5, 3.5)
5.66, d (3.5)
3.96, q (6.5)
1.42, d (6.5)
4.62, dd (10.0, 8.0)
5.42, dd (10.0, 3.2)
4.25, dd (4.0, 0.8)
3.82, td (8.0, 0.8)
1.54, d (8.0)
4.44, dd (9.2, 8.0)
4.14, dd (9.2, 3.2)
4.09, dd (3.2, 0.8)
3.85, td (8.0, 0.8)
1.64, d (8.0)
6.28 d (8.0)
4.23 t (8.0)
3″
4″
5″
4.28 t (8.8)
4.35 t (8.8)
4.04 ddd (9.2, 4.4, 2.4)
4.47 dd (12.0, 2.8)
4.39 dd (12.0, 4.4)
1.95
6″a
6″b
OCOCH3
OCOCH3
21.4, CH3
171.2, C
1.82, s
21.1, CH3
171.0, C
a
1
NMR was recorded at 500 MHz for H and 125 MHz for 13C.
together with a methyl signal at δ 17.8, are due to the sugar
moieties.
configuration (AC) of the new compounds and at the same
time to verify that they are ent-kaurenes.
In the case of ent-kaurenoic acid derivatives the C-15
configuration has been assigned by NOE effects between H-15/
H-9 or H-15/H-14 for the α- and β-epimers, respectively,10
although these interactions were not observed in 1−6. Analysis
of the multiplicity of the H-15 signal has also been used to
assign the configuration at C-15. Nevertheless, inspection of the
1H NMR data of related molecules reveals that the multi-
plicities for H-15 have been reported either as a broad singlet or
as triplets or a doublet of doublets, thus causing conflicting
assignments of the configuration at C-15.11 Consequently, it
was essential to unequivocally assign the C-15 absolute
Comparison of the calculated and experimental vibrational
circular dichroism (VCD) spectra is a sensitive technique for
determining the AC of a single stereogenic center in the
presence of several centers and has allowed the determination
of the AC of a significant number of natural products.12 VCD of
natural product ester derivatives has advantages over the
analysis of free alcohols or acids, which often show
intermolecular solute−solute associations that confuse compar-
ison of experimental and density functional theory (DFT)-
calculated spectra,13 and therefore 8 was selected as a truncated
model compound.
C
DOI: 10.1021/acs.jnatprod.5b00488
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
Table 3. Relative Energies and Conformational Populations of 8 and 11
a
b
c
d
e
d
OPT
f
d
conformer
ΔEMMFF94
%
MMFF94
ΔESP
%
SP
ΔEOPT
%
ΔGB3PW91
%
B3PW91
8a
0.00
0.04
0.00
0.14
51.8
0.42
0.00
0.24
0.00
33.0
67.0
39.8
60.2
0.34
0.00
0.30
0.00
36.0
64.0
37.4
62.6
0.70
0.00
0.00
0.06
23.5
76.5
52.5
47.5
8b
48.2
11a
11b
56.0
44.0
a
b
c
Molecular mechanics energy relative to 73.03 and 72.74 kcal/mol for 8 and 11, respectively. Molecular mechanics population in %. Single-point
d
e
relative energy; the lowest energy values for 8 and 11 were −751 396.8 and −751 396.5 kcal/mol, respectively. Conformational population. Energy
f
of the optimized structures; data are relative to −751 439.7 kcal/mol for 8 and −751 439.1 kcal/mol for 11. Free energy relative to −751 135.1 kcal/
mol for 8 and −751 133.9 kcal/mol for 11.
Acid hydrolysis of 1 with aqueous 5% HCl did not afford
target compound 9. Instead, the keto acid 10 was obtained, in
addition to D-glucose and L-fucose, which were identified as
their trimethylsilyl derivatives by GC and the positive (+35.4)
and negative (−33.2) specific rotations of their acetate
derivatives, respectively. It is known that ent-kaurenes with a
15β-hydroxy group undergo a Wagner−Meerwein rearrange-
ment when heated with mineral acid to afford (16R)-ent-
kauran-15-one (10), while the epimeric 15α-hydroxy group is
stable under these reaction conditions.14 This result suggested
the β-orientation of the fucosyloxy moiety at C-15 in 1.
With a view to obtaining 9 and to prevent the proton-
catalyzed Wagner−Meerwein rearrangement of 1 to 10, the
oxidation/elimination sequence15 was selected. Alkaline hydrol-
ysis of 1 gave fucosyl derivative 4, which when treated with
NaIO4 and KOH gave desacetylxylopic acid (9),16 which
without purification was subjected to acetylation followed by
esterification with diazomethane to give methyl xylopate (8).
