Cytotoxic and NF-κB inhibitory sesquiterpene lactones from Piptocoma rufescens

Article abstract of DOI:10.1016/j.tet.2012.01.061

Six new (1-6) and eight known germacranolide-type sesquiterpene lactones, along with several known phenylpropanol coumarates and methylated flavonoids, were isolated from the leaves of Piptocoma rufescens, collected in the Dominican Republic. The new compounds were identified by analysis of their spectroscopic data, with the molecular structure of 3 being established by single-crystal X-ray diffraction. The absolute configurations of the sesquiterpene lactones isolated were determined from their CD and NOESY NMR spectra, together with the analysis of Mosher ester reactions. Bioassay screening results showed the majority of the sesquiterpene lactones isolated (1-13) to be highly cytotoxic toward the HT-29 human colon cancer cell line, with the most potent compound being 15-deoxygoyazensolide (10, IC₅₀, 0.26 μM). In addition, several of the sesquiterpene lactones exhibited NF-κB (p65) inhibitory activity.

Full text of DOI:10.1016/j.tet.2012.01.061

Tetrahedron 68 (2012) 2671e2678
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journal homepage: www.elsevier.com/locate/tet
Cytotoxic and NF-kB inhibitory sesquiterpene lactones from Piptocoma rufescens
a
b
c
c
c
~
~
ꢀ
Yulin Ren , Ulyana Munoz Acuna , Francisco Jimenez , Ricardo García , Melciades Mejía ,
Heebyung Chai a, Judith C. Gallucci d, Norman R. Farnsworth e y, Djaja D. Soejarto e
,
,
,f
Esperanza J. Carcache de Blanco a b, A. Douglas Kinghorn a
,
,
*
a Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
b Division of Pharmacy Practice and Administration, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
c
ꢀ
Jardín Botanico Nacional ‘Dr. Rafael Ma. Moscoso’, Santo Domingo, Dominican Republic
d Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA
e Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at
Chicago, Chicago, IL 60612, USA
f Botany Department, Field Museum of Natural History, Chicago, IL 60605, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new (1e6) and eight known germacranolide-type sesquiterpene lactones, along with several known
phenylpropanol coumarates and methylated flavonoids, were isolated from the leaves of Piptocoma ru-
fescens, collected in the Dominican Republic. The new compounds were identified by analysis of their
spectroscopic data, with the molecular structure of 3 being established by single-crystal X-ray diffraction.
The absolute configurations of the sesquiterpene lactones isolated were determined from their CD and
NOESY NMR spectra, together with the analysis of Mosher ester reactions. Bioassay screening results
showed the majority of the sesquiterpene lactones isolated (1e13) to be highly cytotoxic toward the HT-
Received 5 December 2011
Received in revised form 18 January 2012
Accepted 19 January 2012
Available online 28 January 2012
Keywords:
Piptocoma rufescens
Goyazensolide-type sesquiterpene lactones
Germacranolide-type sesquiterpene
lactones
29 human colon cancer cell line, with the most potent compound being 15-deoxygoyazensolide (10, IC50
0.26 M). In addition, several of the sesquiterpene lactones exhibited NF- B (p65) inhibitory activity.
Ó 2012 Elsevier Ltd. All rights reserved.
,
m
k
Cytotoxicity for HT-29 human colon cancer
cells
NF-kB (p65) inhibition assay
1. Introduction
2. Results and discussion
Piptocoma is a small genus of the plant family Asteraceae that
occurs in tropical and sub-tropical regions of the Western Hemi-
sphere.1 There are no previous reports on the chemical constituents
of any member of this genus, including the type species, Piptocoma
rufescens Cass. As part of search for new natural product anticancer
agents from diverse organisms,2 a leaf crude methanol extract of P.
rufescens collected in the Dominican Republic was found to be cy-
totoxic toward the HT-29 human colon cancer cell line. Using col-
umn chromatography, several new (1e6) and known cytotoxic
sesquiterpene lactones, together with other known non-cytotoxic
compounds, were isolated from this species. The isolation, struc-
tural characterization, and bioactivity evaluation of these isolates
are reported herein.
A methanol extract of the ground leaves of P. rufescens was par-
titioned with n-hexane and then chloroform, with these extracts
subjected to cytotoxicity testing using the HT-29 human colon
cancer cell line. The active n-hexane-soluble extract was separated
by column chromatography over silica gel to afford two new com-
pounds, rufesolides A (3) and B (4), and two known compounds,
ereglomerulide (7)3 and 15-deoxygoyazensolide (10).4 In turn,
separation of the chloroform-soluble extract of this plant part yiel-
ded four active fractions. Purification of the first fraction gave a new
compound, rufesolide C (5), and five known compounds, 15-
deoxygoyazensolide (10),4 20,30-dihydro-15-deoxygoyazensolide
(11),5 lychnopholide (12),6 4,5-dihydrolychnopholide (13),7 and
eremantholide C.8 Separation of the second fraction produced three
new compounds, rufescenolides A and B (1 and 2) and rufesolide D
(6), and a known compound, 5-epiisogoyazensolide (9).9 Work up of
the third fraction afforded three known compounds, goyazensolide
* Corresponding author. Tel.: þ1 614 247 8094; fax: þ1 614 247 8081; e-mail
addresses:
kinghorn.4@osu.edu,
kinghorn@pharmacy.ohio-state.edu
(A.D.
(8),10
coumarate,3 and 40-methoxy-5,7,30-trihydroxyflavone.11 Finally,
[3-(3,5-dimethoxy-4-hydroxyphenyl)]-1-propanol-trans-
Kinghorn).
y
Deceased September 10, 2011.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.01.061

2672
Y. Ren et al. / Tetrahedron 68 (2012) 2671e2678
separation of the fourth fraction yielded a further quantity of
bearing goyazensolide-type sesquiterpene lactones, with differ-
ences evident at their C-4 and C-5 positions. Replacement of the
observed 13C NMR signals at dC 130.4 (C-4) and 135.0 (C-5) for 104
with those at dC 37.5 (C-4) and 74.6 (C-5) for 1 (Table 2) indicated
the presence of a 5-hydroxy substituent for the latter compound.
This was evidenced by the molecular formula of C19H22O7 for 1
(C19H20O6 for 10) indicated by the mass spectrum, and supported
by HMBC correlations between H-2/C-10, H-4/C-2 and C-6, H-15/C-
goyazensolide (8),10 in addition to 3-(4-hydroxyphenyl)-1-
propanol-trans-coumarate,12
40-methoxy-3,5,7,30-tetrahydroxy-
flavone,11 3-methoxy-5,7-40-trihydroxyflavone,11 and 3,40-dime-
thoxy-5,7-30-trihydroxyflavone.11 The structures of the cytotoxic
sesquiterpene lactones isolated (1e13) are shown in Fig.1, and those
of the inactive compounds are presented in Fig. S10 (Supplementary
data).
