Terpenoids from Melampodium perfoliatum

Article abstract of DOI:10.1021/acs.jnatprod.6b00321

The phytochemical study of the aerial parts of Melampodium perfoliatum afforded six melampolides (1, 3, 5-8), a eudesmanolide (9), two diterpene lactones (10, 11), and two ent-kaurane derivatives (12, 13), together with the known melampodin (2) and polymatin A (4). The structures of the compounds were elucidated by physical data analysis and chemical reactions. Compounds 2, 4, 5, and 10 exhibited dose-dependent anti-inflammatory activity on the 12-O-tetradecanoylphorbol-13-acetate-induced ear edema model, with ID₅₀ values of 1.14, 0.56, 1.15, and 1.49 μmol/ear, respectively, compared to the reference compound indomethacin (0.24 μmol/ear).

Full text of DOI:10.1021/acs.jnatprod.6b00321

Article
pubs.acs.org/jnp
Terpenoids from Melampodium perfoliatum
†
,†
†
L. Villasen
or,‡
Amira Arciniegas, Ana-L. Per
́
ez-Castorena,* Antonio Nieto-Camacho, Jose
́
̃
and Alfonso Romo de Vivar†
†
‡
Instituto de Química and Instituto de Biología, Universidad Nacional Auton
́
oma de Mex
́
ico, Universidad Nacional Auton
ico
́
oma de
Mexico, Circuito Exterior, Ciudad Universitaria, Coyoacan 04510, Ciudad de Mex
́
́
́
ico, Mex
́
*
S Supporting Information
ABSTRACT: The phytochemical study of the aerial parts of
Melampodium perfoliatum afforded six melampolides (1, 3, 5−8), a
eudesmanolide (9), two diterpene lactones (10, 11), and two ent-
kaurane derivatives (12, 13), together with the known
melampodin (2) and polymatin A (4). The structures of the
compounds were elucidated by physical data analysis and chemical
reactions. Compounds 2, 4, 5, and 10 exhibited dose-dependent
anti-inflammatory activity on the 12-O-tetradecanoylphorbol-13-
acetate-induced ear edema model, with ID50 values of 1.14, 0.56,
1.15, and 1.49 μmol/ear, respectively, compared to the reference
compound indomethacin (0.24 μmol/ear).
1
1
he genus Melampodium (Asteraceae, Heliantheae, Mel-
similar to those of melampodin (2). The resonances at δH
6.23 (d, J = 3.2 Hz, H-13a), 5.66 (d, J = 3.2 Hz, H-13b), 2.59
(dd, J = 10.0, 1.6 Hz, H-7), and 5.21 (t, J = 10.0 Hz, H-6) and
at δ 120.9 (CH , C-13), 169.0 (C, C-12), 134.4 (C, C-11),
T
ampodinae) comprises 40 annual and perennial species
widespread in tropical and subtropical regions of Mexico and
Central America, with five species found in southwestern
C
2
1
United States and three in Colombia and Brazil. This genus
50.7 (CH, C-7), and 75.7 (CH, C-6) were indicative of an α-
methylene-γ-lactone moiety. An angeloyloxy residue was
deduced from the resonances of two methyl groups at δH
1.91 (quint, J = 1.2 Hz, H-5′) and 1.99 (dq, J = 7.2, 1.2 Hz, H-
can be characterized by the presence of melampolide-type
2
3
germacranolides; however, flavonoids and some diterpe-
4
,5
noids have also been reported. Melampolides are known to
present a wide variety of biological activities including
9
4′) coupled to that of a methine proton at δ 6.13 (qq, J = 7.2,
H
6
7
8
antitumor, antidiabetic, anti-inflammatory, and fungicidal.
1.2 Hz, H-3′). In the HMBC spectrum, a correlation of H-8 (δ
H
The aim of this paper is to contribute to the chemistry of the
genus studying the aerial parts of Melampodium perfoliatum and
to search for anti-inflammatory compounds. In previous studies,
6.27, dd, J = 8.4, 1.6 Hz) with C-1′ (δ 167.4) permitted the
C
assignment of this ester group at C-8. Correlations of H-2 (δH
3.62, dd, J = 3.6, 2.4 Hz) and H-3 (δ 3.63, brd, J = 3.6 Hz)
H
5
Bohlmann and co-workers reported the presence of kaurenoic
with C-4 and C-1 defined the 2,3-epoxide function, and cross-
peaks of H-1 and H-9 with C-14 established a C-14
methoxycarbonyl group. A hydroxy group was assigned to C-
acid derivatives, polyacetylenes, and enhydrin in the roots of
M. perfoliatum, and the effect of its crude syrups on fall
10
armyworm was investigated by Fischer and co-workers.
9 by the COSY correlation between H-9 (δ 4.04, d, J = 8.4
H
Herein the isolation and characterization of 11 new
Hz) and H-8. The 1(10)E,4E-cyclodecadiene, i.e., melampolide
terpenoidssix melampolides (1, 3, 5−8), one eudesmanolide
skeleton, was inferred from the NOESY correlations of H-1
(
9), two diterpene lactones (10, 11), and two ent-kaurene
with the C-14 methoxy group and H-5 and of H -15 with H-6.
3
derivatives (12, 13)in addition to the known melampodin
In the same spectrum, correlations of H-8 with H-7, as well as
their small coupling constant J7,8 = 1.6 Hz, indicated that they
are cofacial and α-oriented, based upon the biogenetic
1
1,12
13
(2)
and polymatin A (4) are reported. The structures of
these compounds were defined by physical data analysis and
chemical reactions. The anti-inflammatory activity of extracts
and more abundant compounds 2−5, 8, 10, and 12 was
evaluated using the 12-O-tetradecanoylphorbol-13-acetate
14
considerations regarding the orientation of H-7. The large
J6,7 = 10.0 Hz and J8.9 = 8.4 Hz coupling constants defined the
β-orientation of H-6 and H-9 and disclosed a trans-fused
lactone ring. Compound 1 has, therefore, the same relative
(TPA)-induced edema model of acute inflammation.
