Phytochemistry 71 (2010) 104–109

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Antibacterial clerodane diterpenes from Goldenrod (Solidago virgaurea)

Courtney M. Starks *, Russell B. Williams, Matt G. Goering, Mark O’Neil-Johnson, Vanessa L. Norman,
Jin-Feng Hu, Eliane Garo, Grayson W. Hough, Stephanie M. Rice, Gary R. Eldridge
Lead Discovery and Rapid Structure Elucidation Group, Sequoia Sciences Inc., 1912 Innerbelt Business Center Drive, St. Louis, MO 63114, United States

a r t i c l e i n f o a b s t r a c t

Article history: Nine clerodane diterpenes, solidagoic acids C-I (1–7), cleroda-3,13(14)-dien-16,15:18,19-diolide (8) and
Received 23 December 2008 cleroda-3,13(14)-dien-15,16:18,19-diolide (9) were isolated and characterised from the ethanol–ethyl
Received in revised form 7 August 2009 acetate (1:1) extract of Solidago virgaurea. The structures were determined by NMR spectroscopic analy-
Available online 24 October 2009
sis. Several displayed moderate antibacterial activity against Staphylococcus aureus.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Goldenrod (or woundwort)
Solidago virgaurea
Asteraceae
Clerodane
Diterpene
Solidagoic acid
Antibacterial
Staphylococcus aureus

1. Introduction 2. Results and discussion

Antibacterial resistance among pathogenic bacteria is increas-
ing, creating a need for new antibiotics in the drug development
pipeline (Levy and Marshall, 2004; Taubes, 2008). Natural prod-
ucts will likely be a major source of the chemical diversity needed
to thwart multiple resistance mechanisms (Payne et al., 2007). In
the course of our search for new antibacterial compounds from a
taxonomically diverse plant collection, we applied a high-
throughput natural product discovery approach (Eldridge et al.,
2002) to Goldenrod (or woundwort) Solidago virgaurea L. (Astera-
ceae). This plant is one of approximately 80 species of Solidago
from North America, and has become naturalised in Europe (Mab-
berley, 1997). It is now cultivated for local medicinal use in East-
ern Europe (Sztefanov et al., 2002). Solidago species were an early
focus of studies on the Asteraceae diterpenes, and many clerod-
anes have been isolated from the genus (Seaman, 1990). Clerod-
anes oxygenated at the six-position have been isolated
previously from S. virgaurea (Goswami et al., 1984). Here we de-
scribe two known (1, 9) and seven new (2–8) clerodanes from S.
virgaurea.
* Corresponding author. Tel.: +1 314 373 5181x107; fax: +1 314 373 5186.
E-mail address: cstarks@sequoiasciences.com (C.M. Starks).
An organic extract of S. virgaurea was subjected to normal phase
flash chromatography and reversed phase HPLC to yield two
known (1, 9) and seven new (2–8) cis-clerodane diterpenes
(Fig. 1). Compounds 1, 2, 5, 7, 8, and 9 were isolated in microscale
quantities using our high-throughput natural products isolation
methods (Eldridge et al., 2002). Scaling up the purification led to
the isolation of compounds 3, 4, and 6, as well as additional quan-
tities of 1, 2, 7, 8, and 9 (5 was not found in the scaled-up isolation).
Structures were determined using data acquired from 6500 lg of
sample in a capillary microcoil NMR probe. Several of these com-
pounds displayed moderate antibacterial activity against S. aureus
(Table 1).
The 1H NMR spectrum of 1 displayed one doublet (d 0.79) and
two singlet (d 0.96, 1.54) methyl signals, downfield signals at d
7.13 and 5.50, and a number of multiplets between d 1.3 and 2.5
(Table 2). This pattern was indicative of a clerodane-type diterpene
with one modified methyl group. The 1D and 2D NMR spectra were
consistent with the structure shown for 1, a cis-clerodane with an a,
b-unsaturated c-lactone side chain. This compound had been re-
ported previously as a synthetic derivative of solidagoic acid A
(Anthonsen et al., 1973), which bears a b-furanoethyl side chain.
The optical rotation of 1 (½a25 D 50) closely matches that reported
for solidagoic acid A in the same solvent ([a]D 58). Here, we assign
1 the name solidagoic acid C and present more complete NMR spec-
troscopic data than was available in the original publication.