O
O
O
O
O
1
1
14
O 14
2
3
2
3
14
14
2
6
1
7
15
O
10
15
2
6
1
7
O
10
9
10
3
5
3
5
10
4
9
4
5
O
9
8
15
4
5
O
4'
4'
9
15
4
8
8
4'
8
4'
6
7
6
7
2'
O
O
2'
1'
1'
O
HO
HO
O
1'
2'
3'
13
13
13
1'
2'
3'
13
O
O
3'
O
3'
O
O
O
11
11
O
11
12
12
12
O
11
12
5'
O
O
5'
O
5'
O
1
2
3
4
O
O
O 14
O
O
O
14
H3CO
OH
H3CO
OH
2
2
O
10
O
1
1
3
4
5
10
3
4
15
9
9
15
O
O
8
5
4'
4'
2'
8
6
7
6
7
O
O
O
O
1'
2'
3'
1'
13
13
O
O
O
O
11
11
O
O
O
O
3'
12
12
O
5'
O
O
5'
7
6
5
8
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
10
11
13
12
9
Fig. 1. Structures of the cytotoxic sesquiterpene lactones (1e13) isolated from P. rufescens.
Compound 1 was isolated as an amorphous white powder with
a molecular formula of C19H22O7, as determined by HRESIMS (m/z
385.1253 [MþNa]þ, calcd for 385.1263). It showed UV (lmax 212 and
3 and C-5, and H-5/C-7, and the proton-proton spin system from H-
4/H-5, H-5/H-6, H-6/H-7, H-7/H-8 to H-8/H2-9 established from the
1He1H COSY NMR spectrum (Fig. 2).
The relative configurations at C-4, C-5, C-6, C-7, C-8, and C-10 of
1 were deduced by analysis of the NOESY 2D NMR spectrum (Fig. 3),
and the absolute configuration at C-7 of 1 was determined by
analysis of its CD spectrum. Negative Cotton effects at 232 and
264 nm observed in the CD spectrum (Fig. 4) supported a 7R ab-
solute configuration, consistent with previous studies demon-
262 nm) and IR [nmax 3466 (hydroxy), 1766 and 1634 (
a
,b
-un-
saturated g-lactone), 1712 and 1634 (a,b-unsaturated ester), 1712
and 1588 (dihydrofuran-3-one ring) cmꢀ1] absorptions typical for
a furan ring-containing germacranolide, with such compounds also
known as furanoheliangolides or goyazensolides.13 This pre-
liminary structural assignment was supported in the 1H NMR
spectrum by a three-proton methyl singlet at dH 1.47, a three-proton
methyl doublet at dH 1.36, two doublets at dH 5.50 and 6.23 for two
terminal olefinic protons (H-13), and a broad singlet at dH 5.78 for
a vinylic proton H-2 (Table 1). In the 13C NMR spectrum, signals
were observed for a carbonyl group at dC 204.7, an oxygen-
substituted vinylic group at dC 106.2 and 192.0, a lactone carbonyl
group at dC 168.2, and three oxygen-bearing carbons at dC 74.6, 80.7,
and 71.0 (Table 2).10
strating that sesquiterpene lactones with
methylene- -lactone ring connected between the C-6 and C-12
positions with an -configuration for H-7 (7R) show negative Cot-
ton effects at ca. 220 and 260 nm in their CD spectra, arising from
the and n/ transitions of the lactone ring,
respectively.14,15
a trans-fused a-
g
a
p/p*
p*
To determine the absolute configuration at C-5, the modified
Mosher ester NMR method was applied to 1. Following a literature
procedure, the (S)-MTPA ester of 1 (1s) was prepared by reaction of 1
An oxygen bridge between C-3 and C-10 was deduced from the
HMBC correlations from H-2 to C-10, H-4 to C-2, and H-15 to C-3
(Fig. 2). In addition, the 1H and 13C NMR spectra of 1 revealed the
presence of a methacrylate group, characterized by a methyl singlet
at dH 1.81 and two broad singlets at dH 5.53 and 5.99 in the 1H NMR
spectrum, and four signals that appeared at dC 166.9 (C-10),135.3 (C-
20), 126.6 (C-30), and 17.9 (C-40) in the 13C NMR spectrum.10 This
methacrylate group was assigned to C-8, as supported by a HMBC
correlation between H-8 and C-10 (Fig. 2).
and (R)-(ꢀ)-
and the (R)-MTPAesterof1 (1r) wasprepared by reaction of1 and (S)-
-methoxy-
-(trifluoromethyl)phenylacetyl chloride.16 Analysis
a-methoxy-a-(trifluoromethyl)phenylacetyl chloride,
(þ)-a
a
of 1H NMR chemical shift differences (DdSeR) between 1s and 1r
revealed negativevalues for H-2, H-5, H-9, H-13a, H-14, and H-15, and
positive values for H-6, H-8, H-13b, H-30, and H-40 (Fig. 5), which in-
dicateda Sconfigurationfor C-5.16 Inturn, the absoluteconfigurations
at C-4, C-6, C-8, and C-10 were determined as R, S, S, and R, re-
spectively, by analysis of the NOESY correlations between H-5/H-14,
H-6/H-8 and H-15, H-7/Hb-9 and H-14, and H-8/H-15 (Fig. 3). Thus,
Comparison of the 1H and 13C NMR data of 1 with those of 104
(Tables 1 and 2) indicated both compounds to be methacryloxy-

Y. Ren et al. / Tetrahedron 68 (2012) 2671e2678
2673
Table 1
1H NMR data of compounds 1e6 in CDCl3
a
Position
1b
2b
3b
4b
5c
6c
2a
2b
3a
5.78 br s
5.78 s
2.74 m
2.59 m
2.01 m
3.07 m
3.38 m
5.74 br s
2.79 dd (9.9, 19.8)
3.22 dd (4.5, 19.0)
3.67 m
2.45 dd (2.9, 16.6)
2.66 dd (3.9, 16.6)
3.70 m
3b
1.58 m
4
5a
5b
3.39 m
4.34 m
3.36 m
4.32 m
2.01 m
1.73 m
1.67 m
2.14 m
2.03 m
1.45 m
2.13 m
2.19 dd (4.2, 16.5)
1.12 m
2.60 br s
6
7
4.49 dd (5.0, 9.5)
3.35 m
4.51 dd (5.0, 9.4)
3.36 m
4.30 m
2.97 m
4.19 m
3.27 m
4.45 m
2.88 m
4.68 m
2.86 m
8
9a
9b
13a
13b
14
4.34 m
4.32 m
2.33 m
2.45 t (13.4)
6.23 d (3.2)
5.47 d (2.7)
1.47 s
4.94 m
2.21 m
2.59 m
6.27 d (2.2)
5.63 d (1.5)
1.77 s
5.22 m
2.00 m
2.43 t (12.0)
6.35 br s
5.74 br s
1.79 s
4.98 m
2.14 m
2.61 dd (9.0, 17.1)
6.29 br s
5.67 br s
1.88 s
4.88 dt (3.1, 11.4)
2.86 m
3.00 m
6.24 br s
5.64 br s
2.34 dd (1.5, 14.2)
2.43 dd (11.9, 12.9)
6.23 d (3.2)
5.50 d (2.7)
1.47 s
1.34 s
15
1.36 d (7.0)
5.53 br s
5.99 br s
1.38 d (7.7)
6.10 q (8.6)
1.02 d (6.4)
6.14 q (6.5)
1.86 s
6.13 q (6.0)
1.13 d (7.0)
6.17 q (7.0)
1.06 d (6.3)
6.09 dq (1.3, 7.2)
30a
30b
40
1.81 s
1.75 s
1.87 d (8.6)
1.84 s
1.98 d (6.5)
1.77 s
1.98 d (6.0)
1.82 s
1.97 d (7.0)
3.34 s
1.86 d (1.3)
1.98 dd (1.3, 7.2)
3.37 s
50
OMe-3
OAc-10
OH-10
2.03 s
2.08 s
2.01 br s
3.81 s
a
Assignments based on analysis of 2D NMR spectra.