11,12
configuration as described for melampodin (2),
thus
RESULTS AND DISCUSSION
possessing a 10-membered ring system in which the preferred
■
conformation has C-14 and C-15 in an anti-arrangement,
Perfoliatin A (1) has a molecular formula of C H O
according to its HRDARTMS and C NMR data. The IR
2
1
24
8
15
1
3
typical of melampolides. Additionally, the NOESY spectrum
−
1
spectrum showed the presence of hydroxy (3480 cm ),
−1
conjugated γ-lactone (1764 cm ), and α,β-unsaturated ester
Received: April 8, 2016
−1
(
1715 cm ) fuctionalities. Its NMR data (Table 1) were
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
Table 1. NMR Spectroscopic Data (400 MHz, CDCl ) for Compounds 1, 3, and 5
3
a
1
3
5
position
1
δH (J in Hz)
6.92, d (2.4)
δC
δH (J in Hz)
7.02, d (2.0)
δC
δH (J in Hz)
6.86, dd (10.5, 7.5)
δC
134.8, CH
56.3, CH
139.2, CH
56.5, CH
144.5, CH
25.8, CH2
2
2
3
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
2
3
4
5
1
2
3
4
5
a
3.62, dd (3.6, 2.4)
3.64, dd (3.6, 2.4)
2.50, dddd (13.5, 7.5, 6.0, 2.5)
2.26, dddd (13.5, 12.5, 10.5, 2.5)
2.37, ddd (13.5, 6.0, 2.5)
2.04, brt (13.5)
b
a
3.63, brd (3.6)
60.3, CH
3.77, d (3.3)
60.6, CH
36.8, CH2
b
131.3, C
122.9, CH
75.7, CH
50.7, CH
70.8, CH
72.0, CH
134.5, C
134.4, C
169.0, C
120.9, CH2
135.5, C
122.4, CH
75.2, CH
50.0, CH
69.2, CH
72.8, CH
127.3, C
134.0, C
168.6, C
121.2, CH2
137.8, C
126.4, CH
73.1, CH
51.3, CH
71.0, CH
75.3, CH
133.5, C
134.8, C
169.1, C
121.0, CH2
5.32, dq (10.0, 1.6)
5.21, t (10.0)
5.33, dq (10.4, 1.2)
5.26, t (10.4)
4.95, brd (10.5)
5.09, t (10.5)
2.64, brd (10.0)
6.29, dd (8.5, 2.0)
3.96, m
2.59, dd (10.0, 1.6)
6.27, dd (8.4, 1.6)
4.04, d (8.4)
2.72, dd (10.4, 1.2)
6.78, dd (8.8, 1.2)
5.40, d (8.8)
0
1
2
3a
3b
4
5
′
′
′
′
′
″
″
″
″
″
6.23, d (3.2)
5.66, d (3.2)
6.27, d (3.2)
5.72, d (3.2)
6.24, d (3.5)
5.64, d (3.5)
166.7, C
165.3, C
17.4, CH3
168.6, C
59.2, C
166.9, C
16.8, CH3
169.7, C
59.6, C
2.09, d (1.6)
17.2, CH3
167.4, C
2.18, d (1.2)
1.91, d (1.0)
126.6, C
6.13, qq (7.2, 1.2)
1.99, dq (7.2, 1.2)
1.91, quint (1.2)
140.4, CH
15.9, CH3
20.5, CH3
3.06, q (5.2)
1.22, d (5.2)
1.52, s
60.0, CH
13.7, CH3
19.3, CH3
169.6, C
59.5, C
3.05, q (5.5)
1.28, d (5.5)
1.57, s
59.9, CH
13.7, CH3
19.2, CH3
3.02, q (5.2)
1.16, d (5.2)
1.48, s
60.0, CH
13.3, CH3
18.7, CH3
52.9, CH3
OCH3
3.85, s
52.8, CH3
3.83, s
3.81, s
52.9, CH3
a
5
00 MHz.
12
also showed cross-peaks between H -15, H-9, and H-3,
absolute configuration of melampodin (2) has been reported,
3
indicating the α-orientation of the epoxide function. The
and since the ECD spectrum of 1 was similar to that of 2, the
B
DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
positive Cotton effect at λ 245 nm permitted definition of the
absolute configuration of perfoliatin A as [2R, 3S, 6R, 7S, 8S,
Nevertheless, the ECD spectrum of these compounds did not
show the same pattern as that of melampodin; therefore their
absolute configurations were not defined.
9S, 1(10)E, 4E].
Perfoliatin B (3) has a molecular formula of C H O as
determined by HRDARTMS and C NMR data. The IR and
NMR data for compound 3 were similar to those of compound
Perfoliatin F (8) showed a molecular formula of C H O
on the basis of HRDARTMS and C NMR spectra. H and
C NMR spectroscopic data of 8 were comparable to those of
2
6
30 11
26 32 11
1
3
13
1
13
1
except for the presence of typical NMR resonances (Table 1)
5, with the exception of the resonances of H-5 (δ 2.67, d, J =
H
of two 2,3-epoxyangeloyloxy units. In the HMBC spectrum,
correlations between H-8 (δ 6.78, dd, J = 8.8, 1.2 Hz) and H-9
9.5 Hz), C-5 (δ 62.7), H -15 (δ 1.71, s), C-15 (δ 17.4), and
C
3
H
C
H
C-4 (δ 59.2), assigned to a 4,5-epoxide function and those of a
C
(
δH 5.40, d, J = 8.8 Hz) with C-1′ and C-1″ permitted the
second epoxyangeloyloxy group located at C-9 by the HMBC
cross-peaks. The NOESY spectrum (Figure 1) showed
assignment of these ester groups to C-8 and C-9, respectively.
The NOESY spectrum of perfoliatin B confirmed the same
relative configuration as for compounds 1 and 2, and since the
ECD spectrum showed the same pattern as that of melampodin
correlations of H-1 with H-2α (δ 2.45, dddd, J = 13.5, 7.0,
H
7.0, 2.0 Hz), of H-5 with H-7 and H-3α (δ 1.24, brd, J = 13.5
H
Hz), of H-9 with H-6 and H-2β (δ 2.99, m), and of H -15
H
3
(2), the absolute configuration of 3 could be defined as [2R, 3S,
with H-9 and H-6, indicating the trans-4,5-epoxide function and
a relative configuration similar to that of melampodin. The
ECD spectrum of perfoliatin F exhibited the same pattern as
that of compound 2; therefore it possesses a [4R, 5R, 6R, 7S,
6R, 7S, 8S, 9S, 1(10)E, 4E].
Perfoliatin C (5) showed a molecular formula of C H O
21
26
8
1
3
1
by analysis of its HRDARTMS and C NMR data. In the H
NMR data of compound 5 (Table 1) the resonances of the 2,3-
epoxy group, present in melampodin, were replaced by those of
H -2 (δ 2.50, dddd, J = 13.5, 7.5, 6.0, 2.5 Hz, H-2a and 2.26,
dddd, J = 13.5, 12.5, 10.5, 2.5 Hz, H-2b) and H -3 (δ 2.37,
8
S, 9S, 1(10)E] absolute configuration.
De-O-acetylmeleucanthin (9) exhibited a molecular formula
13
2
H
of C H O according to its HRDARTMS and C NMR data.
21 24 8
2
H
The IR spectrum revealed the presence of hydroxy (3522
−
1
−1
−1
ddd, J = 13.5, 6.0, 2.5 Hz, H-3a and 2.04, brt, J = 13.5 Hz, H-
b). The NOESY spectrum of 5 exhibited essentially the same
cm ), γ-lactone (1770 cm ), and ester (1713 cm )
functionalities. Analysis of the 1D and 2D NMR spectra
showed the presence of an α-methylene-12,6-γ-lactone moiety
3
correlations as those of compounds 1 and 3; therefore its
relative configuration should be the same. The [6R, 7S, 8S, 9S,
(
Table 2), as in compounds 1−8. The olefinic methines H-1 at
1(10)E, 4E] absolute configuration was deduced from the
δ 5.29 (brd, J = 9.5 Hz), H-2 at δ 5.93 (dd, J = 9.5, 5.5 Hz,),
H
H
similarity of the ECD spectrum of 1, perfoliatin C, with that of
melampodin.
Perfoliatin D (6) and perfoliatin E (7) showed IR and NMR
spectroscopic data closely related to those of 5, and their
molecular formulas were established as C H O and
C H O , respectively, by HRDARTMS and C NMR data.