0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.09.032
 C.M. Starks et al. / Phytochemistry 71 (2010) 104–109 105

12 R1 R replaced by a two proton resonance at d 4.51. These differences

20 indicated that 2 contains an angelate group attached at C-18. Com-
parison of the chemical shift of H-30 (6.04) with literature values
H H (Nair and Adams, 1960) confirmed that the esterified group was
1 17
an angelate, rather than the isomeric tiglate. The 1H signal at d
5 7
3 4.51 showed HMBC correlations to the angelate carbonyl (d
COOH 169.8) and to C-4 (d 137.0). Examination of the 2D NMR spectro-
18 R2 19 O O scopic data indicated that the clerodane core was the same as in 1.
The molecular formula of 3 was established as C20H28O5 based
1 R1 = A; R2 = CH3 8R=A
9R=B on HRESIMS. The 1H spectrum resembled that of 1, but lacked
2 R1 = A; R2 = CH2OAng
3 R1 = C; R2 = CH3 the oxygenated methylene signal at d 4.72. Instead, the NMR spec-
4 R1 = C; R2 = CH2OAng troscopic data indicated two doubly oxygenated methines (d 6.03,
5 R1 = D; R2 = CH3 99.0 and d 6.07, 98.7) whose proton signals together integrated to
6 R1 = E; R2 = CH3 one proton. The H-14 resonance also appeared as two singlets (d
7 R1 = E; R2 = CH2OAng 6.84, 6.91) that integrated to a single proton. These observations
solidagoic acid A R1 = F; R2 = CH3 were consistent with an analog of 1 hydroxylated at C-15 to form
solidagoic acid B R1 = F; R2 = CH2OAng a hemiacetal. The sample contained a mixture of C-15 epimers that
were not separated by HPLC. This mixture of epimers gave rise to
two sets of signals for H-14 and H-15, as well as for the H-17
A 15 B O C OH methyl protons.
O The NMR spectra of 4 resembled those of 3, but included signals
13 O
16 O for an angelate group. An angelate-containing structure was con-
sistent with the molecular formula of C25H34O7 established by
O
O HRESIMS. As in 2, the C-18 methyl group in 4 is replaced by an oxy-
genated methylene (two signals, d 4.46, 4.47). Additional support
D F
OCH3 E O for attachment of the angelate group at C-18 was provided by
ROESY correlations from the H-18 methylene protons to H-3 (d
O O O 5.90) and H-6b (d 2.37). Like 3, compound 4 was isolated as a pair
of C-15 epimers, giving rise to two sets of signals for H-14, H-15,
OH H-17 and H-18.
O
HRESIMS of 5 gave a molecular formula of C21H30O5, consistent
with a structure having an additional methoxyl group with respect
4'
O to 1. NMR spectroscopic data for 5 were obtained in CD3OD, and its
Ang = 1
H spectrum closely resembled the spectrum of 1 obtained in
1' 3'
CD3OD (Table S1, Supplementary data), except for the presence
5' of two singlets at d 3.51 and 3.53, which together integrated to
3H. In addition, the H-15 methylene signal in 1 was replaced by
Fig. 1. Structures of compounds 1–9 and solidagoic acids A and B. a resonance at d 5.81 (1H), and the H-14 signal moved slightly up-
field, appearing as two singlets (d 6.89, 6.91) that together inte-
grated to 1H. This pattern suggested that like 3 and 4, 5 also
Table 1
Antibacterial activity of clerodanes 1–9 against S. aureus.
existed as a mixture of C-15 isomers, this time with a methoxyl
group at C-15. The attachment point for the methoxyl group was
Compound MIC (lg/ml) verified by HMBC correlations from the methoxyl protons to C-
1 60 15 (d 104.7), and from H-15 to the methoxyl carbon (d 56.7).
2 30 HRESIMS of 6 indicated a structure isomeric with 3. Compared
3 >64
4 nda
with the other compounds examined, the H-14 and C-14 reso-
5 49 nances in 6 are shifted upfield (d 5.84, 118.1), consistent with
6 >64 placement of the butenolide carbonyl at C-15 (Table 3). Chemical
7 37 shifts for C-16 (d 100.5), and H-16 (d 5.93, 6.02, 1H total) are con-
8 43
sistent with the hemiacetal structure shown, comprising a mixture
9 >64
Vancomycin 2 of epimers at C-16. Unlike in 3, the hemiacetal protons do not show
allylic coupling to H-12.
a
Not determined.
HRESIMS of 7 indicated a structure isomeric with 4, and the 1H
and 13C chemical shifts suggested the same hemiacetal-containing
butenolide structure as in 6. This butenolide structure is further
To further verify the presence of the C-19 carboxylate moiety, supported by HMBC correlations from the H-16 protons at d 5.90
we prepared the methyl ester derivative of 1. The location of the to the C-15 carbonyl (d 174.7). Like 2 and 4, compound 7 has an
introduced methyl group was confirmed by HMBC correlations to angelate group attached at C-18. This structure is the butenolide
C-19 (d 179.4) from H-10 (d 2.31) and from the methoxy protons analog of solidagoic acid B (Anthonsen et al., 1973), and its optical
(d 3.56). The 1H signal for H-18 moved upfield upon methylation rotation (½a25
D 23) is comparable to that reported for solidagoic
(from d 1.53 to 1.44), suggesting interaction between Me-18 and acid B in the same solvent ([a]D 28).
the carboxylate oxygens in 1. The molecular formula of 8 was determined by HRESIMS to be
HRESIMS indicated a molecular formula of C25H34O6 for com- C20H26O4, indicating one more degree of unsaturation than in 1.
pound 2. The 1H spectrum of 2 resembled that of 1, with the addi- The 1H spectrum showed two doublets at d 3.90 and 4.47, and
tion of a quartet of quartets at d 6.04 and methyl signals at d 1.97 the HSQC indicated that they were attached to the same carbon
(d) and 1.89 (s). In addition, the olefinic C-18 methyl signal in 1 was (d 77.8). These methylene protons showed an HMBC correlation
106 C.M. Starks et al. / Phytochemistry 71 (2010) 104–109