b
c
Data (
Data (
d) measured at 400 MHz.
d
) measured at 300 MHz; s¼singlet, br s¼broad singlet, d¼doublet, dd¼double doublet, t¼triplet, dt¼double triplet, q¼quartet, dq¼double quartet, m¼multiplet. J
values are presented in Hz and are omitted if the signals overlapped as multiplets. The overlapped signals were assigned from 1Hꢀ H COSY, HSQC, and HMBC spectra without
1
designating multiplicity.
Table 2
13C NMR data of compounds 1e6 in CDCl3
a
Position 1b
2b
3b
4b
5c
6b
1
204.7 C
204.8 C
208.5 C
207.2 C
207.6 C
214.5 C
2
106.2 CH 106.2 CH 36.0 CH2 38.6 CH2 41.7 CH2 38.5 CH2
3
4
5
6
7
8
192.0 C
37.5 CH
74.6 CH
80.7 CH
51.8 CH
71.0 CH
192.0 C
37.5 CH
74.7 CH
80.7 CH
51.8 CH
70.7 CH
29.6 CH2 122.3 CH 79.7 CH
29.6 CH 133.6 C 29.5 CH
38.6 CH2 36.1 CH2 33.0 CH2 36.6 CH2
79.4 CH
30.8 CH
78.0 CH
47.1 CH
67.1 CH
78.4 CH
47.7 CH
65.5 CH
78.4 CH
46.4 CH
66.3 CH
76.8 CH
44.3 CH
70.0 CH
9
45.3 CH2 45.4 CH2 43.8 CH2 43.5 CH2 43.9 CH2 38.8 CH2
10
11
12
13
14
15
10
89.7 C
132.8 C
168.2 C
89.8 C
133.1 C
168.2 C
84.0 C
134.5 C
169.7 C
84.8 C
133.9 C
169.9 C
83.3 C
134.2 C
169.0 C
78.8 C
135.0 C
168.6 C
125.2 CH2 125.1 CH2 124.8 CH2 122.8 CH2 124.1 CH2 126.7 CH2
20.9 CH3 21.0 CH3 21.1 CH3 21.3 CH3 21.9 CH3 28.6 CH3
9.4 CH3
166.9 C
135.3 C
126.6 CH2 141.1 CH 140.7 CH 140.8 CH 140.9 CH 139.9 CH
17.9 CH3 20.0 CH3 20.4 CH3 20.4 CH3 20.4 CH3 20.5 CH3
15.7 CH3 15.9 CH3 16.1 CH3 16.0 CH3 15.8 CH3
Fig. 2. COSY and the key HMBC correlations of 1.
9.4 CH3
167.2 C
126.3 C
24.0 CH3 25.8 CH3 20.4 CH3 20.5 CH3
166.2 C
126.7 C
166.3 C
126.7 C
166.4 C
126.7 C
166.3 C
126.9 C
20
30
40
50
OAc
169.1 C
21.1 CH3 21.9 CH3 21.0 CH3
58.0 CH3 58.2 CH3
169.1 C
169.6 C
OMe-3
a
Assignments based on analysis of 2D NMR spectra.
b
c
Data (
Data (
d
) measured at 100.6 MHz.
d
) measured at 75.5 MHz; CH3, CH2, CH, and C multiplicities determined by
DEPT 90, DEPT 135, and HSQC experiments.
the new compound 1 was determined as (4R,5S,6S,7R,8S,10R)-1-oxo-
3,10-epoxy-5-hydroxy-8-methacryloyloxygermacra-2,11(13)-dien-
6,12-olide,17 and has been assigned the trivial name, rufescenolide A.
Compound 2 was isolated as an amorphous white powder with
a molecular formula of C20H24O7, 14 (CH2) units greater than that of
1, as determined by HRESIMS (m/z 399.1404 [MþNa]þ, calcd for
399.1420). The closely related UV and IR spectra of 1 and 2 indicated
the latter compound to be also a goyazensolide-type sesquiterpene
lactone. Direct comparison of the 1H and 13C NMR spectra of 1 and 2
showed that both compounds contain identical sesquiterpene
Fig. 3. Selected NOESY correlations of 1.
lactone units but different ester residues at their C-8 positions.
Compound 2 was assigned as having an angelate group, which
displayed a methyl singlet at dH 1.75, a methyl doublet at dH 1.87,
and a vinylic quartet at dH 6.10 in the 1H NMR spectrum (Table 1).6

2674
Y. Ren et al. / Tetrahedron 68 (2012) 2671e2678
Fig. 4. CD (A) and UV (B) spectra of 1 (dark blue), 2 (pink), 3 (gray blue). The data were obtained in MeOH corrected by subtracting a spectrum of the appropriate solution in the
absence of the samples recorded under identical conditions.
Fig. 5. DdSeR values for the Mosher esters of 1.
In turn, five signals for this ester group appeared at dC 167.2 (C-10),
Fig. 6. COSY and the key HMBC correlations of 3.