The absolute configuration of 8 and of its C-15 epimer 11,
prepared from an authentic sample of grandifloric acid, was
obtained by comparing the experimental and calculated IR and
VCD spectra. A molecular mechanics conformational search
using the MMFF94 force field provided 12 minimum energy
conformers for each molecule in a 10 kcal/mol energy window.
Two conformers of each epimer with energy in the 0.2 kcal/
mol gap (Table 3) accounted for 99.99% of the conformational
populations of 8 and 11. These four conformations were
submitted to single-point calculations with the B3PW91 hybrid
functional and the DGDZVP basis set. Since conformers 8a and
8b, as well as 11a and 11b, remained within similar energy
ranges, they were optimized at the same level of theory (Table
3). The dipole and rotational strengths, calculated at the
B3PW91/DGDZVP level, were converted into molar absorp-
tivity of each conformer using Lorentzian functions with a
bandwidth of 6 cm−1. Finally, the Boltzmann-weighted factor
for each conformer obtained with the Gibbs free energy was
D
DOI: 10.1021/acs.jnatprod.5b00488
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Journal of Natural Products
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used to generate the calculated IR and VCD spectra. The
solvent-subtracted and calculated IR and VCD spectra for 8 and
11 are shown in Figures 1 and 2, respectively. Table 4 shows
Table 4. Confidence Level Data for the IR and VCD Spectra
of 8 and 11 and Cross-Comparisons
a
b
c
d
e
f
compound
anH
SIR
SE
S−E
ESI
C (%)
8
0.972
0.974
0.969
0.976
95.1
97.3
94.2
93.1
86.1
83.9
67.0
67.7
17.7
18.5
24.8
68.8
65.5
42.2
42.8
100
100
82
11
g
8E vs 11C
11E vs 8C
h
24.9
85
a
b
c
Anharmonicity factor. IR spectral similarity. VCD spectral similarity
d
for the correct enantiomer. VCD spectral similarity for the incorrect
e
enantiomer. Enantiomer similarity index, calculated as SE − S−E
.
f
g
Confidence level for the stereochemical assignments. Experimental 8
h
vs calculated 11 spectra comparison. Experimental 11 vs calculated 8
spectra comparison.
same confidence level. As also shown in Table 4, cross-
comparisons of the calculated and experimental spectra of 8
and 11 and vice versa show high values of spectra similarity of
the IR and VCD spectra, but the enantiomer similarity index
(ESI) and the confidence level are lower than those of the
correct configuration, thus eliminating any doubt of the
assignment of the 15R absolute configuration of 8. It is
important to note that the VCD spectra of 8 and 11 did not
exhibit VCD exciton coupling19 of the carbonyl group signals.
The configuration at C-15 in compound 8 was also
supported by the NOE effect between H-15 at δ 5.15 t (2.5)
and H-14 at δ 4.04 d (12.0). Thus, the structure of 1 was
defined as ent-15β-(β-L-fucosyloxy)kaur-16-en-19-oic acid β-D-
glucopyranosyl ester.
Compound 2 displayed an [M + Na]+ ion at m/z 691.3306,
as determined by HRESIMS, which supports the molecular
formula C34H52O13. The 1H and 13C NMR spectra (Table 1) of
2 displayed features similar to those of 1, except for the
Figure 1. Comparison of the experimental and calculated, at the
B3PW91/DGDZVP level, IR and VCD spectra of 8.
1
presence of a sharp methyl singlet at δ 1.81 in the H NMR
spectrum and two extra carbon resonances (δ 171.4 and 21.1)
in the 13C NMR spectrum, associated with the presence of an
1
acetate group. The H NMR chemical shifts of the sugar
moieties of 2 were comparable with those of 1, except for the
deshielding of H-4′ at δ 5.64 (dd, 3.6, 1.2 Hz) indicative of the
esterification at C-4′. The HMBC correlations between the
signal at δ 5.64 (H-4′) and the methyl protons at δ 1.81 with
the carbon resonance at δ 171.4 revealed the presence of an
acetate group at C-4′. Consequently the structure of 2 was
defined as ent-15β-(4-acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic
acid β-D-glucopyranosyl ester.