Table 2
1
H NMR spectroscopic data for compounds 1–5 (600 MHz)a.

Position 1b 2b 3b 4b 5c
d d d
1a 1.51 1.57 m 1.50 1.56 1.57d
1b 1.73 br d (12.6) 1.78 br d (12.9) 1.71 br d (12.3) 1.77 br d (12.3) 1.75 br d (12.9)
2a 2.08d 2.19d 2.06d 2.18d 2.06d
2b 2.08d 2.19d 2.06d 2.18d 2.06d
3 5.50 br s 5.92 br s 5.48 br s 5.90 br s 5.46 br s
6a 1.40 td (13.3, 4.5) 1.51m 1.40d 1.52d 1.43d
6b 2.31d 2.41d 2.29d 2.37d 2.32d
7a 1.32d 1.34 m 1.33 m 1.34 m 1.32 m
7b 1.59d 1.65d 1.61d 1.64d 1.67d
8 1.63d 1.65d 1.64d 1.65d 1.68d
10 2.32d 2.39d 2.28d 2.34d 2.30d
11a 1.34d 1.26 td (13.6, 5.0) 1.40d 1.40 td (13.5, 5.0) 1.43d
11b 1.55d 1.69d 1.62d 1.64d 1.60d
12a 2.13 td (13.9, 3.7) 2.15 td (13.6, 4.6) 2.09d 2.10 2.08d
12b 2.45 br t (13.8) 2.49 br t (13.5) 2.40 br t (14.2) 2.41 br t (13.8) 2.42 m
14 7.13 s 7.18 s 6.84 s, 6.91 se 6.84 s, 6.92 se 6.89 s, 6.91 se
15 4.72 s 4.76 s 6.03 s, 6.07 se 6.03, 6.08d,e 5.81 s
15-OMe 3.51 s, 3.53 se
16
17 0.79 d (6.5) 0.80 d (5.6) 0.80, 0.82d,e 0.80, 0.81d,e 0.82, 0.83d,e
18 1.54 s 4.51 s 1.52 s 4.46, 4.47d,e 1.53 br s
20 0.96 s 0.98 s 0.93 s 0.95 s 0.97 s
30 6.04 qq (7.2, 1.8) 6.05 d
40 1.97 br d (7.3) 1.95 d (7.4)
50 1.89 br s 1.87 s
a
The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
b
NMR data in CDCl3.
c
NMR data in CD3OD.
d
Multiplicity was not determined due to overlapping signals.
e
Two sets of signals were observed, due to the mixture of epimers.