126.3 (C-20), 141.1 (C-30), 20.0 (C-40) and 15.7 (C-50) in its 13C NMR
spectrum (Table 2).6 The NOESY correlations between H-40/H-30
and H-30/H-50 indicated a trans configuration for the C-40 and C-50
methyl groups (Fig. S8, Supplementary Data). The closely compa-
rable 1H and 13C NMR data (Tables 1 and 2), specific rotation values,
and the CD and NOESY NMR spectra of 1 and 2 (Fig. 4 and Fig. S8,
Supplementary data) suggested that both compounds have the
same absolute configurations at their C-4, C-5, C-6, C-7, C-8, and C-
10 stereogenic centers. Therefore, compound 2 (rufescenolide B)
was determined as (4R,5S,6S,7R,8S,10R)-1-oxo-3,10-epoxy-5-
hydroxy-8-angeloyloxygermacra-2,11(13)-dien-6,12-olide.17
Compound 3, obtained as colorless needles (mp 190e191 ꢁC),
showed UV (lmax 216 nm) and IR [nmax 1769 (
a,b-unsaturated g-
lactone), 1738 (ester) 1716 and 1698 ( ]
a
,b
-unsaturated ester) cmꢀ1
absorptions typical for a germacranolide.3 The positive HRESIMS
exhibited a sodiated molecular ion peak at m/z 429.1890 (calcd
429.1889 for C22H30O7Na), indicating 8ꢁ of unsaturation. Among
these, four resulted from carbonyl groups, two from carbon-carbon
double bonds, and the remaining two from a bicyclic system of
a germacranolide.
A ketone group could be located at the C-1 position of 3, as
indicated by the HMBC correlations between H2-2 and H2-9/C-1
(Fig. 6). A saturated ten-membered ring was suggested from the
long 1Hꢀ1H COSY sequence represented by H2-2/H2-3/H-4 (H-15)/
Fig. 7. ORTEP plot for the molecular structure of 3 drawn with 50% probability dis-
placement ellipsoids (oxygen atoms are red, carbon atoms are blue, and the small
white circles represent hydrogen atoms, which are drawn with an artificial radius).
H2-5/H-6/H-7/H-8/H2-9.
A cyclic lactone containing an exo-
methylene group could be proposed at the C-6 and C-7 positions,
as supported by HMBC correlations between H-13/C-7 and C-12
(Fig. 6). An angelate group was linked to the C-8 position, as
supported by the HMBC correlation between H-8/C-10 (Fig. 6). In
addition, an acetoxy group was proposed at C-10, which was found
to be the sole oxygen-substituted quaternary carbon in the
molecule.
almost planar trans-fused a-methylene-g-lactone ring and a ten-
membered ring adopting a half chair-boat-chair conformation,
with C-1, C-2, C-3, C-4, C-5, C-9, C-10 forming a half chair confor-
mation, C-1, C-3, C-4, C-5, C-6, C-8, C-9, and C-10 forming a boat
conformation, and C-4, C-5, C-6, C-7, C-8, C-9, and C-10 comprising
a chair conformation (Fig. 8). The axial proton H-7 is coupled with
two trans-axial protons, H-6 and H-8, with Ha-2, H-6, H-8, H-14,
The complete structure of 3 was established by single-crystal X-
ray diffraction analysis (Fig. 7), which confirmed the structure de-
termined by analysis of the NMR spectroscopic data. The crystal
structure of 3 showed that this molecule contains two rings, an
and H-15 being b-configured, and Hb-2, H-4 and H-7 being a-
configured, as indicated by its NOESY correlations (Fig. 9). An
angelate residue is bonded to the C-8 position, and the carbonyl
group is oriented cis to H-8 and in a cis relationship with the

Y. Ren et al. / Tetrahedron 68 (2012) 2671e2678
2675
the C-6, C-7, C-8, and C-10 stereogenic centers for both compounds.
Therefore, compound (rufesolide B) was determined as
4
(6R,7S,8S,10R)-1-oxo-8-angeloyloxy-10-acetoxygermacra-
3(4),11(13)-dien-6,12-olide.17
Compound 5 was isolated as an amorphous white powder with
a molecular formula of C23H32O8, 30 mass units more than that of 3,
consistent with the presence of a methoxy group at the C-3 posi-
tion, as indicated by a singlet at dH 3.34 displayed in the 1H NMR
spectrum and a signal at dC 58.0 that appeared in the 13C NMR
spectrum of 5. This was supported by observed HMBC correlations
between the protons of the methoxy group and C-3, H-5/C-3, and
H-15/C-3 and C-5 (Fig. S7, Supplementary data). The consistent
NOESY and CD data of 5 with those of 3 (Figs. S8 and S9, Supple-
mentary data) suggested the same absolute configurations at the C-
6, C-7, C-8, and C-10 stereogenic centers for both compounds, and
3R and 4R absolute configurations were determined by the NOESY
correlations between H-3/H3-15, H-6/Ha-2, H-8/Ha-2 and H-14
(Fig. S8, Supplementary data). Therefore, compound 5 (rufesolide
C) was determined as (3R,4R,6R,7S,8S,10R)-1-oxo-3-methoxy-8-
angeloyloxy-10-acetoxygermacra-11(13)-en-6,12-olide.17
Fig. 8. View of the central portion of compound 3 showing the conformation of the 10-
membered ring.
Compound 6 was isolated as an amorphous white powder with
a molecular formula of C21H30O7, 41 mass units (COCH2) less than
that of 5, consistent with being a deacetyl derivative of the latter
compound. This was supported by two signals at dC 21.0 and 169.6
for an acetyl group in the 13C NMR spectrum of 5 being absent in the
analogous data for 6. Comparison of the NMR data of 6 with litera-
ture values indicated this compound to be a new isomer of 3-
methoxy-8b
-angeloyloxyternifolin.19 The CD spectrum of 6 consis-
tent with those of 3e5 (Fig. S9, Supplementary data) indicated a 7S
absolute configuration, and 3R,4R,6R,8S, and 10R absolute configu-
rations were supported by NOESY correlations between Ha-2/H-6
and H-8, H-3/H3-15, H3-15/Ha-5, and Ha-5/H-6 (Fig. S8, Supple-
mentary data). Therefore, compound 6 (rufesolide D) was de-
termined as (3R,4R,6R,7S,8S,10R)-1-oxo-3-methoxy-8-angeloyloxy-
10-hydroxygermacra-11(13)-en-6,12-olide.17
The structures of the known compounds (Fig. S10, Supplemen-
tary data) isolated from P. rufescens, including several sesquiter-
pene lactones, phenylpropanol coumarates, and methylated
flavonoids, were determined by analysis of their spectroscopic data
and comparison of these data with reference values,3e12 and the
assignments of their 1H and 13C NMR spectroscopic data are listed
in the Tables S1eS5 (Supplementary data). The absolute configu-
rations of the known sesquiterpene lactones were determined by
comparison of their NOESY NMR and CD spectra with those of 1e6
(Figs. S8 and S9, Supplementary data).