In perfoliatin D, additional resonances of an angeloyloxy
residue and of an oxymethine at δ 4.60 (brd, J = 4.4 Hz) were
observed. The angeloyloxy moiety was assigned to C-9 by
analysis of the HMBC spectrum, and the oxymethine to C-3 by
the COSY correlation between H-3 (δ 4.60, brd, J = 4.4 Hz)
and H -2 (δ 2.86, m). Oxymethine resonances in the NMR
spectrum of 7 at δ 5.07 (dd, J = 6.0, 5.2 Hz), 4.65 (d, J = 5.2
Hz), and 4.85 (d, J = 8.4 Hz) were assigned to H-2, H-3, and
H-9, respectively, on the basis of COSY and HMBC
correlations. Similarly, an angeloyloxy group was located at
C-8. In the NOESY spectrum of perfoliatin D (Figure 1), a
and H-3 at δ 5.69 (d, J = 5.5 Hz) were assigned by the cross-
H
peaks observed in the COSY experiment. An epoxyangeloyloxy
moiety at C-8 (δ 72.5) was deduced by the HMBC correlation
C
between H-8 (δ 5.67, t, J = 3.0 Hz) and C-1′ (δ 168.9), and a
H
C
2
6
32 10
hydroxy group was assigned to C-9 (δ 70.7) by the COSY
C
1
3
2
1
26
9
correlation between H-9 (δ 4.39, d, J = 3.0 Hz) and H-8. The
H
eudesmanolide skeleton was deduced from the HMBC
H
correlations of H-1 and H-9 with C-5 (δ 42.5) and of H-5
C
(
1
δH 3.04, d, J = 11.5 Hz) with the C-14 carbonyl carbon (δC
71.3). The relative configuration of 9 was defined by the
H
NOESY correlations of H-7 with H-5 and H-8 that suggested
2
H
their α-orientation, based upon biogenetic considerations
14
H
regarding the orientation of H-7. H-8 (J7,8 = 3.0 Hz) and
H-9 (J8,9 = 3.0 Hz) should be α- and β-equatorially oriented,
respectively, as defined by correlations of H-9 with H-1 and H-
16
8
. The coupling constants of the CH -13 (d, J = 3.0 Hz) and
2
the J6,7 value of 11.5 Hz indicated a trans-fused γ-lactone moiety
with a dihedral angle between H-6 and H-7 of around 170°
reminiscent of the β-axial orientation of H-6. The C-14
methoxycarbonyl group was considered β-oriented on bio-
14
genetic grounds.
Compound 10 was obtained as a colorless oil. Its molecular
formula, C H O , was assigned on the basis of HRDARTMS
20
30
7
and 1 C NMR spectroscopic data. The IR absorption bands at
3
1
3
468, 1712, 1685, and 1619 cm− were indicative of hydroxy,
1
3
carbonyl, and conjugated carbonyl functionalities. In the
C
NMR spectrum, the signals of two carbonyls and two vinylic
carbons accounted for three of the six indices of hydrogen
deficiency, indicating a tricyclic structure. The H NMR
Figure 1. NOESY correlations of compounds 6−8.
1
spectrum showed a vinylic proton (δ 6.08, hept, J = 1.2 Hz,
H
correlation of H-3 with H -15 indicated the α-orientation of the
hydroxy group. In perfoliatin E, NOESY correlations of H-1
H-14), which correlated in the HMBC spectrum with C-16 (δC
20.9), C-17 (δ 27.8), and C-13 (δ 196.6). Correlations of the
3
C
C
with H-2 and of H-3 with H -15 defined the β- and α-
later with H -12 (δ 3.60, d, J = 17.2 Hz, H-12a and 2.38, d, J =
3
2 H
orientations of the hydroxy groups at C-2 and C-3, respectively.
Additional NOESY correlations (Figure 1) showed that 6 and 7
had similar relative configurations to compounds 1−3 and 5.
17.2 Hz, H-12b), observed in the same experiment, defined the
presence of a methylpentenoyl moiety ((CH ) -C
3
2
CHCOCH −). The NMR signals at δ 172.1 (C-18) and δ
2
C
H
C
DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. NMR Spectroscopic Data (400 MHz, CDCl ) for Compounds 6−8
3
a
6
7
8
position
1
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
7.06, dd (9.6, 8.0)
2.86, m
145.5, CH
32.2, CH2
6.94, d (6.0)
143.9, CH
74.7, CH
7.17, dd (10.5, 7.5)
2.99, m
149.9, CH
24.6, CH2
2
2
3
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
2
3
4
5
1
2
3
4
5
a
5.07, dd (6.0, 5.2)
b
a
2.45, dddd (13.5, 7.0, 7.0, 2.0)
2.36, ddd (13.5, 7.0, 2.0)
1.24, brd (13.5)
4.60, brd (4.4)
74.0, CH
4.65, d (5.2)
76.3, CH
35.3, CH2
b
141.3, C
123.2, CH
74.9, CH
50.7, CH
70.7, CH
70.7, CH
131.6, C
134.2, C
169.1, C
121.9, CH2
141.3, C
125.8, CH
75.2, CH
51.6, CH
71.5, CH
71.3, CH
135.5, C
134.8, C
169.2, C
121.3, CH2
59.2, C
5.54, brd (11.2)
5.25, t (10.4)
2.88, m
5.30, brd (10.8)
5.26, t (10.8)
2.67, d (9.5)
4.29, t (9.5)
3.02, m
62.7, CH
75.8, CH
45.5, CH2
70.7,CH
71.6, CH
129.8, C
133.2, C
167.9, C
122.9, CH2
2.74, brd (10.8)
6.33, dd (8.4, 1.6)
4.85, d (8.4)
6.79, dd (8.4, 1.6)
5.36, d (8.4)
6.81, dd (8.5, 1.0)
5.90, d (8.5)
0
1
2
3a
3b
4
5
′
′
′
′
′
″
″
″
″
″
6.29, d (3.2)
5.79, d (3.2)
6.32, d (1.6)
5.68, d (1.6)
6.34, d (3.0)
5.85, d (3.0)
165.6, C
15.9, CH3
168.5, C
59.4, C
166.9, C
165.2, C
17.4, CH3
168.5, C
59.3, C
2.01, brs
2.03, d (1.2)
18.3, CH3
167.7, C
1.71, s
127.0, C
3.02, q (5.2)
1.16, d (5.2)
1.43, s
58.9, CH
13.7, CH3
19.1, CH3
166.8, C
126.5, C
141.2, CH
15.9, CH3
20.5, CH3
52.2, CH3
6.13, qq (7.2, 1.2)
1.99, dq (7.2, 1.2)
1.88, quint (1.2)
139.9, CH
15.9, CH3
20.6, CH3
3.04, m
60.0, CH
13.4, CH3
18.8, CH3
169.5, C
59.6, C
1.19, d (5.5)
1.47, s
6.17, qd (6.0, 1.2)
1.95, dq (6.0, 1.2)
1.81, quint (1.2)
3.79, s
3.04, m
60.0, CH
13.5, CH3
19.1, CH3
52.6, CH3
1.18, d (5.5)
1.52, s
OCH3
3.82, s
52.5, CH3
3.83, s
a
5
00 MHz.
4
.22 (dd, J = 8.0, 6.5 Hz, H-6) indicated the presence of a
3.77, dd, J = 8.0, 7.6 Hz) and H-2 (δ 4.08, dd, J = 7.6, 3.6 Hz)
and for the signals attributable to the diastereotopic methyl
H
17,18
saturated 18,6-ζ-lactone unit.
In the HMBC spectrum,
correlations of H-12 with C-18, C-11, and C-10; of H -8 with
groups of the acetonide moiety (δ 1.56, s, H -2′ and δ 1.42,
2
H
3
H
13
C-10, C-9, and C-7; and of H -19 with C-8, C-7, and C-6
s, H -3′). C NMR data were in agreement with structure 11.