to a carbonyl at d 171.9 (Fig. 2), and there were no 1H signals for
either an H-18 or an H-19 methyl group. These observations sug-
gested that C-18 and C-19 are attached via a five-membered lac-
tone ring, with the carbonyl at either C-18 or C-19. The
methylene protons exhibited clear HMBC correlations to C-6 (d
32.4), placing them at C-19 and the carbonyl at C-18. The location
of the carbonyl at C-18 is further evidenced by the upfield shift of
H-6b (d 1.60) and the downfield shift of H-3 (d 6.77) compared
with the other compounds presented here. The 1D and 2D NMR
spectroscopic data indicated that the remainder of the structure
was identical to 1.
HRESIMS indicated that 9 was isomeric with 8. The NMR spectra
for the two compounds differed significantly only in the butenolide
ring. In 9, H-14 and C-15 resonate further upfield (d 5.80, 116.4)
than in 8 (d 7.15, 145.7), indicating that C-15, rather than C-16,
is oxidised to the carbonyl. Consistent with this, the butenolide
methylene protons show allylic coupling to H-12 in 8 but not in
9. Compound 9 was originally isolated from S. gigantea (Jurenitsch
et al., 1988), and more complete NMR spectroscopic data are pre-
sented here.
The relative stereochemistry of 1–9 was determined by compar-
ison to solidagoic acids A and B (Anthonsen et al., 1973), and was
supported by ROESY correlations and by the chemical shifts of
O et al., 1976).
the C-17, C-18, and C-20 methyl groups. The cis-decalin ring fusion
was established by the downfield chemical shift of C-20 (d 26.4–
27.2); in trans-clerodanes this chemical shift is observed at d 17–
19, while in cis-clerodanes it is observed at d 21–29 (Manabe and
Nishino, 1986). An H-20 proton resonance downfield from that of
H-17 is also indicative of a cis ring fusion (Nogueira et al., 2001);
in 1–9, H-20 resonates at d 0.93–0.98, whereas H-17 resonates at
d 0.79–0.85. A trans relationship between the C-17 and C-20
methyl groups is supported by the difference in their 13C shifts;
for trans-oriented methyl groups in a cis-decalin system, the ex-
pected DdC between C-17 and C-20 is 11, whereas for cis-oriented
methyl groups DdC is 2 (Nogueira et al., 2001). In compounds 1–9,
DdCs between C-17 and C-20 range from 10.1 to 11.3. Finally, the
configuration at C-9 was supported by ROESY correlations in 8 be-
tween H-19 (d 3.90, 4.47) and H-11a (d 1.26). The corresponding
ROESY correlation was also observed in 9.
The absolute stereochemistry of compounds 1–9 was assigned
by analogy with solidagoic acids A and B (Anthonsen et al.,
1973). Anthonsen et al. initially assigned the 5a, 10a-cis stereo-
chemistry to solidagoic acids A and B based on circular dichroism
measurements and comparison with the known compound plat-
hyterpenone. The absolute stereochemistry of the 5a, 10a-cis-
clerodanes was later confirmed by X-ray crystallographic analysis
of a bromine-containing derivative of solidagolactone VIII (Nishino
et al., 1984). Solidagoic acid A has been distinguished from 5b, 10b-
cis analogs by comparison of their optical properties (McCrindle