All isolated goyazensolides and germacranolides (1e13) dis-
played negative Cotton effects around 220 and 260 nm in their CD
spectra and UV absorption maxima around 215 nm (Fig. S9, Sup-
plementary data), indicating the absolute configuration for the C-7
position.14,15 Additionally, the goyazensolides (1e2 and 8e13) all
showed positive Cotton effects around 210 and 310 nm in their CD
spectra and UV absorption maxima around 260 nm (Fig. S9, Sup-
plementary data), which disappeared in the CD and UV spectra of
the germacranolides (3e7), indicating that these positive Cotton
Fig. 9. Selected NOESY correlations of 3.
conjugated carbon-carbon double bond at the C-20 and C-30 posi-
tions. The two methyl groups of C-40 and C-50 are trans (Fig. 7).
Even though the conformation of the ring system and relative
configuration of 3 have been established by X-ray analysis (Figs. 7
and 8), it is not possible to determine its absolute configuration
in this manner. Refinement of the Flack parameter to determine the
absolute configuration was inconclusive, as its value was 0.04(60).18
The small value of 0.04 indicates that the absolute configuration of
the rufesolide A (3) molecule is probably correct, but the large error
value of 0.60 gives little confidence in this number. As a result, it
was not possible to determine the absolute configuration of 3 with
the Flack parameter obtained. The Cotton effects displayed in the
CD spectrum of 3 were sufficient to make such a determination.
Negative Cotton effects at 223 and 272 nm in the CD spectrum
(Fig. 4) of 3 implied a a
-configuration for H-7 (7S configuration),14,15
and the 4S, 6R, 8S, and 10R configurations thus were established
relative to 7S by X-ray crystallographic analysis and supported
by NOESY correlations between Ha-2/H-6, Hb-2/H-7, H-6/H-8
and H-15 (Fig. 9). Thus, the structure of 3 was determined
as
(4S,6R,7S,8S,10R)-1-oxo-8-angeloyloxy-10-acetoxygermacra-
11(13)-en-6,12-olide.17 This germacranolide was accorded the
trivial name, rufesolide A.
effects are indicative of exciton coupling arising from the
a,b-un-
saturated carbonyl chromophore at C-1020 for an
a
-configuration
Compound 4 was isolated as an amorphous white powder with
a molecular formula of C22H28O7, from the HRESIMS sodiated molec-
ular ion peak at m/z 427.1715, two hydrogen atoms less than that of 3.
for H-14 (a 10R configuration for compounds 1e2 and 8e13).
All compounds obtained in this study from P. rufescens were
tested in terms of their cytotoxicity against the HT-29 human colon
cancer cell line, using paclitaxel as the positive control. Both the
goyazensolides (1e2 and 8e13) and germacranolides (3e7)
showed potent cytotoxicity toward this cell line (Table 3), with 15-
deoxygoyazensolide (10) being the most active compound with an
1
This compound exhibited closely comparable H and 13C NMR reso-
nances to those of 3, except for the signals for an extra carbon-carbon
double bond at the C-3 and C-4 positions, as assigned in Tables 1 and 2
by HMBC correlations between H-2/C-1, H-5/C-3 and C-7, and H-15/C-
3 and C-5 (Fig. S7, Supplementary data). The closely comparable
NOESY NMR and CD spectra of 4 with those of 3 (Figs. S8 and S9,
Supplementary data) indicated the same absolute configurations at
IC50 value of 0.26
in this assay system. All the cytotoxic sesquiterpene lactones con-
tain a -methylene- -lactone ring at the C-6 and C-7 positions and
mM. However, all other compounds were inactive
a
g

2676
Y. Ren et al. / Tetrahedron 68 (2012) 2671e2678
Table 3
Cytotoxicity and NF-
Biosciences, Uppsala, Sweden. For visualization of TLC plates, sul-
k
B p65 inhibition of 1e13a
furic acid reagent was used. All procedures were carried out using
anhydrous solvents purchased from commercial sources and
employed without further purification.
Compound
Cytotoxicityb
NF-k
B p65 inhibitionc
1
2
3
4
5
6
7
8
1.9
6.6
3.0
4.0
1.4
1.0
1.2
0.56
0.28
0.26
0.58
1.4
NTd
NTd
>10
>10
2.1
3.2. Plant material
The leaves of P. rufescens Cass. (Asteraceae) were collected from
>10
>10
3.8
a shrub 1.5 m tall with violet stamens, at Villa Mella (18ꢁ390 N;
ꢁ
0
ꢀ
69 58 E; 150 m), on the road to Yamasa, in a forest on serpentine
rock, Sierra Prieta, Distrito Nacional, Dominican Republic, in Janu-
9
NTd
3.2
10
11
12
13
ꢀ
ary, 1996. A voucher herbarium specimen (Jimenez & Mejía 2040)
NTd
2.9
was identified by F. J., R. G., and D. D. S. and has been deposited both
ꢀ
0.56
>10
at the Herbarium of the Jardín Botanico Nacional ‘Dr. Rafael Ma.
a
Moscoso’, Santo Domingo, Dominican Republic, and at the John G.
Searle Herbarium of the Field Museum of Natural History, Chicago,
IL, under the accession number FM2169750.
All data presented as 95% confidence intervals obtained by non-linear regression
in these assays.
b
Data presented as IC50 values (
m
M) toward HT-29 cells, with paclitaxel used as
a positive control (IC50 1 nM).
c
Data presented as IC50 values (mM), with rocaglamide used as positive control
3.3. Extraction and isolation
(IC50 0.075
mM).
d
Not tested due to sample limitations.
The ground leaves of P. rufescens (470 g) were extracted with
MeOH (4 Lꢂ5) at room temperature. The solvent was evaporated in
vacuo. The resultant dried MeOH extract (90 g, 19.2%) was sus-
pended in 10% H2O in MeOH (700 mL) and partitioned with n-
hexane (500, 500, and 400 mL) to yield a n-hexane-soluble residue
(18.0 g, 3.8%). A volume of 100 mL of H2O was added to the
aqueous-MeOH layer and this was then partitioned with CHCl3
(500, 500, and 400 mL) to afford a chloroform-soluble extract (8.4 g,
1.8%), which was washed with 1% aqueous solution of NaCl to
partially remove any plant polyphenols present. Both the n-hex-
ane- and chloroform-soluble residues showed inhibitory activity
toward the HT-29 human colon cancer cell line in a cytotoxicity
assay.
a a,b-unsaturated ester group at the C-8 position, which have been
reported as key structural requirements for sesquiterpene lactones
to mediate cytotoxicity.21 To further investigate of the bioactivities
of the isolates, all compounds obtained in sufficient quantity in this
study, except for 1, 2, 9, 11, eremantholide C, and 40-methoxy-5,7,30-
trihydroxyflavone, were tested in a NF-kB (p65) inhibition assay,
using rocaglamide as the positive control. Compounds 5, 8, 10, and
12 were active (Table 3), with all other compounds tested being
inactive (IC50 value>10 mM).