Compound 3, as in the case of 2, showed an [M + Na]+ ion
at m/z 691.3306, which together with the 13C NMR data
1
indicates the molecular formula C34H52O13. The H and 13C
NMR spectra (Table 1) showed resonances similar to those of
2, except for the chemical shifts of H-3′ and H-4′. While in 3
the H-3′ signal was deshielded to δ 5.42 (dd, 10.0, 3.2 Hz), H-
4′ was shielded as compared with compound 2, indicating the
presence of the acetate group at C-3′ in 3, and therefore 3 is a
regioisomer of 2 possessing the ent-15β-(3-acetoxy-β-L-
fucosyloxy)kaur-16-en-19-oic acid β-D-glucopyranosyl ester
structure.
The HRESIMS data of 4 showed an [M + Na]+ ion at m/z
487.2667 (calculated for C26H40O7 + Na, 487.2672), which
supported the molecular formula C26H40O7. The 13C NMR
spectrum of 4 showed 26 carbon signals, 20 of them due to the
Figure 2. Comparison of the experimental and calculated, at the
B3PW91/DGDZVP level, IR and VCD spectra of 11.
the data obtained using the CompareVOA software.17 For
compound 8 the IR and VCD spectra similarities, SIR and SE,18
are 95.1 and 86.1, respectively, with a confidence level of 100%,
while for compound 11 the values are 97.3 and 83.9 with the
1
aglycone moiety and six due to a sugar unit. The H and 13C
NMR data (Table 2) of 4 were similar to those of 1, but lacked
the glucose moiety resonances. Therefore, the structure of 4
E
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J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
was obtained from fraction D3, 2 (100 mg), 3 (25 mg), and 4 (15 mg)
were obtained from fractions D8, D11, and D13, respectively, D15
(4.7 mg) gave a mixture of 5 and 6, L-chiro-inositol9 (150 mg)
crystallized from fraction F, fraction G (30 mg) was subjected to silica
gel TLC eluting with n-BuOH/2-propanol/formic acid/H2O
(48:17:18:17) to give chlorogenic acid8 (15 mg), and astragalin7 (25
mg) was isolated from fraction I (67 mg) in the same way.
was established as ent-15β-(β-L-fucosyloxy)kaur-16-en-19-oic
acid.
A minor amount of a mixture of 5 and 6 (4.5 mg) was
1
isolated. Their H and 13C NMR data (Table 2 for 5 and
Experimental Section for 6) were similar to those of 2 and 3,
respectively, except for the absence of the glucose moiety
resonances at C-19. Although 5 and 6 were isolated as natural
products, they could not be fully identified. Thus, enzymatic
hydrolysis of a mixture of 1−3 using β-glucosidase from
almonds gave a sufficient quantity of 5 for its complete
characterization, as shown in the Experimental Section and in
Table 2.
ent-15β-(β-L-Fucosyloxy)kaur-16-en-19-oic acid β-D-glucopyra-
nosyl ester (1): white powder; mp 145−150 °C; [α]25 −31.6 (c
D
0.01, MeOH); IR (KBr) νmax 3370, 2924, 2856, 1732, 1655, 1061
cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+
649.3222 (calculated for C32H50O12 + Na, 649.3200).
ent-15β-(4-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid β-D-
glucopyranosyl ester (2): white powder; mp 150−152 °C; [α]25
D
The antiprotozoal activity of 1−4 was tested on Entamoeba
histolytica and Giardia lamblia trophozoites (Table 5). In
−46.6 (c 0.01, MeOH); IR (KBr) νmax 3373, 2925, 2857, 1724, 1655,
1602, 1240, 1070 cm−1; 1H and 13C NMR, see Table 1; HRESIMS m/
z [M + Na]+ 691.3316 (calculated for C34H52O13 + Na, 691.3306).
ent-15β-(3-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid β-D-
Table 5. In Vitro Antiprotozoal Activity of Compounds 1−4
glucopyranosyl ester (3): white powder; mp 145−148 °C; [α]25
D
a
IC50 μM (CI)
−33.0 (c 0.01, MeOH); IR (KBr) νmax 3691, 3597, 2929, 1856, 1734,
1602, 1022 cm−1; 1H and 13C NMR, see Table 2; HRESIMS m/z [M
+ Na]+ 691.3316 (calculated for C34H52O13 + Na, 691.3306).