O 2.1. Concluding remarks

H HMBC
ROESY Two known and seven new clerodane diterpenes were isolated
from S. virgaurea. Seven of these possess a carboxylic acid at C-19, a
motif that is relatively uncommon among the clerodanes. The
O O moderate antibacterial activity displayed by these clerodanes sug-
gests that they may provide a starting point for the synthesis of
Fig. 2. Selected HMBC and ROESY correlations for 8. more active compounds.
 C.M. Starks et al. / Phytochemistry 71 (2010) 104–109
3. Experimental
3.1. General experimental procedures
NMR spectra were acquired at 600 MHz on a Bruker Avance
600 MHz spectrometer equipped with a 5 ll CapNMR capillary
microcoil probe with a 1.5 ll active volume (Magnetic Resonance
Microsensors, Savoy, IL). For each compound, 1H, gCOSY, ROESY,
HSQC, and HMBC spectra were acquired; 13C chemical shifts were
obtained from the HSQC and HMBC spectra. Spectra were recorded
in CDCl3 unless noted, and chemical shifts are reported with re-
spect to the residual non-deuterated solvent signal. Additional
chemical shifts recorded in CD3OD are in the Supplementary data.
HRESIMS was done on a Waters LCT time-of-flight mass spectrom-
eter with an electrospray interface and either berberine or chloro-
genic acid as internal standards. UV kmax values were taken from
the diode array detector during semipreparative HPLC purification
in 40–50% ACN with 0.05% TFA.
3.2. Plant material
Air-dried aerial parts of S. virgaurea (1 kg) were obtained from
American Mercantile Corporation (Memphis, TN). The material
was harvested in July in southeastern Bulgaria. Dr. Wendy Apple-
quist at Missouri Botanical Garden confirmed that the plant’s mor-
phology was consistent with S. virgaurea. A voucher specimen (S-
1857) was retained at Sequoia Sciences.
3.3. Extraction and isolation
High-throughput isolation of 1, 2, 5, 7, 8, and 9 was carried out
with published methods (Eldridge et al., 2002). Purification was
then scaled up to obtain additional material. Ground plant material
(1000 g) was extracted for 3 days with 5 L of EtOH–EtOAc (1:1, v/
v). The dried extract (90 g) was then subjected to silica gel CC with
the following eluents: hexane–EtOAc (3:1), hexane–EtOAc (1:1),
EtOAc, EtOAc–MeOH (7:3), and EtOAc–MeOH (1:1). Fractions
determined by HPLC to contain clerodanes were pooled and dried;
this material was again purified further on silica gel CC with the
following eluents: hexane–EtOAc (1:1), hexane–EtOAc (1:3),
EtOAc, and EtOAc–MeOH (1:1). Clerodane-containing fractions
were pooled (5.2 g), applied to C18 solid phase extraction car-
tridges (Phenomenex, 3  5 g), and eluted with MeOH–H2O (1:1,
v/v), MeOH–H2O (4:1, v/v), and MeOH. Material (3.3 g) eluting in
the MeOH–H2O (4:1, v/v) fraction was then separated by prepara-
tive reversed phase HPLC in 50 mg portions to yield fractions 1–5.
Preparative HPLC was performed using a Thermo Electron Betasil
C18 column (5 lm particle size, 100  21.2 mm) with a flow rate
of 20 ml/min. The column was eluted with CH3CN–H2O (15:85, v/
v) for 2 min, followed by a 34 min gradient to CH3CN–H2O (3:1,
v/v); the solvents contained 0.05% (v/v) TFA.
Compounds were isolated from fractions 1–5 by multiple semi-
preparative HPLC injections using either a Thermo Electron Fluo-
phase PFP column (5 lm particle size, 250  7.7 mm) with a flow
rate of 3 ml/min, or a Thermo Electron Hypersil Gold C18 column
(5 lm particle size, 250  10 mm) with a flow rate of 4 ml/min;
the solvents contained 0.05% (v/v) TFA. A splitter was used to di-
vert 10% of the eluent to a Sedere Sedex 75 ELSD detector, while
the rest passed through a diode array UV detector and was then
collected. The amount of each compound isolated was estimated
using HPLC/ELSD as previously described (Hu et al., 2005). Fraction
1 was applied to a Fluophase column with a 35–45% CH3CN–H2O
gradient to yield 8 (4.9 mg) and 9 (9.5 mg). Fraction 2 was sub-
jected to Hypersil Gold CC with a 40–50% CH3CN–H2O gradient
to yield 3 (4.1 mg) and 6 (4.4 mg), whereas fraction 3 was purified
107
by Hypersil Gold CC with a 45–58% CH3CN–H2O gradient to yield 1
(11.6 mg). Further purification of fraction 4 was achieved using
Hypersil Gold CC with a 50–58% CH3CN–H2O gradient to yield 4
(2.3 mg) and 7 (2.9 mg), whereas fraction 5 was purified using a
Fluophase column with a 35–45% CH3CN–H2O gradient to yield 2
(4.6 mg). Compound 5 was not detected during the scaled-up iso-
lation. Isolated compounds displayed >90% purity as estimated by
examination of the NMR spectra.
3.4. Bacterial assay
S. aureus strain 25923 was acquired from ATCC. An overnight
culture of S. aureus was diluted 1:100 in media (tryptic soy broth
with 0.5% D-glucose and 3% NaCl) and incubated at 37 °C until it
reached an O.D600 of 0.4. The resulting culture was diluted 1:10
in media and placed in a 96-well plate (100 lL/well). Test com-
pounds were dissolved in DMSO and then diluted in media and
added to the wells at a series of concentrations (100 lL/well, 3–6
wells per concentration per compound). Vancomycin was used as
a positive control. The plates were incubated for 24 h at 37 °C,
and growth inhibition was determined by the change in O.D600 at
the end of the incubation period compared to control wells with-
out compound. MICs are reported as the minimum concentration
that resulted in 90% inhibition.
3.5. Solidagoic acid C (1)
White solid; ½a25
D 50 (c 0.083, EtOH); UV kmax 217 nm; ESIMS
m/z 331 [MH], 355 [M+Na]+; For 1H NMR spectroscopic data see
Table 2; 13C NMR (CDCl3) d: 182.8 (C-19), 177.0 (C-16), 146.9 (C-
14), 137.2 (C-4), 136.5 (C-13), 125.1 (C-3), 71.9 (C-15), 52.9 (C-5),
43.6 (C-10), 39.6 (C-9), 38.4 (C-8), 31.6 (C-11), 30.6 (C-6), 29.4
(C-7), 28.1 (C-20), 27.8 (C-2), 21.3 (C-12), 20.8 (C-1), 20.4 (C-18),
17.1 (C-17).
3.6. Solidagoic acid D (2)
White solid; ½a25D 23 (c 0.083, EtOH); UV kmax 216 nm; HRE-
SIMS m/z 431.2461 [M+H]+ (calcd. for C25H35O6, 431.2434); For
1
H NMR (CDCl3) spectroscopic data, see Table 2; 13C NMR (CDCl3)
d: 169.8 (C-10 ), 147.6 (C-14), 139.4 (C-30 ), 137.0 (C-4), 136.5 (C-
13), 129.2 (C-20 ), 129.1 (C-3), 71.8 (C-15), 65.7 (C-18), 51.3 (C-5),
43.7 (C-10), 40.0 (C-9), 38.1 (C-8), 31.7 (C-11), 30.9 (C-6), 29.1
(C-7), 28.1 (C-20), 27.6 (C-2), 22.0 (C-50 ), 21.1 (C-12), 20.7 (C-1),
17.0 (C-40 ), 16.8 (C-17).
3.7. Solidagoic acid E (3)
White solid; ½a25
D 50 (c 0.083, EtOH); UV kmax 222 nm; HRE-
SIMS m/z 349.1992 [M+H]+ (calcd. for C20H29O5, 349.2015); For
1
H NMR (CDCl3) spectroscopic data, see Table 2; 13C NMR (CDCl3)
d: 181.6 (C-19), 146.1 (C-14), 144.9 (C-14), 139.8 (C-13), 137.6
(C-4), 124.8 (C-3), 99.0 (C-15), 98.7 (C-15), 52.7 (C-5), 43.5 (C-
10), 39.0 (C-9), 38.2 (C-8), 30.5 (C-6), 30.0 (C-11), 29.4 (C-7), 27.7
(C-2), 27.6 (C-20), 20.9 (C-12), 20.7 (C-1), 20.3 (C-18), 17.0 (C-17).
3.8. Solidagoic acid F (4)
White solid; ½a25
D 17 (c 0.083, EtOH); UV kmax 227 nm; HRE-
SIMS m/z 447.2381 [M+H]+ (calcd. for C25H35O7, 447.2383); For
1
H NMR (CDCl3) spectroscopic data, see Table 2; 13C NMR (CDCl3)
d: 181.4 (C-19), 146.4 (C-14), 145.0 (C-14), 129.4 (C-3), 99.1 (C-
15), 98.9 (C-15), 65.8 (C-18), 51.7 (C-5), 43.7 (C-10), 38.5 (C-9),
38.1 (C-8), 31.3 (C-6), 30.3 (C-11), 29.5 (C-7), 27.9 (C-20), 27.7
(C-2), 21.0 (C-12), 20.8 (C-1), 17.1 (C-17).
108 C.M. Starks et al. / Phytochemistry 71 (2010) 104–109