Several goyazensolides and germacranolides, including goya-
zensolide (8), lychnopholide (12), and parthenolide, have been re-
ported previously to exhibit cytotoxicity toward a panel of human
cancer cell lines.22 Recently, goyazensolide (8) and 15-
deoxygoyazensolide (10), together with other analogues, have been
The active n-hexane-soluble extract (18.0 g, IC50<10
mg/mL)
was subjected to passage over a silica gel column (4.5ꢂ45 cm),
eluted with gradient mixtures of n-hexane/acetone (100:1/1:1;
500 mL each). The eluates were pooled by TLC analysis to give
investigated for their NF-k kB inhibitory
B inhibitory activity.23 The NF-
activity of compounds 5 and 12 has not been reported previously.
In summary, a number of germacranolide-type sesquiterpene
lactones, phenylpropanol coumarates, and methylated flavonoids
were isolated and characterized from the leaves of P. rufescens for
the first time in this study. Based on the results obtained, both the
goyazensolides and germacranolides can be regarded as charac-
teristic chemical components and the cytotoxic principles of this
species.
thirteen combined fractions. Of these, fraction 9 (IC50<2
mg/mL)
was chromatographed further over silica gel column
a
(2.5ꢂ20 cm), eluted with gradient mixtures of n-hexane/acetone
(20:1/3:1, 200 mL each), and then purified by separation over
a Sephadex LH-20 column (2.5ꢂ25 cm), by elution with CH2Cl2/
MeOH (1:1), affording rufesolide
deoxygoyazensolide (10, 50 mg). Fractions 10e12 (IC50<1
B
(4, 6.0 mg) and 15-
g/mL)
m
were combined and separated by silica gel chromatography,
eluted with n-hexane/acetone (5:1), and then purified by passage
over a Sephadex LH-20 column, by elution with CH2Cl2/MeOH
(1:1), to afford rufesolide A (3, 11.0 mg) and ereglomerulide (7,
45 mg). Compound 3 was recrystallized from a mixture of n-
hexane and acetone to give fine needles suitable for single-crystal
X-ray analysis.
3. Experimental
3.1. General experimental procedures
Melting points were measured using a Fisher Scientific appa-
ratus and are uncorrected. Specific rotation values were obtained
on a PerkineElmer model 343 polarimeter. UV spectra were
recorded on a Hitachi U2910 UV spectrophotometer. CD measure-
ments were performed using a JASCO J-810 spectropolarimeter. IR
spectra were recorded on a Nicolet 6700 FT-IR spectrometer. 1H and
13C, DEPT, HSQC, HMBC, NOESY, and COSY NMR spectra were
recorded at room temperature on Bruker Avance DPX-300, DRX-
400, DRX-600, or DRX-800 MHz NMR spectrometers, with TMS as
internal standard. ESIMS and HRESIMS were measured on
a LCTeTOF mass spectrometer in the positive-ion mode. Column
chromatography was conducted using silica gel (65ꢂ250 or
230ꢂ400 mesh, Sorbent Technologies, Atlanta, GA). Analytical and
preparative thin-layer chromatograph (TLC) systems were per-
formed on precoated silica gel 60 F254 plates (Sorbent Technologies,
Atlanta, GA). Sephadex LH-20 was purchased from Amersham
The active chloroform-soluble extract (8.0 g, IC50<2 mg/mL) was
subjected to passage over a silica gel column (4.5ꢂ45 cm), and
eluted with gradient mixtures of n-hexane/acetone (100:1/1:1;
500 mL each). Fractions were pooled by TLC analysis to yield nine
combined fractions. Of these, fractions 1e4 were deemed cytotoxic
toward the HT-29 cell line (IC50<1 mg/mL). Fraction 1 was chro-
matographed over a silica gel column, and eluted with gradient
mixtures of n-hexane/acetone (10:1/3:1), to yield two combined
sub-fractions. The first sub-fraction was chromatographed over
silica gel using n-hexane/acetone (5:1) as solvent, and then purified
by separation over a Sephadex LH-20 column, eluted with CH2Cl2/
MeOH (1:1), affording rufesolide
C (5, 13.0 mg) and 15-
deoxygoyazensolide (10, 550 mg). The second sub-fraction was
separated by silica gel chromatography, eluted with a gradient
mixture of n-hexane/acetone (5:1/1:1), and then purified by

Y. Ren et al. / Tetrahedron 68 (2012) 2671e2678
2677
passage over a Sephadex LH-20 column, eluted with CH2Cl2/MeOH
(1:1), to afford 20,30-dihydro-15-deoxygoyazensolide (11, 3.0 mg),
lychnopholide (12, 5.0 mg), 4,5-dihydrolychnopholide (13, 2.0 mg),
and eremantholide C (1.0 mg).
3.3.1.2. Preparation of the (R)-MTPA ester (1r) of rufescenolide A
(1). Using the same procedure as described for 1s, the (R)-MTPA
ester derivative of 1 (1r) was produced by the reaction of 1 (1.5 mg),
(S)-(þ)-
(5
L), and deuterated pyridine (0.5 mL). 1H NMR data of 1r
(400 MHz, pyridine-d5):
a
-methoxy-
a
-(trifluoromethyl)phenylacetyl
chloride
Fraction 2 was chromatographed over a silica gel column, and
eluted with gradient mixtures of n-hexane/acetone (10:1/1:1),
to yield two combined sub-fractions. The first sub-fraction was
chromatographed over silica gel using n-hexane/acetone (3:1) as
solvent, and then finally purified by passage over a Sephadex LH-
20 column, eluted with CH2Cl2/MeOH (1:1), affording rufesce-
nolide A (1, 10.0 mg) and rufescenolide B (2, 1.0 mg). The second
sub-fraction was separated by silica gel chromatography, eluted
by n-hexane/acetone (3:1), and then purified by passage over
a Sephadex LH-20 column, eluted with CH2Cl2/MeOH (1:1), to
afford rufesolide D (6, 8.0 mg) and 5-epiisogoyazensolide (9,
2.0 mg).
Fraction 3 was chromatographed over silica gel, eluted by n-
hexane/acetone (3:1), and then purified over a Sephadex LH-20
column, with CH2Cl2/MeOH (1:1) as eluent, affording goyazenso-
lide (8, 15 mg), [3-(3,5-dimethoxy-4-hydroxyphenyl)]-1-propanol-
trans-coumarate (6.0 mg), and 40-methoxy-5,7,30-trihydroxy-
flavone (1.0 mg).