3
3
established the connectivity of the ζ-lactone moiety and the
attachment of the methylpentenoyl moiety to C-11. A 10,11-
epoxide function was deduced from the chemical shifts of H-10
Compound 12 was obtained as colorless prisms, mp 245−
246 °C. The molecular formula C H O , determined by
20
34
3
HRFABMS and 13C NMR, indicated an index of hydrogen
13
(
δ 3.44, dd, J = 3.2, 2.4 Hz), C-10 (δ 62.8), and C-11 (δ
deficiency of four. In the C NMR spectrum the resonances of
three methyls, nine methylenes (one oxygenated), four
methines (one oxygenated), and four nonprotonated carbons
(one oxygenated) and the absence of resonances of carbonyl
and olefinic groups suggested a tetracyclic structure of the
kaurane type with a primary, a secondary, and a tertiary
H
C
C
61.0). The tetrahydropyran portion was evidenced by the
chemical shifts of C-3 (δ 85.7) and C-7 (δ 84.0), by the
C
C
COSY correlations of H-6 with H-5 and of H-5 with H-4, and
by the HMBC correlations of H-5 with C-6 and C-7 and of H3-
20 with C-3. Finally, the HMBC spectrum showed correlations
1
of H -1 (δ 3.74, dd, J = 11.2, 6.8 Hz, H-1a and 3.66, dd, J =
oxygenated carbon. In the H NMR spectrum the oxy-
2
H
1
1.2, 3.6 Hz, H-1b) with C-2 and C-3 and of H-2 (δ 3.56, dd,
methylene resonances at δ 3.69 (d, J = 11.0 Hz) and 3.58
H
H
J = 6.8, 3.6 Hz) with C-3, C-4, and C-20, in accordance with
structure 10. The relative configuration of the tricyclic ring
system in compound 10 was deduced by the NOESY
correlations of H -19 with H-6, H -20, and H-9b and of H-
(d, J = 11.0 Hz) were assigned to H -17 by their correlations
2
with C-15, C-16, and C-13 in the HMBC spectrum. Likewise,
the oxymethine resonance at δH 3.32 (t, J = 3.0 Hz) was
attributed to H-3 by its cross-peaks with C-1 and C-5, and a
third hydroxy group was located at C-16 (δC 82.8) by the
correlations of this carbon with H-17, H-14, and H-15. The α-
equatorial orientation of H-3 was deduced from the small
3
3
10 with H-12b and H-9b, indicating that they are cofacial.
Compound 11 is an artifact emanating from the isolation and
purification processes of compound 10. The molecular formula
13
C H O was deduced from analysis of HRDARTMS and
C
coupling constants of H -2/H-3 (3.0 Hz). The C-16 hydroxy
2
3
34
7
2
NMR spectroscopic data. The IR absorptions at 1712, 1686,
group was assigned an α-orientation via comparison of the
and 1620 cm− indicated carbonyl group and double-bond
1
chemical shifts of C-16 and C-17 with reported data.
19,20
The
1
functionalities, but no hydroxy groups. Its H NMR spectrum
NOESY interactions of H -20 and H -18α indicated that they
3 3
was highly similar to that of compound 10, except for the
paramagnetic shift of H -1 (δ 3.99, dd, J = 8.0, 3.6 Hz and
are cofacial and on the opposite side from H-9 and H -19,
which correlated with H-5 in the same spectrum. Thus, the
3
2
H
D
DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
structure of compound 12 is defined as 3β,16α,17-trihydroxy-
Compound 13 has a molecular formula of C H O as
2
6
44
8
2
1
13
ent-kaurane. The (3S,5S,8S,9R,10S,13R,16R) absolute config-
uration of 12 was established by X-ray crystallographic
determined by HRFABMS and C NMR data. The NMR
spectroscopic data of compound 13 were similar to those of 12
(Table 3), except for the signals of a sugar moiety. This sugar
was identified as β-D-glucopyranose by the axial−axial coupling
constants of H-1′, H-2′, H-3′, and H-4′ and by the isolation of
2
2
analysis (Figure 2).
2
5
D-glucose, [α] +56 (c 0.15, H O), following hydrolysis of 13.
D
2
In the 13C NMR spectrum of compound 13, C-3 appeared
deshielded (δ 87.5) and correlated with the anomeric proton
C
of the glucose (δ 4.29, d, J = 8.0 Hz) in the HMBC spectrum,
H
indicating that compound 13 is the 3-O-β-D-glucopyranosyl
derivative of 12. Hydrolysis of glucoside 13 also afforded
compound 14, which exhibited a molecular formula of
1
3
C H O according to its HRDARTMS and C NMR data.
20
32
2
−1
In the IR spectrum of 14 absorptions at 3476 and 1720 cm
1
indicated the presence of hydroxy and carbonyl groups. The H
NMR spectrum was similar to that of 12, with the additional
resonance of a formyl group at δH 9.65; this resonance was
assigned to H-17 by its correlation with C-16 in the HMBC
Figure 2. ORTEP drawing of compound 12.
spectrum. As in compound 12, H-3 showed (δ 3.42 t, J = 3.0
H
Hz) the same coupling constants, indicating its α-equatorial
Table 3. NMR Spectroscopic Data (500 MHz, CD OD) for Compounds 12−14
3
a
b
1
2
13
14
c
position
δH (J in Hz)
1.49, m
δC
δC
δH (J in Hz)
δC
δH (J in Hz)
1.55, m
δC
1
1
2
2
3
4
5
6
6
7
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
a
34.5, CH2
33.9, CH2
26.4, CH2
1.48, m
35.3, CH2
33.3, CH2
b
a
1.23, brt (13.5)
1.98, brt (13.5)
1.48, m
1.33, m
1.88, m
1.25, m
26.3, CH2
25.3, CH2
1.96, brt (13.5)
1.53, m
25.3, CH2
b
3.32, t (3.0)
76.8, CH
38.1, C
75.1, CH
38.0, C
3.35, m
87.5, CH
39.3, C
3.42, t (3.0)
76.0, CH
37.5, C
1.29, dd (12.0, 1.5)
1.46, m
50.0, CH
21.1, CH2
49.0, CH
20.5, CH2
1.35, m
1.50, m
1.39, m
1.57, m
50.4, CH
20.9, CH2
1.27, m
1.48, m
1.32, m
1.49, m
48.9, CH
20.3, CH2
a
b
a
1.37, m
1.60, m
43.2, CH2
42.7, CH2
43.1, CH2
40.8, CH2
b
1.52, m
45.8, C
44.9, C
45.8, C
45.9, C
1.10, d (7.0)
58.0, CH
40.3, C
57.0, CH
39.6, C
1.10, d (6.0)
57.9, CH
40.2, C
1.17, brd (7.5)
55.8, CH
39.1, C
0
1
1.59, m
19.3, CH2
27.3, CH2
18.7, CH2
26.9, CH2
1.61, m
1.61, m
19.3, CH
27.4, CH2
1.66, m
18.5, CH2
31.1, CH2
2a
2b
3
1.63, brd (9.0)
1.53, m
1.66, m
1.56, m
2.03, m
46.4, CH
38.2, CH2
46.1, CH
37.8, CH2
2.01, m
46.5, CH
38.2, CH2
2.56, m
37.8, CH
37.7, CH2
4a
4b
5a
5b
6
1.93, brd (12.0)
1.59, m
1.92, d (11.2)
1.60, m
1.89, brd (12.9)
0.84, brd (12.9)
1.51, d (14.5)
1.38, d (14.5)
54.0, CH2
54.0, CH2
1.52, m
54.1, CH2
1.73, dd (13.5, 5.7)
40.4, CH2
1.38, m
82.8, C
81.5, C
82.8, C
2.62, brd (5.7)
9.65, s
53.6, CH
203.8, C
7a
7b
8
3.69, d (11.0)
3.58, d (11.0)
0.83, s
66.7, CH2
66.4, CH2
3.69, d (11.6)
3.58, d (11.6)
0.84, s
66.9 CH2
22.6, CH3
29.1, CH3
18.3, CH3
22.4, CH3
29.4, CH3
18.0,CH3
22.7, CH3
29.1, CH3
18.5, CH3
106.4, CH
75.8, CH
78.3, CH
71.7, CH
77.6, CH
62.8, CH2
0.84, s
0.95, s
1.01, s
22.1, CH3
28.5, CH3
17.3, CH3
9
0.90, s
0.99, s
0
1.06, s
1.11, s
′
′
′
′
4.29, d (8.0)
3.20, t (8.8)
3.32, t (8.8)
3.28, t (8.8)
3.21, m
′
H6′a
H6′b
3.82, dd (12.0, 2.5)
3.65, dd (12.0, 5.0)