Table 3
1
H NMR spectroscopic data for compounds 6–9 (CDCl3, 600 MHz)a.

Position 6 7 8 9
b b b
1a 1.52 1.58 1.72 1.70b
1b 1.72 br d (12.9) 1.78 br d (11.5) 1.72b 1.70b
2a 2.08b 2.20b 2.21b 2.19b
2b 2.08b 2.20b 2.41 dq (20.2, 3.4) 2.41 br d (19.7)
3 5.49 br s 5.92 br s 6.74 br t (3.4) 6.71 br s
6a 1.43b 1.55b 1.48b 1.48b
6b 2.31b 2.42 d (13.2) 1.60b 1.60b
7a 1.35b 1.40 m 1.39b 1.41b
7b 1.65b 1.68b 1.39b 1.41b
8 1.66b 1.68b 1.66b 1.67b
10 2.27b 2.35 t (13.3) 1.63b 1.56b
11a 1.50b 1.53, 1.46b,c 1.26 m 1.29 td (13.0, 3.4)
11b 1.61b 1.67, 1.66b,c 1.47b 1.53b
12a 2.12, 2.29b,c 2.13, 2.32b,c 2.18b 2.29b
12b 2.65 br t (15.4), 2.50 br t (14.2)b,c 2.67, 2.52b,c 2.18b 2.33b
14 5.84 s 5.87 s 7.15 s 5.80 s
15 4.76 s
16 5.93 s, 6.02 sc 5.90, 6.04 4.72 s
17 0.83, 0.84b,c 0.85, 0.84b,c 0.81 d (6.7) 0.82 d (6.5)
18 1.54 s 4.49 s
19a 3.90 d (8.2) 3.72 d (7.9)
19b 4.47 d (8.2) 4.46 d (7.9)
20 0.94 s 0.96 s 0.94 s 0.92 s
30 6.05 br q (6.9)
40 1.97 dq (7.2, 1.0)
50 1.88 br s
a
The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
b
Multiplicity was not determined due to overlapping signals.
c
Two sets of signals were observed, due to the mixture of epimers.