Fraction 4 was chromatographed over silica gel, eluted with
a gradient mixture of n-hexane/acetone (50:1/1:1), to produce
three combined sub-fractions. The precipitate from the first sub-
fraction was triturated and purified over a Sephadex LH-20 col-
umn, using CH2Cl2/MeOH (1:1) for elution, to afford 3,40-dime-
thoxy-5,7-30-trihydroxyflavone (31 mg). The mother liquor was
evaporated in vacuo, and the residue was chromatographed over
silica gel, eluted with n-hexane/acetone (3:1), and then purified
over a Sephadex LH-20 column, using CH2Cl2/MeOH (1:1) for
elution, to furnish goyazensolide (8, 90 mg). The second sub-
fraction was chromatographed over silica gel, eluted by n-hex-
ane/acetone (3:1), and then separated over Sephadex LH-20, using
CH2Cl2/MeOH (1:1) for elution, to yield 3-(4-hydroxyphenyl)-1-
propanol-trans-coumarate (15.0 mg). The third sub-fraction was
separated by silica gel, eluted by n-hexane/acetone (1:1), and then
purified finally over a Sephadex LH-20 column, using CH2Cl2/
MeOH (1:1) for elution, furnishing 40-methoxy-3,5,7,30-tetrahy-
droxyflavone (2.0 mg) and 3-methoxy-5,7-40-trihydroxyflavone
(13.0 mg).
m
d
6.38 (1H, d, J¼2.81 Hz, H-13a), 6.05 (1H,
br s, H-30), 6.04 (1H, s, H-2), 6.00 (1H, dd, J¼6.67, 8.86 Hz, H-5), 5.65
(1H, d, J¼2.35 Hz, H-13b), 5.47 (1H, br s, H-30), 4.77 (1H, dd, J¼4.02,
8.82 Hz, H-6), 4.67 (1H, d, J¼12.45 Hz, H-8), 3.88 (2H, m, H-7 and H-
4), 2.74 (1H, t, J¼13.08 Hz, H-9), 2.50 (1H, d, J¼12.74 Hz, H-9), 1.75
(3H, s, H-40), 1.56 (3H, s, H-14), 1.35 (3H, d, J¼6.94 Hz, H-15).
3.3.2. Rufescenolide B (2). Amorphous colorless powder (n-hex-
ane); ½a 2D0
ꢃ
þ40 (c 0.06, MeOH); ½a D20
þ33.3 (c 0.06, CH2Cl2); UV
ꢃ
(MeOH) lmax (log ε) 216 (3.94), 262 (3.79) nm; CD (MeOH, nm) lmax
(
D
ε) 209 (þ1.1), 229 (ꢀ4.1), 270 (ꢀ3.3), 312 (þ2.1); IR (dried film)
nmax 3473, 1770, 1710, 1589, 1454 cmꢀ1 1H and 13C NMR data, see
;
Tables 1 and 2; HRESIMS m/z: 399.1404 [MþNa]þ (calcd for
C20H24O7Na, 399.1420).
3.3.3. Rufesolide A (3). Colorless needles (n-hexane/acetone); mp
a 20
a 20
ꢁ
190e191 C; ½ ꢃD þ7.7 (c 0.09, MeOH); ½ ꢃD þ1.0 (c 0.1, CH2Cl2); UV
(MeOH) lmax (log ε) 216 (3.92) nm; CD (MeOH, nm) lmax ε) 223
(ꢀ4.1), 272 (ꢀ0.7), 292 (ꢀ1.1); IR (dried film) nmax 1769, 1738, 1716,
(D
1698, 1461 cmꢀ1 1H and 13C NMR data, see Tables 1 and 2; HRE-
;
SIMS m/z: 429.1890 [MþNa]þ (calcd for C22H30O7Na, 429.1889).
3.3.4. X-ray crystal data of rufesolide A (3). Data for a small colorless
needle of 3 were collected by synchrotron radiation at the Advanced
Light Source at Lawrence Berkeley National Laboratory.24 Crystal
data: molecular formula C22H30O7, MW¼406.46, orthorhombic,
ꢁ
ꢁ
space group P212121, a¼5.5228(2) A, b¼17.4103(7) A,
c¼22.1641(10) A, V¼2131.2(2) A , Z¼4, Dcalcd¼1.267 gm/cm3, 150 K,
3
ꢁ
ꢁ
size 0.01ꢂ0.02ꢂ0.09 mm3, Bruker APEXII CCD diffractometer with
l
¼0.8856 A, 5.42ꢄ2
q
ꢄ67.36ꢁ (Table S6, Supplementary data). Data
ꢁ
were collected using the APEX2 software and processed with SAINT
within APEX2.25 Corrections for absorption and beam corrections are
based on the multi-scan technique as implemented in SADABS.25 The
structure was solved by direct methods in SHELXS-97.26 Full-matrix
least-squares refinements based on F2 were performed in SHELXL-
97,26 as incorporated in the WinGX package.26 The final refinement
cycle was based on 4356 intensities and 267 variables and resulted in
agreement factors of R1(F)¼0.060 and wR2(F2)¼0.088. For the subset
3.3.1. Rufescenolide A (1). Amorphous colorless powder (n-hex-
ane); ½a 2D0
ꢃ
þ95 (c 0.12, MeOH); ½a D20
ꢃ
þ132.0 (c 0.1, CH2Cl2); UV
of data with I>2s(I), the R1(F) value is 0.040 for 3464 reflections.
(MeOH) lmax (log ε) 212 (3.87), 262 (3.81) nm; CD (MeOH, nm) lmax
Refinement of the Flack parameter18 to determine the absolute
configuration was not conclusive, as its value was 0.04 (60). The CIF
file of the X-ray data of 3 has been deposited in the Cambridge
Crystallographic Data Centre (deposition no.: CCDC 852357).
(D
ε) 210 (þ7.8), 232 (ꢀ1.8), 264 (ꢀ4.0), 312 (þ5.3); IR (dried film)
nmax 3466, 1766, 1712, 1634, 1588, 1453 cmꢀ1; 1H and 13C NMR data,
see Tables 1 and 2; HRESIMS m/z: 385.1253 [MþNa]þ (calcd for
C19H22O7Na, 385.1263).