a
b
c
4
00 MHz. CDCl . Pyridine-d .
3
5
E
DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
orientation. The NOESY correlations for rings A, B, and C
were similar to those of compound 12, suggesting that 14 is the
dehydration product of 12. Finally, the NOESY correlation
between H-16 and H-12 defined the α-orientation of the formyl
group.
Germany). Flash column chromatography (FCC) was performed on
silica gel 230−400 (Macherey-Nagel, Du
̈
ren, Germany). Analytical
TLC was carried out on Si gel 60 GF or RP-18W/UV (10−40
254
254
̈
μm, Macherey-Nagel, Duren, Germany), and preparative TLC on Si
gel GF254 layer thickness 2.0 mm or RP-18W/UV layer thickness 1.0
254
11,12
mm, using 10 × 20 cm plates.
Plant Material. Melampodium perfoliatum (Cav.) Kunth was
The known compounds melampodin (2)
A (4) were identified by comparison of their physical and
spectroscopic features with reported data.
and polymatin
1
3
collected in Ozumba County, Mexico State, Mex
013 and authenticated by one of the authors (J.L.V.). A voucher
specimen (MEXU 1369108) was deposited at the Herbario del
Instituto de Biologia, UNAM, Mexico.
́
ico, in September
2
The phytochemical study of M. perfoliatum afforded
melampolides and kaurane derivatives, as main secondary
metabolites, in accordance with previous reports on species of
́
́
Extraction and Isolation. Dried and ground aerial parts (2 kg)
were extracted successively with petroleum ether, acetone, and MeOH
at room temperature. The petroleum ether extract (14 g) was
fractionated by VCC eluted with a petroleum ether−EtOAc gradient
system. Fractions A−E were obtained with mixtures (19:1, 4:1, 7:3,
1:1) and EtOAc, respectively. Fraction A afforded 150 mg of a mixture
of β-sitosterol and stigmasterol. Fraction B (430 g) was further purified
using an FCC eluted with petroleum ether−acetone (4:1) to obtain
mixtures B and B . Mixture B (46 mg) was purified by preparative
2
−5
the genus Melampodium.
Inspired by the anti-inflammatory
8,23
activity reports displayed for this type of metabolites,
the
anti-inflammatory activity of extracts and more abundant
compounds 2−5, 8, 10, and 12 was tested using the TPA-
2
4
induced ear acute inflammation model. Evaluations in
primary screening were performed at doses of 1 mg/ear.
Petroleum ether, acetone, and MeOH extracts displayed mild
activity, with edema inhibitions of 54.61 ± 7.29%, 36.93 ±
1
2
1
TLC [CH Cl −acetone (49:1) × 4] to obtain polymatin A (4, 10 mg).
2
2
2
1
.66%, and 20.72 ± 2.25%, respectively. Compounds 3, 8, and
Mixture B (28 mg), by preparative TLC (CH Cl −acetone, 19:1 ×
2
2
2
2, with less than 50% of edema inhibition, were considered
3), afforded de-O-acetylmeleucanthin (9, 12 mg). Fraction C (712
mg) was subjected to FCC (petroleum ether−acetone, 3:1) to yield
not active. Compounds 2, 4, 5, and 10 exhibited a dose-
dependent anti-inflammatory activity, with ID50 values of 1.14,
fractions C and C . Fraction C (113 mg) was purified by preparative
1 2 1
TLC [petroleum ether−acetone (4:1) × 3] to afford C and C ; C
0
.56, 1.15, and 1.49 μmol/ear, respectively, with activity lower
1a
1b
1a
(
32 mg) was purified by preparative RPTLC (MeOH−H O, 1:1) to
than that of the reference compound, indomethacin (0.24
μmol/ear). The presence of the α-methylene-γ-lactone moiety
seems to be one of the structural requirements for anti-
inflammatory activity of sesquiterpene lactones, and there is
some evidence of a positive effect on activity by the presence of
an acetoxy group at C-9 and of a methoxycarbonyl group at C-
2
afford perfoliatin A (1, 5 mg), while C1 (52 mg), by preparative
b
RPTLC (MeOH−H O, 1:1), yielded perfoliatin D (6, 5 mg) and
2
25
perfoliatin F (8, 4 mg). Preparative RPTLC (MeOH−H O, 1:1) of
2
fraction C (68 mg) afforded 10 mg of 8. Fraction D (860 mg) was
2
purified by two successive FCCs eluted with petroleum ether−acetone
(
7:3) to afford melampodin (2, 25 mg) and mixture D . Mixture D
1 1
2
6
1
0. Our results on the TPA model showed that the most
was purified by preparative TLC (petroleum ether−acetone, 7:3) to
afford perfoliatin C (5, 40 mg). Fractions E (1.7 g) after two successive
FCCs eluted with petroleum ether−acetone (7:3) and CH Cl −
active compound was polimatin A (4) (ID50 0.56 μmol/ear),
whose ability to inhibit NO production has been already
2
2
2
6
reported. The activity decreased with the epoxidation of the
acetone (4:1), respectively, afforded compound 10 (15 mg) and a less
polar compound (11, 9 mg) formed during the purification process.
The acetone extract (17 g) was fractionated by VCC using a
petroleum ether−acetone gradient system as eluent. Fractions eluted
with petroleum ether−acetone (19:1) yielded mixture F, the fraction
eluted with petroleum ether−acetone (17:3) afforded mixtures G and
H, and those obtained with petroleum ether−acetone (7:3) yielded
mixture J. From mixture F, 120 mg of a mixture of β-sitosterol and
stigmasterol was isolated. Mixture G (1.1 g) was purified by FCC
angeloyloxy group at C-8 in compound 5 (ID 1.15 μmol/ear),
5
0
and the presence of a 2,3 epoxy moiety did not seem to affect
the edema inhibition since compounds 2 and 5 had nearly the
same ID50 values (ID50 1.14 and 1.15 μmol/ear, respectively).
In addition, when an epoxyangeloyloxy group at C-9 was
present, there was loss of activity, as in compound 3, suggesting
that ester groups bigger than acetoxy at C-9 had a negative
effect on edema inhibition.
eluted with petroleum ether−acetone (4:1) to obtain mixtures G and
1
G . Mixture G (180 mg), by FCC (CH Cl −acetone 19:1) followed
2
1
2
2
EXPERIMENTAL SECTION
of preparative TLC (CH Cl −acetone, 19:1), afforded 5 (20 mg).