3.9. Solidagoic acid G (5)
White solid; UV kmax 209 nm; ESIMS m/z 361 [MH]; HRESIMS
m/z 361.1981 [MH] (calcd. for C21H29O5, 361.2015); For 1H NMR
spectroscopic data, see Table 2; 13C NMR (CD3OD) d: 180.9 (C-19),
174.3 (C-16), 144.1 (C-14), 140.6 (C-13), 137.9 (C-4), 124.4 (C-3),
104.7 (C-15), 56.7 (C-15-OMe), 52.4 (C-5), 43.9 (C-10), 39 (C-9,
overlapped), 38.4 (C-8), 30.8 (C-6), 30.7 (C-11), 29.3 (C-7), 27.4
(C-2), 27.2 (C-20), 20.6 (C-1), 20.5 (C-12), 19.4 (C-18), 16.2 (C-17).
3.10. Solidagoic acid H (6)
White solid; ½a25
D 0 (c 0.083, EtOH); UV kmax 226 nm; HRESIMS
m/z 349.2007 [M+H]+ (calcd. for C20H29O5, 349.2015); For 1H NMR
(CDCl3) spectroscopic data, see Table 3; 13C NMR (CDCl3) d: 174.8
(C-15), 137.2 (C-4), 125.0 (C-3), 118.1 (C-14), 100.5 (C-16), 52.8
(C-5), 43.8 (C-10), 39.0 (C-9), 38.3 (C-8), 30.5 (C-6), 29.8 (C-11),
29.5 (C-7), 27.7 (C-2), 27.7 (C-20), 23.6 (C-12), 20.8 (C-1), 20.3
(C-18), 17.0 (C-17).
spectroscopic data, see Table 3; 13C NMR (CDCl3) d: 176.0 (C-16),
171.9 (C-18), 145.7 (C-14), 136.9 (C-3), 136.2 (C-4), 135.7 (C-13),
77.8 (C-19), 71.6 (C-15), 44.6 (C-5), 43.5 (C-10), 39.0 (C-9), 37.9
(C-8), 32.4 (C-6), 32.0 (C-11), 28.3 (C-7), 27.8 (C-2), 27.1 (C-20),
21.5 (C-12), 20.3 (C-1), 17.0 (C-17).
3.13. Cleroda-3,13(14)-dien-15,16:18,19-diolide (9)
White solid; ½a25
D 14 (c 0.083, EtOH); UV kmax 240 nm; HRE-
SIMS m/z 331.1929 [M+H]+ (calcd. for C20H27O4, 331.1909); For
1
H NMR spectroscopic data, see Table 3; 13C NMR (CDCl3) d:
175.7 (C-15), 172.8 (C-13), 171.9 (C-18), 137.2 (C-3), 135.8 (C-4),
116.4 (C-14), 77.5 (C-19), 74.5 (C-16), 44.5 (C-5), 43.7 (C-10),
39.3 (C-9), 37.8 (C-8), 32.3 (C-6), 31.6 (C-11), 28.2 (C-7), 27.6 (C-
2), 26.9 (C-20), 24.7 (C-12), 20.1 (C-1), 16.8 (C-17).
3.14. Methylation of 1