ent-15β-(β-L-Fucosyloxy)kaur-16-en-19-oic acid (4): white pow-
compound
Entamoeba histolytica
Giardia lamblia
1
2
3
4
43.3 (43.6−43.1)
49.5 (50.1−49.0)
52.7 (52.9−52.3)
73.5 (73.9−72.8)
2.18 (2.2−2.14)
0.23 (0.58−0.17)
41.9 (42.2−41.5)
69.5 (69.9−69.2)
48.9 (49.3−48.7)
98.5(98.7−97.8)
0.83 (0.87−0.82)
1.22 (1.57−0.81)
der; mp 142−146 °C; [α]25 −36.7 (c 0.01, MeOH); IR (KBr) νmax
D
3368, 1731, 1706, 1655, 1231 cm−1; 1H and 13C NMR data, see Table
2; HRESIMS m/z [M + Na]+ 487.2667 (calculated for C26H40O7 +
Na, 487.2672).
c
emetine
c
metronidazole
ent-15β-(4-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid (5):
white powder; mp 149−152 °C; [α]25 −28.0 (c 0.01, MeOH); IR
a
D
Results are expressed as mean (n = 6), CI = 95% confidence intervals;
1
(KBr) νmax 3596, 1735, 1693, 1449, 1370, 1242 cm−1; H and 13C
c
correlation coefficient >0.9500 and p < 0.05. Positive controls.
NMR data, see Table 2; DART-MS m/z [M + H]+ 507.
ent-15β-(3-Acetoxy-β-L-fucosyloxy)kaur-16-en-19-oic acid (6): 1H
NMR partial data extracted from a 5 and 6 mixture (pyridine-d5, 500
MHz) δ 2.61 (1H, s, H-13), 5.88 (1H, s, H-17a), 5.11 (1H, s, H-17b),
1.32 (3H, s, CH3-18), 1.21 (3H, s, CH3-20), 1.96 (3H, s, OCOCH3),
4.92 (1H, d, J = 7.6 Hz, H-1′), 4.65 (1H, dd, J = 10.0, 7.6 Hz, H-2′),
5.46 (1H, dd, J = 10.0, 3.2 Hz, H-3′), 4.28 (1H, d, J = 4.0 Hz, H-4′),
3.88 (1H, qd, J = 6.4, 0.8 Hz, H-5′), 1.58 (3H, d, J = 6.4 Hz, CH3-6′);
13C NMR (CDCl3, 125 MHz) δ 41.1 (CH, C-13), 106.6 (CH2, C-17),
29.7 (CH3, C-18), 180.7 (C, C19), 16.6 (CH3, C-20), 171.2 (C,
OCOCH3), 21.4 (CH3, OCOCH3), 107.1 (CH, C-1′), 72.8 (CH, C-
2′), 78.6 (C, C-3′), 70.5 (CH, C-4′), 71.6 (CH, C-5′), 17.6 (CH3, C-
6′).
general, all compound showed moderate activity against both
protozoa with IC50 values ranging from 43.3 to 73.5 μM for E.
histolytica and from 41.9 to 98.5 μM for G. lamblia.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured on a Fisher-Johns apparatus and are uncorrected. IR spectra
were obtained on a Bruker Tensor 27 spectrometer. VCD data were
acquired on a BioTools dualPEM ChiralIR FT-VCD spectropho-
tometer (Jupiter, FL, USA). The 1D and 2D NMR experiments were
1
performed on a Bruker Advance III spectrometer at 400 MHz for H
Paniculoside V (7): white powder; mp 168−172 °C (reported6
173−175 °C); 13C NMR data were essentially the same as reported;6
1H NMR (pyridine-d5, 400 MHz) δ 1.82 (1H, d, J = 12.4, H-1a), 0.83
(1H, td, J = 12.4, 3.2, H-1b), 2.19 (1H, m, H-2a), 1.38 (1H, m, H-2b),
2.34 d (1H, d, J = 12.8, H-3a), 0.95 (1H, dd, J = 13.6, 4.4, H-3b), 1.30
(1H, m, H-5), 2.43 (1H, m, H-6a), 2.08 (1H, m, H-6b), 2.44 (1H, m,
H-7a), 1.48 (1H, m, H-7b), 1.92 (1H, m, H-9), 1.90 (1H, m, H-11a),
1.43 (1H, m, H-11b), 1.62 (1H, m, H12a), 1.46 (1H, m, H-12b), 2.57
(1H, br s, H-13), 2.22 (1H, m, H-14a), 1.00 (1H, m, H-14b), 4.11
(1H, t, J = 2.4, H-15), 5.89 (1H, br s, H-17a), 5.09 (1H, dd, J = 1.6,
0.8, H-17b), 1.24 (3H, s, CH3-18), 1.29 (3H, s, CH3-20), 5.04 (1H, d,
J = 7.6, H-1′), 4.12 (1H, t, J = 8.8, H-2′), 4.27 (1H, m ov, J = 9.2, H-
3′), 4.25 (1H, m ov, H-4′), 3.92 (1H, ddd, J = 8.8, 4.8, 2.8, H-5′), 4.54
(1H, dd, J = 11.2, 2.8, H-6′a), 4.42 (1H, dd, J = 12.0, 4.0, H-6′b), 6.27
(1H, d, J = 8.0, H-1″), 4.21 (1H, t, J = 8.8, H-2″), 4.28 (1H, t, J = 8.4,
H-3″), 4.34 (1H, t, J = 9.2, H-4″), 4.03 (1H, ddd, J = 9.2, 4.4, 2.4, H-
5″), 4.46 (1H, dd, J = 12.0, 2.4, H-6″a), 4.38 (1H, dd, J = 12.0, 4.8, H-
6″b).