2
2
■
Mixture G (352 mg) was subjected to two successive FCCs eluted
2
General Experimental Procedures. Melting points were
determined on a Fisher-Johns melting point apparatus and are
uncorrected. Optical rotations were obtained on a PerkinElmer 343
polarimeter. UV and IR spectra were recorded on a Shimadzu UV
with CH Cl −acetone (19:1) and petroleum ether−acetone (7:3) to
2
2
obtain polymatin A (4, 5 mg) and perfoliatin B (3, 5 mg). Mixture H
(
7
1.6 g) yielded, after purification by FCC (petroleum ether−acetone,
:3), compound 12 (35 mg) and mixture H . Purification of H via
1
60U and a Bruker Tensor 27 spectrometer, respectively. ECD were
1
1
FCC (CH Cl −acetone, 19:1) afforded 2 (40 mg). Mixture J (690
obtained on a Jasco J-720 spectropolarimeter. X-ray diffraction analysis
was performed on a Bruker D8 Venture diffractometer with Cu Kα
radiation. 1D and 2D NMR spectra were obtained on a Bruker Avance
III 400 MHz or a Varian-Unity Inova 500 MHz spectrometer with
tetramethylsilane (TMS) as internal standard. For DARTMS a JEOL
AccuTOF JMS-T100LC DART was used. FABMS data were obtained
on a JEOL MStation JMS-700 mass spectrometer operated with an
acceleration voltage of 10 kV, with samples desorbed from a
nitrobenzyl alcohol matrix using 6 kV xenon atoms. HRFABMS
were performed at 10 000 resolution using electric field scans and
polyethylene glycol ions (Fluka 200 and 300) as the reference material.
GC analysis was performed on an Agilent 6890 GC system with an
AT5 column (30 m × 0.25 mm, 0.1 μm film thickness) using a
temperature gradient starting at 100 °C, which was raised to a final
temperature of 240 °C in 10 min. Column chromatography (VCC)
was carried out under vacuum on silica gel G 60 (Merck, Darmstadt,
2
2
mg) after two consecutive FCCs (CH Cl −acetone, 4:1, and
2
2
petroleum ether−acetone, 1:1), followed of a preparative RPTLC
(
(
MeOH−H O, 1:1), gave perfoliatin E (7, 5 mg). The MeOH extract
2
40 g) was fractionated in a VCC using as eluent an EtOAc−MeOH
gradient system. Fractions eluted with EtOAc afforded 250 mg of β-
sitosterylglucopyranoside and fraction K (1.2 g). Purification of K via
FCC (EtOAc−MeOH, 49:1) yielded 35 mg of compound 13. Sucrose
(120 mg) was also obtained from fractions eluted with EtOAc−MeOH
(7:3) from the main column.
Perfoliatin A (1): white, amorphous powder; [α]25
+109 (c 0.1,
D
−4
CHCl
); UV (MeOH) λmax (log ε) 207 (4.43) nm; ECD (0.19 × 10
) νmax 3480, 1764,
1715 cm ; H NMR and C NMR data, see Table 1; DARTMS m/z
3
M, MeOH) [ϕ]214 −9262, [ϕ]246 +2862; IR (CHCl
3
−1
1
13
+
+
405 [M + H] ; HRDARTMS m/z 405.15480 [M + H] (C H O
2
1
25
8
requires 405.15494).
F
DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Melampodin (2): colorless prisms (hexane−acetone); mp 210−212
NMR (CDCl , 100 MHz) δ 196.6 (C, C-13), 172.1 (C, C-18), 157.2
(C, C-15), 123.3 (CH, C-14), 85.7 (C, C-3), 84.0 (C, C-7), 82.3 (CH,
3
°
C; [α]25 +151 (c 0.1, MeOH); UV (MeOH) λ (log ε) 207 (4.41)
D
max
−4
nm; ECD (0.23 × 10 M, MeOH) [ϕ] −13 318, [ϕ] +4121; IR
CHCl ) ν 3486, 1764, 1720 cm ; DARTMS m/z 421 [M + H] ;
HRDARTMS m/z 421.15037 [M + H] (C H O requires
C-6), 76.5 (CH, C-2), 63.4 (CH , C-1), 62.8 (CH, C-10), 61.0 (C, C-
214
245
2
−1
+
(
11), 49.6 (CH , C-12), 34.8 (CH , C-4), 31.4 (CH , C-8), 27.8 (CH ,
3
max
2
2
2
3
+
2
1
25
9
C-17), 26.4 (CH , C-5), 22.6 (CH , C-9), 22.3 (CH , C-20), 22.1
2 2 3
+
4
21.14986).
(CH , C-19), 20.9 (CH , C-16); DARTMS m/z 383 [M + H] ;
3 3
Perfoliatin B (3): white, amorphous powder; [α]25D +35 (c 0.1,
+
HRDARTMS m/z 383.20673 [M + H] (C H O requires
20
31
7
−4
CHCl ); UV (MeOH) λ (log ε) 206 (4.18) nm; ECD (0.46 × 10
383.20698).
1,2-Acetonide-3,7:10,11-diepoxy-1,2,dihydroxy-13-oxo-14-phyt-
3
max
M, MeOH) [ϕ] −8819, [ϕ] +4132; IR (CHCl ) ν 1772, 1747,
2
14
248
3
max
−1
1
13
25
1
5
715 cm ; H NMR and C NMR data, see Table 1; DARTMS m/z
19 [M + H] ; HRDARTMS m/z 519.18708 [M + H] (C H O
en-18,6-olide (11): colorless oil; [α]
−24 (c 0.1, CHCl ); UV
D
3
+
+
26
31 11
(MeOH) λmax (log ε) 205 (3.37), 235 (3.73) nm; IR (CHCl ) ν
1712, 1686, 1620 cm ; H NMR (CDCl , 400 MHz) δ 6.06 (1H,
3 max
−1 1
requires 519.18664).
3
Polymatin A (4): colorless gum; [α]25D +35 (c 0.1, CHCl ); UV
3
hept, J = 1.2 Hz, H-14), 4.47 (1H, t, J = 6.8 Hz, H-6), 4.08 (1H, dd, J =
7.6, 3.6 Hz, H-2), 3.99 (1H, dd, J = 8.0, 3.6 Hz, H-1a), 3.77 (1H, dd, J
= 8.0, 7.6 Hz, H-1b), 3.70 (1H, d, J = 17.2 Hz, H-12a), 3.28 (1H, dd, J
= 2.8, 2.4 Hz, H-10), 2.51 (1H, m, H-9a), 2.37 (1H, d, J = 17.2 Hz, H-
12b), 2.30 (1H, m, H-9b), 2.17 (1H, m, H-5a), 2.13 (3H, d, J = 1.2 Hz,
H-16), 2.04 (1H, m, H-5a), 2.01 (1H, m, H-8a), 1.95 (2H, m, H-4a,
H-5b), 1.89 (3H, d, J = 1.2 Hz, H-17), 1.85 (1H, m, H-8b), 1.67 (1H,
m, H-4b), 1.56 (3H, s, H-2′), 1.42 (3H, s, H-3′), 1.33 (3H, s, H-19),
(
[
MeOH) λmax (log ε) 207 (4.38) nm; ECD (0.20 × 10−4 M, MeOH)
−
1
ϕ]216 −6035, [ϕ] +486; IR (CHCl ) ν 3469, 1767, 1717 cm ;
247
3
max
+
DARTMS m/z 391 [M + H] ; HRDARTMS m/z 391.17522 [M +
+
H] (C H O requires 391.17568).