3.11. Solidagoic acid I (7)
White solid; ½a25
D 15 (c 0.083, EtOH); UV kmax 215 nm; ESIMS
m/z 445 [MH], 464 [M+NH4]+, 469 [M+Na]+, 347 [MOAng];
HRESIMS m/z 445.2227 [MH] (calcd. for C25H33O7, 445.2226);
For 1H NMR (CDCl3) spectroscopic data, see Table 3; 13C NMR
(CDCl3) d: 174.7 (C-15), 169.4 (C-10 ), 139.9 (C-30 ), 136.2 (C-4),
129.8 (C-3), 128.9 (C-20 ), 118.5 (C-14), 118.1 (C-14), 101.9 (C-16),
101.6 (C-16), 65.9 (C-18), 43.7 (C-10), 39.0 (C-9), 38.0 (C-8), 31.3
(C-6), 30.6 (C-11), 30.0 (C-11), 29.5 (C-7), 27.8 (C-2), 27.9 (C-20),
24.3 (C-12), 23.5 (C-12), 22.0 (C-50 ), 20.7 (C-1), 17.2 (C-40 ), 17.0
(C-17).
3.12. Cleroda-3,13(14)-dien-16,15:18,19-diolide (8)
White solid; ½a25
D +5 (c 0.083, EtOH); UV kmax 240 nm; HRESIMS
m/z 331.1896 [M+H]+ (calcd. for C20H27O4, 331.1909); For 1H NMR
Compound 1 (750 lg) was dissolved in dry MeOH (400 ll) and
treated overnight with an excess of trimethylsilyldiazomethane
(Presser and Hüfner, 2004). After quenching with HOAc, the prod-
uct was dried and purified by semipreparative HPLC to yield the
19-methyl ester (200 lg): white solid; UV kmax 210 nm; HRESIMS
m/z 347.2234 [M+H]+ (calcd. for C21H31O4, 347.2222); 1H NMR
(CD3OD) d: 7.27 (1H, s, H-14), 5.46 (1H, br s, H-3), 4.83 (2H, s, H-
15), 3.56 (3H, s, OMe), 2.34 (1H, m, H-6b), 2.31 (1H, m, H-10),
2.30 (1H, m, H-12b), 2.07 (3H, overlapped, H-2a, H-2b, H-12a),
1.76 (1H, br d, J = 12.6, H-1b), 1.68 (1H, m, H-8), 1.64 (1H, m, H-
7b), 1.57 (1H, m, H-1a), 1.47 (2H, overlapped, H-6a, H-11b), 1.44
(3H, s, Me-18), 1.35 (1H, m, H-7a), 1.31 (1H, m, H-11a), 0.98 (3H,
s, Me-20), 0.84 (3H, d, J = 6.5, Me-17); 13C NMR (CD3OD) d: 179.4
(C-19), 147.0 (C-14), 137.8 (C-4), 136.1 (C-13), 124.5 (C-3), 72.5
(C-15), 52.7 (C-5), 52.6 (C-19-OMe), 43.8 (C-10), 38 (C-9, over-
lapped), 38.2 (C-8), 30.4 (C-6), 30.2 (C-11), 29.3 (C-7), 27.5 (C-2),
27.2 (C-20), 20.7 (C-1), 20.4 (C-12), 19.3 (C-18), 16.2 (H-17).
 C.M. Starks et al. / Phytochemistry 71 (2010) 104–109
Acknowledgements
We acknowledge the scientific collaboration with Advanced
Chemistry Development, Inc., (ACD Labs) and the use of the ACD/
SpecManager and ACD/Structure Elucidator. We thank Damon Ar-
ney at American Mercantile for providing a voucher specimen of S.
virgaurea. Jiangnan Peng kindly measured optical rotations.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2009.09.032.
References
Anthonsen, T., Henderson, M.S., Martin, A., Murray, R.D.H., McCrindle, R., McMaster,
D., 1973. Constituents of Solidago species. Part IV. Solidagoic acids A and B,
diterpenoids from Solidago gigantea var. serotina. Can. J. Chem. 51, 1332–1345.
Eldridge, G.R., Vervoort, H.C., Lee, C.M., Cremin, P.A., Williams, C.T., Hart, S.M.,
Goering, M.G., O’Neil-Johnson, M., Zeng, L., 2002. High-throughput method for
the production and analysis of large natural product libraries for drug discovery.
Anal. Chem. 74, 3963–3971.
Goswami, A., Barua, R.N., Sharma, R.P., Baruah, J.N., Kulanthaivel, P., Herz, W., 1984.
Clerodanes from Solidago virgaurea. Phytochemistry 23, 837–841.
Hu, J.F., Garo, E., Yoo, H.D., Cremin, P.A., Zeng, L., Goering, M.G., O’Neil-Johnson, M.,
Eldridge, G.R., 2005. Application of capillary-scale NMR for the structure
determination of phytochemicals. Phytochem. Anal. 16, 127–133.
109
Jurenitsch, J., Maurer, J., Rain, U., Robien, W., 1988. Diterpenebutenolides in Solidago
gigantea. Phytochemistry 27, 626–627.
Levy, S.B., Marshall, B., 2004. Antibacterial resistance worldwide: causes, challenges
and responses. Nat. Med. 10, S122–129.
Mabberley, D.J., 1997. The Plant-book: A Portable Dictionary of the Vascular Plants.
Cambridge University Press, Cambridge.
Manabe, S., Nishino, C., 1986. Stereochemistry of cis-clerodane diterpenes.
Tetrahedron 42, 3461–3470.
McCrindle, R., Nakamara, E., Anderson, A.B., 1976. Constituents of Solidago species.
Part VII. Constitution and stereochemistry of the cis-clerodanes from Solidago
arguta Ait. and of related diterpenoids. J. Chem. Soc., Perkin Trans. 1, 1590–
1597.
Nair, M.D., Adams, R., 1960. Stereochemistry of necic acids. J. Am. Chem. Soc. 82,
3786–3787.
Nishino, C., Manabe, S., Kazui, M., 1984. Piscicidal cis-clerodane diterpenes from
Solidago altissimi L: absolute configurations to 5, 10-cis-clerodanes. Tetrahedron
Letters 25, 2.
Nogueira, R.T., Shepherd, G.J., Laverde Jr., A., Marsaioli, A.J., Imamura, P.M., 2001.
Clerodane-type diterpenes from the seed pods of Hymenaea courbaril var.
stilbocarpa. Phytochemistry 58, 1153–1157.
Payne, D.J., Gwynn, M.N., Holmes, D.J., Pompliano, D.L., 2007. Drugs for bad bugs:
confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discovery
6, 29–40.
Presser, A., Hüfner, A., 2004. Trimethylsilyldiazomethane – a mild and efficient
reagent for the methylation of carboxylic acids and alcohols in natural products.
Monatshefte für Chemie 135, 1015–1022.
Seaman, F., 1990. Diterpenes of Flowering Plants: Compositae (Asteraceae).
Springer-Verlag, New York.
Sztefanov, A., Petheö, F., Bernáth, J., Sárdi, E., 2002. Comparative analysis of
Hungarian Solidago virga-aurea populations. Acta Horticulturae (ISHS) 576, 53–
55.
Taubes, G., 2008. The bacteria fight back. Science 321, 356–361.