Methyl xylopate (8): A solution of 4 (17 mg) in H2O (2.0 mL) was
treated with 23 mg of NaIO4 and stirred at rt for 18 h. Then, 20 mg of
KOH was added and the reaction mixture refluxed for 1 h. After
addition of H2O (2.0 mL) the pH was adjusted to 4−5 with HOAc,
and the resulting mixture was extracted with EtOAc (3 × 10 mL). The
EtOAc fraction was washed with H2O (25 mL), dried over anhydrous
Na2SO4, and evaporated under reduced pressure to yield 9, which
without purification was subjected to esterification with diazomethane,
followed by acetylation with pyridine and Ac2O to give 8 (1 mg),
and 100 MHz for 13C and on a Varian Unity Inova 500 MHz for H
1
and 125 MHz for 13C. Chemical shifts were referenced to TMS, and J
values are given in Hz. The HRESIMS data were recorded on a Waters
Synap G2S HDMS Q-TOF mass spectrometer. The DART-MS data
were obtained on a Jeol AccuTOF JMS-T100LC mass spectrometer.
Preparative TLC was carried out on precoated Macherey Nagel Sil G/
UV254 plates of 1.0 mm thickness. Silica gel 230−400 mesh
(Macherey-Nagel), Sephadex LH-20 (Pharmacia Biotech), and
octadecyl-functionalized silica gel (Sigma-Aldrich) were used for
column chromatography.
Plant Material. A. cylindrica was collected from Tenancingo, State
of Mexico, Mexico, in February 2012. The plant material was identified
by Jose Luis Villasenor, and a voucher specimen (MEXU-1333 472)
̃
was deposited at the National Herbarium (MEXU) of the Instituto de
́
Biologia, UNAM, Mexico City, Mexico.
Extraction and Isolation. The dried and powdered leaves of A.
cylindrica (100 g) were added to boiling H2O (1 L × 3) and filtered
immediately. The infusion was filtered, and after cooling to room
temperature it was lyophilized to yield 33 g of residue. This material
was fractionated over a Sephadex LH-20 column, using MeOH as
eluent, to give nine fractions (A−I). Fraction D (950 mg) was
subjected to flash column chromatography on octadecyl-functionalized
silica gel, with an isocratic mobile phase of MeOH/H2O (1:3, v/v)
with a 5.0 mL min−1 flow rate, to obtain 50 fractions (25 mL each),
which were combined into 15 major fractions (D1−D15) by TLC
evaluation. D1 (35 mg) was subjected to silica gel CC eluting with
EtOAc/MeOH/H2O (200:16:7), affording 7 (25 mg). Pure 1 (30 mg)
F
DOI: 10.1021/acs.jnatprod.5b00488
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
which was identified by NMR spectroscopic comparison with literature
data.20
were compared using the CompareVOA program (Biotools). All
Gaussian calculations were carried out using a server node with 16
processors at 2.60 GHz and 16 Gb of RAM.
Antiprotozoal Assays. The antiprotozoal activity of isolated
compounds against Entamoeba histolytica HM1-IMSS and Giardia
lamblia IMSS:1090:1 strains was determined using the reported
method.26 The resulting data are given in Table 5.