21
27
7
25
Perfoliatin C (5): white, amorphous powder; [α]
+11 (c 0.1,
D
−
4
CHCl ); UV (MeOH) λ (log ε) 206 (4.11) nm; ECD (0.49 × 10
3
max
M, MeOH) [ϕ]2 −4641, [ϕ] +436; IR (CHCl ) ν 3499, 1764,
13
246
3
max
−
1
1
13
1
1
.22 (3H, s, H-20); 13C NMR (CDCl , 100 MHz) δ 196.8 (C, C-13),
71.7 (C, C-18), 156.8 (C, C-15), 123.2 (CH, C-14), 109.4 (C, C-1′),
1
747, 1718 cm ; H NMR and C NMR data, see Table 1;
DARTMS m/z 407 [M + H] ; HRDARTMS m/z 407.17049 [M +
H] (C H O requires 407.17059).
Perfoliatin D (6): white, amorphous powder; [α]
3
+
+
21
27
8
84.1 (C, C-7), 83.3 (C, C-3), 80.7 (CH, C-2), 79.0 (CH, C-6), 65.7
(CH , C-1), 63.4 (CH, C-10), 61.4 (C, C-11), 50.2 (CH , C-12), 33.3
2
5
D
−16 (c 0.1,
−4
2
2
CHCl ); UV (MeOH) λ (log ε) 205 (4.03) nm; ECD (0.48 × 10
3
max
(CH , C-4), 31.9 (CH , C-8), 27.7 (CH , C-17), 26.3 (CH , C-2′),
2
2
3
3
M, MeOH) [ϕ] −4335, [ϕ] +52; IR (CHCl ) ν 3485, 1764,
2
21
253
3
max
25.8 (CH , C-5), 25.2 (CH , C-3′), 22.9 (CH , C-9), 22.5 (CH , C-
2
3
2
3
−1
1
13
1
5
715 cm ; H NMR and C NMR data, see Table 2; DARTMS m/z
05 [M + H] ; HRDARTMS m/z 505.20773 [M + H] (C H O
2
0), 22.3 (CH , C-19), 20.9 (CH , C-16); DARTMS m/z 423 [M +
3 3
+
+
+
+
26
33 10
H] ; HRDARTMS m/z 423.23836 [M + H] (C H O requires
23 35 7
requires 505.20737).
4
23.23828).
(3S,5S,8S,9R,10S,13R,16R)-3,16,17-Trihydroxy-ent-kaurane (12):
Perfoliatin E (7): white, amorphous powder; [α]25D −4 (c 0.1,
−4
CHCl ); UV (MeOH) λ (log ε) 205 (3.90) nm; ECD (0.47 × 10
25
3
max
colorless prisms (MeOH); mp 245−246 °C; [α]
MeOH); IR (KBr) ν 3263 cm ; H NMR and C NMR see Table
3
−28 (c 0.1,
D
M, MeOH) [ϕ]212 −1141, [ϕ] −1013, [ϕ] ∼0; IR (CHCl ) ν
−1
1
13
222
250
3
max
−1
1
13
max
3
489, 1765, 1715 cm ; H NMR and C NMR data, see Table 2;
+
; HRFABMS m/z 322.2511 [M] (C H O requires 322.2508).
+
20 34 3
DARTMS m/z 423 [M + H] ; HRDARTMS m/z 423.16532 [M +
3
-O-β-D-Glucopyranosyl-3,16,17-trihydroxy-ent-kaurane (13):
+
H] (C H O requires 423.16551).
25
1
21
27
9
white, amorphous powder; [α] −5 (c 0.1, MeOH); H NMR and
C NMR see Table 3; HRFABMS m/z 507.2926 [M + Na]
2
5
D
Perfoliatin F (8): white, amorphous powder; [α]
−43 (c 0.1,
13
+
D
−4
CHCl ); UV (MeOH) λ (log ε) 208 (4.20) nm; ECD (0.38 × 10
3
max
(
C H O Na requires 507.2934).
26
44
8
M, MeOH) [ϕ] −7768, [ϕ] +998; IR (CHCl ) ν 1773, 1758,
2
13
248
3
max
3
β-Hydroxy-ent-kaurane-17-al (14): white, amorphous powder;
−1
1
13
1
5
714 cm ; H NMR and C NMR data, see Table 2; DARTMS m/z
21 [M + H] ; HRDARTMS m/z 521.20196 [M + H] (C H O
[α]25
−68 (c 0.1, CHCl
); IR (CHCl
) νmax 3476, 1720 cm ; H
−1
1
+
+
D
3
3
26 33 11
13
+
NMR and C NMR see Table 3; DARTMS m/z 304 [M] ;
HRDARTMS m/z 305.24897 [M + H] (C20
requires 521.20229).
+
De-O-acetylmeleucanthin (9): white, amorphous powder; [α]25
H
33
O
2
requires
D
3
05.24805).
Crystal Data for Compound 12.
+
238 (c 0.1, CHCl ); UV (MeOH) λ (log ε) 206 (4.04), 269 (3.50)
3 max
20
−
4
C H O H O, M
20 34 3 2 r
340.49,
nm; ECD (0.5 × 10 M, MeOH) [ϕ] −4468, [ϕ] +4497; IR
215
273
−1
1
monoclinic, space group P2 , a = 11.2505(4) Å, α = 90°, b =
1
(
6
5
(
CHCl ) ν 3522, 1770, 1713 cm ; H NMR (CDCl , 500 MHz) δ
3 max 3
7
9
.3824(2) Å, β = 93.6665(14)°, c = 11.4285(4) Å; γ = 90°, V =
3 3
.16 (1H, d, J = 3.0 Hz, H-13a), 5.93 (1H, dd, J = 9.5, 5.5 Hz, H-2),
.69 (1H, brd, J = 5.5 Hz, H-3), 5.67 (1H, t, J = 3.0 Hz, H-8), 5.42
47.26(5) Å , Z = 2, D = 1.194 Mg/m , F(000) = 376; crystal
c
3
dimensions/shape/color 0.269 × 0.132 × 0.050 mm /prism/colorless.
Reflections collected 14 893, independent reflections 3405; final R
indices [I > 2σ(I)] R = 0.0502, wR = 0.1345; R indices (all data) R =
1H, d, J = 3.0 Hz, H-13b), 5.29 (1H, d, J = 9.5 Hz, H-1), 4.55 (1H, t,
J = 11.5 Hz, H-6), 4.39 (1H, d, J = 3.0 Hz, H-9), 3.72 (3H, s, OCH3),
.36 (1H, dq, J = 11.5, 3.0 Hz H-7), 3.04 (H, d, J = 11.5 Hz, H-5), 3.03
1H, q, J = 5.5 Hz H-3′), 2.03 (3H, brs, H-15), 1.53 (3H, s, H-5′),
1
2
3
(
1
(
0
.0530, wR = 0.1383. Absolute structure parameter: −0.1(3).