3.3.5. Rufesolide B (4). Amorphous colorless powder (n-hexane);
3.3.1.1. Preparation of the (S)-MTPA ester (1s) of rufescenolide A
(1). Compound 1 (1.5 mg) was transferred to a NMR tube and
dried under vacuum in an oil pump for 8 h. Deuterated pyridine
½
a 2D0
ꢃ
e46.6 (c 0.09, MeOH); ½a D20
ꢃ
e68.0 (c 0.1, CH2Cl2); UV (MeOH)
ε) 206 (þ10.3), 226
lmax (log ε) 216 (3.94) nm; CD (MeOH, nm) lmax (D
(ꢀ7.7), 279 (ꢀ3.8), 289 (ꢀ4.3); IR (dried film) nmax 1770, 1715, 1669,
(0.5 mL) and (R)-(ꢀ)-
a
-methoxy-
a
-(trifluoromethyl)phenylacetyl
1641, 1581, 1455 cmꢀ1 1H and 13C NMR data, see Tables 1 and 2;
;
chloride (5 L) were transferred into the NMR tube under argon.
m
HRESIMS m/z: 427.1715 [MþNa]þ (calcd for C22H28O7Na, 427.1733).
Then, the NMR tube was shaken and kept at room temperature
for 8 h. The 1H NMR spectrum was measured directly at room
temperature, and the data were assigned based on comparison of
the 1H NMR spectrum of the product with that of 1. 1H NMR data
3.3.6. Rufesolide C (5). Amorphous colorless powder (n-hexane);
½
a 2D0
ꢃ
þ29.3 (c 0.08, MeOH); ½a D20
þ10 (c 0.1, CH2Cl2); UV (MeOH)
ꢃ
lmax (log ε) 219 (4.41) nm; CD (MeOH, nm) lmax
(Dε) 221 (ꢀ4.3), 275
of 1s (400 MHz, pyridine-d5):
d
6.38 (1H, d, J¼2.78 Hz, H-13a),
(-2.1), 292 (ꢀ2.5); IR (dried film) nmax 1768,1716,1461 cmꢀ1; 1H and
13C NMR data, see Tables 1 and 2; HRESIMS m/z: 459.2031 [MþNa]þ
(calcd for C23H32O8Na, 459.1995).
6.05 (1H, br s, H-30), 6.01 (1H, s, H-2), 5.97 (1H, dd, J¼6.70 Hz,
8.70, H-5), 5.67 (1H, d, J¼2.33 Hz, H-13b), 5.48 (1H, br s, H-30),
4.83 (1H, dd, J¼4.00, 9.03 Hz, H-6), 4.68 (1H, d, J¼11.51 Hz, H-8),
3.88 (2H, m, H-7 and H-4), 2.73 (1H, t, J¼13.29 Hz, H-9), 2.49 (1H,
d, J¼12.85 Hz, H-9), 1.76 (3H, s, H-40), 1.55 (3H, s, H-14), 1.17 (3H,
d, J¼6.92 Hz, H-15).
3.3.7. Rufesolide D (6). Amorphous colorless powder (n-hexane);
½
a 2D0
ꢃ
þ66.6 (c 0.09, MeOH); ½a D20
ꢃ
þ65.0 (c 0.1, CH2Cl2); UV (MeOH)
lmax (log ε) 215 (3.95) nm; CD (MeOH, nm) lmax (D
ε) 223 (ꢀ3.8); IR

2678
Y. Ren et al. / Tetrahedron 68 (2012) 2671e2678
(dried film) nmax 3494,1770,1699,1647,1456 cmꢀ1; 1H and 13C NMR
data, see Tables 1 and 2; HRESIMS m/z: 417.1875 [MþNa]þ (calcd for
C21H30O7Na, 417.1889).
References and notes
1. Pruski, J. F. Novon 1996, 6, 96e102.
2. Kinghorn, A. D.; Carcache-Blanco, E. J.; Chai, H.-B.; Orjala, J.; Farnsworth, N. R.;
Soejarto, D. D.; Oberlies, N. H.; Wani, M. C.; Kroll, D. J.; Pearce, C. J.; Swanson, S.
M.; Kramer, R. A.; Rose, W. C.; Fairchild, C. R.; Vite, G. D.; Emanuel, S.; Jarjoura,
D.; Cope, F. O. Pure Appl. Chem. 2009, 81, 1051e1063.
3.4. Cytotoxicity assay
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This investigation was supported by grants U01 CA52956
and P01 CA125066, funded by the National Cancer Institute,
NIH, Bethesda, MD. The leaf sample of Piptocoma rufescens was
collected under a collaborative agreement between the Uni-
ꢀ
versity of Illinois at Chicago and the Jardín Botanico Nacional
‘Dr. Rafael Ma. Moscoso’, Santo Domingo, Dominican Republic.
We thank Dr. Jeanette Krause of the University of Cincinnati for
the X-ray data collection through the SCrALS program. We
thank Dr. Kari Green-Church, of the Mass Spectrometry and
Proteomics of the Campus Chemical Instrument Center (CCIC),
The Ohio State University (OSU), for assistance with the MS
measurements, Dr. Chunhua Yuan (CCIC, OSU) for assistance
with the HMBC and NOESY measurements on 600 and 800 MHz
NMR spectrometers, and Jack Fowble (College of Pharmacy,
OSU) for access to the other NMR instrumentations used in this
investigation.
23. Wagmer, S.; Hofmann, A.; Siedle, B.; Terfloth, L.; Merfort, I.; Gasteiger, J. J. Med.
Supplementary data
Chem. 2006, 49, 2241e2252.
24. Crystallographic data were collected through the SCrALS (Service Crystallog-
raphy at Advanced Light Source) program at the Small-Crystal Crystallography
Beamline 11.3.1 at the Advanced Light Source (ALS), Lawrence Berkeley Na-
tional Laboratory. The ALS is supported by the U.S. Department of Energy, Office
of Energy Sciences Materials Sciences Division, under contract DE-
AC02e05CH11231.
Copies of MS and 1H and 13C NMR spectra of compounds 1e6
and 1H NMR spectra of (S)- and (R)-MTPA esters (1s/1r) of 1; dia-
grams of COSY and the key HMBC and NOESY correlations of
compounds 2, 4e13, and eremantholide C; UV and CD spectra of
compounds 1e13 and eremantholide C; structures of eremantho-
lide C, phenylpropanol coumarates, and methylated flavonoids
obtained from P. rufescens; crystallographic data and the CIF file of
compound 3; and analytical data of the known compounds. Sup-
plementary data associated with this article can be found in the
online version, at doi:10.1016/j.tet.2012.01.061. These data include
MOL files and InChIKeys of the most important compounds de-
scribed in this article.
25. APEX2 v2010.3.0 and SAINT v7.60A for data collection and data processing
programs, respectively. Bruker Analytical X-ray Instruments, Inc., Madison, WI;
SADABS v2008/1 semi-empirical absorption and beam correction program. G.
€
M. Sheldrick, University of Gottingen, Germany.
26. (a) SHELXS-97: Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112e122; (b)
SHELXL-97: Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112e122; (c) WinGX-
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D.; Carcache de Blanco, E. J.; Swanson, S. M.; Kinghorn, A. D. J. Nat. Prod. 2011,
74, 1117e1125.