Alkaline Hydrolysis. A solution of 1 (30 mg) in 10% KOH/EtOH
(5 mL) was heated at 70 °C for 4 h. After acidification with HOAc, the
solvent was removed and the residue was partitioned between n-
BuOH and H2O (10 mL × 3). The n-BuOH layer was concentrated in
vacuo to yield a white powder, which was identified as 4 (18 mg) by
NMR spectroscopic comparison.
Acid Hydrolysis. To a mixture of 2−4 (35 mg) in MeOH (5 mL)
was added 10% HCl (10 mL), and the mixture was refluxed for 8 h,
followed by extraction with CH2Cl2 (3 × 20 mL) to give an aqueous
fraction containing sugars and a CH2Cl2 fraction containing 10, which
was characterized23 by 1H and 13C NMR data. The aqueous phase was
neutralized with 1 N KOH, extracted with n-BuOH (20 mL), and
washed with H2O (3 × 10 mL), and after removal of the solvent under
reduced pressure, the sugars were converted into their silylated
derivatives by treatment with bis(trimethylsilyl)trifluoracetamide and
then analyzed by GC-MS, as described.24
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00488.
■
S
1H, 13C, DEPT 135, HSQC, HMBC, COSY, NMR,
1
ESIMS, and IR spectra for compounds 1 and 4; H and
13C NMR spectra for compounds 2, 3, and 5 and a
mixture of 5 and 6 (PDF)
A solution of 4 (60 mg) in MeOH was subjected to acid hydrolysis
as described above. The aqueous phase was neutralized with Na2CO3
and extracted with n-BuOH (20 mL × 3). After removal of the solvent
under reduced pressure L-fucose was acetylated without isolation and
purified by TLC using hexanes/EtOAc (2:1), affording 3 mg of the
corresponding acetate, which was identified by 1H NMR and the
AUTHOR INFORMATION
Corresponding Author
*Tel: +52-55-5622-4411. Fax: +52-55- 5616-2217. E-mail:
quijano@unam.mx.
■
specific rotation [α]25 −33.2 (c 0.0028, CHCl3).
D
Enzymatic Hydrolysis. To a solution of a mixture of 1−3 (60 mg)
in 20 mL of 0.1 M NaOAc buffer (pH 5.0) was added β-glucosidase
from almonds (12 000 μL, Sigma-Aldrich 49290). The mixture was
stirred at 50 °C for 96 h, followed by extraction with EtOAc (3 × 20
mL), to give an organic fraction containing 4−6, which were identified
by TLC using reference samples. The aqueous phase was extracted
with n-BuOH (3 × 20 mL) and treated as described above to give 5
mg of D-glucose peracetate identified by 1H NMR and specific rotation
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors acknowledge H. Rios, R. Gavino, I. Chav
Quiroz, E. Huerta, A. Pena, R. Patino, L. Velasco, J. Per
■
́
ez, B.
ez, and
̃
́
̃
̃
S. Hernan
Direccion
Comunicacion
Mex
́
dez for collecting NMR, UV, IR, and MS data,
[α]25 +35.4 (c 0.01, CHCl3).
D
́
́
de Com
́
́
puto y de Tecnologias de Informacion
́
y
15-Acetoxykaurenoic Acid Methyl Ester (11): Grandifloric acid,
isolated from Montanoa gigas, showed spectroscopic data identical to
those published.21 Grandifloric acid (15 mg) was treated, using the
esterification and acetylation procedure for 8, to obtain 11 (12 mg).
NMR characterization agreed with the literature.22
de la Universidad Nacional Auton
́
oma de
́
ico, via Grant SC15-1-IG-69, Consejo Nacional de Ciencia
y Tecnologia (CONACYT-Mexico) for financial support via
́
Grants 152994 and 165614 and scholarship 240055, Posgrado
en Ciencias Quimicas, UNAM, and the Direccion
Asuntos del Personal Academ
203510.
́
Astragalin: yellow powder; mp 175−177 °C (reported 177−178
́
General de
ico (DGAPA) for Grant No. IN-
°C); NMR data were essentially the same as reported.7
́
Chlorogenic acid: white powder; mp 203−205 °C (reported 208
°C); NMR data were the same as reported.8
L-chiro-Inositol: colorless crystals; mp 237−241 °C (reported 240−
243 °C); [α]25D −50.0 (c 0.0101, H2O) (reported [α]20D −70 (c 0.55,
H2O)); NMR data were essentially the same as reported.9
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DOI: 10.1021/acs.jnatprod.5b00488
J. Nat. Prod. XXXX, XXX, XXX−XXX