2
1
3
Hydrolysis of Compound 13. Compound 13 (15 mg) was
.25 (3H, d, J = 5.5 Hz H-4′); C NMR (CDCl , 125 MHz) δ 171.3
3
refluxed for 4 h with MeOH (1 mL) and 2 N HCl (1 mL). The
MeOH was evaporated, the reaction mixture was extracted with
C, C-14), 169.3 (C, C-12), 168.9 (C, C-1′), 139.0 (CH, C-4), 133.9
C, C-11), 125.7 (CH, C-2), 122.3 (CH, C-1), 119.6 (CH , C-13),
(
2
CHCl , the aqueous layer was evaporated to obtain an amber residue
1
6
4
1
4
17.4 (CH, C-3), 77.2 (CH, C-6), 72.5 (CH, C-8), 70.7 (CH, C-9),
3
(
(
4 mg), and 0.5 mg of this was silylated using silylating mixture Fluka 1
85434) and analyzed by GC to identify glucose as the sugar present.
0.3 (CH, C-3′), 59.3 (C, C-2′), 54.1 (C, C-10), 52.7 (CH , OCH ),
3
3
4.8 (CH, C-7), 42.5 (CH, C-5), 29.7 (CH , C-15), 19.0 (CH , C-5′),
3
3
+
The remaining 3.5 mg was purified by FCC (EtOAc−MeOH, 7:3) to
3.6 (CH , C-4′); DARTMS m/z 405 [M + H] ; HRDARTMS m/z
05.15455 [M + H] (C H O requires 405.15494).
3
obtain D-glucose ([α]25 +56 (c 0.15, H O)). The organic layer (9 mg)
+
D
2
21
25
8
was purified by FCC (CH Cl −acetone, 19:1) to obtain compound 14
3
,7:10,11-Diepoxy-1,2-dihydroxy-13-oxo-14-phyten-18,6-olide
2
2
25
(
10): colorless oil; [α] −20 (c 0.1, CHCl ); UV (MeOH) λ (log
(3 mg).
D
3
max
ε) 205 (3.65), 235 (3.78) nm; IR (CHCl ) ν 3468, 1712, 1685,
619 cm ; H NMR (CDCl , 400 MHz) δ 6.08 (1H, hept, J = 1.2 Hz,
Evaluation of the Anti-inflammatory Activity. Animals. Male
CD-1 mice weighing 25−30 g were maintained in standard laboratory
conditions in the animal house (temperature 27 ± 1 °C) in a 12/12 h
light−dark cycle, being fed laboratory diet and water ad libitum,
following the Mexican official norm MON-062-Z00-1999.
3
max
−1 1
1
3
H-14), 4.22 (1H, dd, J = 8.0, 6.5 Hz, H-6), 3.74 (1H, dd, J = 11.2, 6.8
Hz, H-1a), 3.66 (1H, dd, J = 11.2, 3.6 Hz, H-1b), 3.60 (1H, d, J = 17.2
Hz, H-12a), 3.56 (1H, dd, J = 6.8, 3.6 Hz, H-2), 3.34 (1H, dd, J = 3.2,
2
2
5
.4 Hz, H-10), 2.44 (2H, m, H-9), 2.38 (1H, d, J = 17.2 Hz, H-12b),
.16 (1H, m, H-4a), 2.13 (3H, d, J = 1.2 Hz, H-16), 2.04 (1H, m, H-
a), 1.98 (1H, m, H-5b), 1.90 (3H, d, J = 1.2 Hz, H-17), 1.86 (2H, m,
TPA-Induced Edema Model. The TPA-induced ear edema assay in
mice was performed for extracts and isolated compounds as previously
reported. Purity of tested compounds was not less than 98% by
HPLC. Indomethacin was used as reference compound. In the primary
2
4
13
H-8), 1.69 (1H, m, H-4b), 1.41 (3H, s, H-19), 1.21 (3H, s, H-20);
C
G
DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
screening extracts and compound were evaluated at doses of 1 mg/ear.
Reported ID50 values are the average of five independent experiments.
Statistical Analysis. All data were represented as percentage mean
(15) Fischer, N. H.; Olivier, E. J.; Fischer, H. D. In Progress in the
Chemistry of Natural Products; Herz, W., Grisebach, H., Kirby, G. W.,
Eds.; Springer-Verlag: Weinheim, 1979; Vol. 38, p 90.
±
standard error of mean (SEM). The statistical analysis used
́ ́
(16) Catalan, C. A. N.; Cuenca, M. R.; Hernandez, L. R.; Joseph-
Student’s t test, whereas analysis of variance (ANOVA) followed by
Dunnett’s test was used to compare several groups with a control.
Values of p ≤ 0.05 and p ≤ 0.01 were considered to be significant.
Nathan, P. J. Nat. Prod. 2003, 66, 949−953.
(17) Bohlmann, F.; Jakupovic, J.; Dhar, A. K.; King, R. M.; Robinson,
H. Phytochemistry 1981, 20, 1081−1083.
(
(
18) Quijano, L.; Fischer, N. H. Phytochemistry 1984, 23, 829−831.
19) Li, X.; Zhang, D.; Onda, M.; Konda, Y.; Iguchi, M.; Harigaya, Y.
ASSOCIATED CONTENT
■
J. Nat. Prod. 1990, 53, 657−661.
*
S
Supporting Information
(20) Hirono, S.; Chou, W.-H.; Kasai, R.; Tanaka, O.; Tada, T. Chem.
Pharm. Bull. 1990, 38, 1743−1744.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00321.
(21) IUPAC. Pure Appl. Chem. 1999, 71, 587−643.
(22) Crystallographic data of structure 12 (CCDC 1511101) has
been deposited with the Cambridge Crystallographic Data Centre.
Copies of the data can be obtained, free of charge, on application to
the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
+ 044(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
Comparative ECD of compounds 1, 3, 5, and 8 with that
1
13
of melampodin (2); H, C, and 2D NMR spectra of
1
13
compounds 1, 3, and 5−14; and H and C NMR
spectra of compounds 2−4 (PDF)
(23) Hueso-Falcon, I.; Cuadrado, I.; Cidre, F.; Amaro-Luis, J. M.;
Ravelo, A. G.; Esteves-Braun, A.; De las Heras, B.; Hortelano, S. Eur. J.
Med. Chem. 2011, 46, 1291−1305.
AUTHOR INFORMATION
(24) Arciniegas, A.; Per
Villasen
32.
25) Dirsch, V. M.; Stuppner, H.; Ellmerer-Mu
M. Bioorg. Med. Chem. 2000, 8, 2747−2753.
26) Hong, S. S.; Lee, S. A.; Han, X. H.; Lee, M. H.; Hwang, J. S.;
́
ez-Castorena, A. L.; Nieto-Camacho, A.;
■
*
5
̃
or, J. L.; Romo de Vivar, A. J. Mex. Chem. Soc. 2009, 53, 229−
ller, E. P.; Vollmar, A.
Corresponding Author
Tel (A.-L. Perez-Castorena): (5255) 56-224412. Fax: (5255)
6-162217. E-mail: alperezc@unam.mx.
2
(
́
̈
(
Notes
Park, J. S.; Oh, K.- W.; Han, K.; Lee, M. K.; Lee, H.; Kim, W.; Lee, D.;
Hwang, B. Y. Chem. Pharm. Bull. 2008, 56, 199−202.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
́
We are indebted to R. Gavin
̃
o, I. Chaves, H. Rıo
o, B. Quiroz, L. Velasco, J. Perez, S. Hernan
, L. Rıos, G. Cortes, and C. Garcıa
́
s, A. Pen
̃
a, E.
dez,
for
Huerta, R. Patin
̃
́
́
C. Mar
́
quez, E. Garcıa
́
́
́
́
technical assistance.
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DOI: 10.1021/acs.jnatprod.6b00321
J. Nat. Prod. XXXX, XXX, XXX−XXX