Metabolites of the smoke tree , Dalea spinosa, potentiate antibiotic activity against multidrug-resistant Staphylococcus aureus

Article abstract of DOI:10.1021/np058057s

Two new 2-arylbenzofuran aldehydes (1 and 2) and three known phenolic compounds (3-5) were isolated from organic extracts of Dalea spinosa. These compounds were evaluated for their intrinsic antimicrobial activity and their ability to perform as multidrug-resistance inhibitors by potentiating the activity of known antimicrobials against a variety of pathogenic microorganisms. Compound 1 and its acetate derivative 6 exhibited no direct antimicrobial activity but enhanced the effect of the weak plant antimicrobial berberine when tested against Staphylococcus aureus. Additional potentiation assays with S. aureus overexpression and knockout isogenic efflux mutants for the NorA pump were done in order to assess whether the potentiating effects were associated with inhibition of this known pump mechanism.

Full text of DOI:10.1021/np058057s

J. Nat. Prod. 2006, 69, 261-264
261
Notes
Metabolites of the “Smoke Tree”, Dalea spinosa, Potentiate Antibiotic Activity against
Multidrug-Resistant Staphylococcus aureus
Gil Belofsky,*,† Roberto Carreno,† Kim Lewis,‡ Anthony Ball,‡ Gabriele Casadei,‡,§ and George P. Tegos
Department of Chemistry and Biochemistry, The UniVersity of Tulsa, Tulsa, Oklahoma 74101, Department of Biology, Northeastern UniVersity,
Boston, Massachusetts 02115, Department of Food Science, School of Veterinary Medicine, UniVersity of Teramo, Teramo, Italy, 64100,
Department of Dermatology, HarVard Medical School, Wellman Laboratories of Photomedicine, Massachusetts General Hospital,
Boston, Massachusetts 02114
ReceiVed May 12, 2005
Two new 2-arylbenzofuran aldehydes (1 and 2) and three known phenolic compounds (3-5) were isolated from organic
extracts of Dalea spinosa. These compounds were evaluated for their intrinsic antimicrobial activity and their ability to
perform as multidrug-resistance inhibitors by potentiating the activity of known antimicrobials against a variety of
pathogenic microorganisms. Compound 1 and its acetate derivative 6 exhibited no direct antimicrobial activity but
enhanced the effect of the weak plant antimicrobial berberine when tested against Staphylococcus aureus. Additional
potentiation assays with S. aureus overexpression and knockout isogenic efflux mutants for the NorA pump were done
in order to assess whether the potentiating effects were associated with inhibition of this known pump mechanism.
As one travels west through the south-central plains of the United
States to Southern California, Dalea spp. generally change in
morphology from herbs to woody shrubs to the largest member of
the genus that grows up to eight meters in height, the “smoke tree”,
Dalea spinosa A. Gray (syn: Psorothamnus spinosus) (Fabaceae).
A methanolic extract of the lower bark of D. spinosa was subjected
to successive fractionation by silica gel and Sephadex LH-20
chromatography. The resulting fractions were examined by 1H NMR
spectroscopy and TLC, to identify compounds with promising
chemical functionalities and good chromatographic resolution. An
aldehyde functionality, later observed to be visible in the HSQC
NMR of the crude extract itself, was present in several fractions,
which led us to focus on these materials, resulting in the isolation
of spinosans A (1) and B (2). Spinosan A is a potent new potentiator
of antibiotic activity against multidrug-resistant (MDR) Staphylo-
coccus aureus. Further chemically guided fractionation led to the
isolation of two known pterocarpans (3 and 4) and a known
isoflavone (5).
The HRESIMS, 13C NMR, and HSQC data for compound 1
indicated a molecular formula of C17H12O6. Distinctive signals were
1
present in the H and 13C NMR spectra (Table 1) for an aldehyde
group, a methylenedioxy functionality, and a methoxy group. Also
present were five aromatic proton signals, three of which exhibited
ortho and meta coupling (8.4 and 2.0 Hz, respectively), indicating
the connection of the trisubstituted benzene ring to C-2. The location
their placement on one trisubstituted benzene ring. Assignment of
of the methoxy group at C-2′, rather than at C-4′, was supported
by the observation of only one NOESY correlation between the
methoxy protons and H-3′. To further confirm the structure of 1,
the acetate derivative 6 was prepared. Complete NMR spectroscopic
data for 6 were acquired (see Experimental Section) that agreed in
all respects with the overall structure assignments for 1. As observed
for 1, the methoxy group exhibited a NOESY correlation to H-3′.
C-4′ was shielded by 8.6 ppm upon acetylation of the attached
oxygen and correlated to H-5′ by HMBC. These data verified the
relative positions of the hydroxy and methoxy groups in spinosan
A (1).
The HRESIMS of 2 revealed a molecular formula of C17H14O5,
requiring one less degree of unsaturation than 1. The formyl group
was present as in 1, but the methylenedioxy group was replaced
the overall substitution pattern of this ring was accomplished in a
straightforward manner from HSQC and HMBC correlations. The
benzofuran ring system, with the methylenedioxy group placed at
C-5,6, was established with the aid of key HMBC correlations from
H-4 to C-3, C-5, and C-6 and from H-7 to C-5, C-6, C-8, and C-9,
1
as well as by comparison of the H and 13C NMR spectra with
known compounds.1-3 Other key HMBC correlations, between H-6′
and C-2 and from the formyl proton to C-2 and C-3, established
* Corresponding author. Tel: 303-381-2069 Fax: 303-381-2074. E-
mail: gbelofsky@efficas.com.
† University of Tulsa.
‡ Northeastern University.
§ University of Teramo.
Massachusetts General Hospital.
10.1021/np058057s CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/12/2006

262 Journal of Natural Products, 2006, Vol. 69, No. 2
Notes
Table 1. NMR Data for Compounds 1 and 2 in Acetone-d6
Table 2. Berberine Potentiation Assay: MICs (µM) of
Berberinea against S. aureus Wild-Type (WT), NorA Knockout
(NorA-), and Overexpression Mutant (NorA+), Alone and in
the Presence of 1, 4, 5, 6, or INF 271 (at molar concentrations
in the range 42-56 µM)
1
2
a
a
position
δC
δHb (mult.; JHH
)
δC
δHb (mult.; JHH
)
2
3
163.6
118.4
163.6
118.0
berberine (alone) +1 +4 +5 +6 +INF 271
3-CHO
4
5
6
187.8 9.98 (s)
101.0 7.51 (s)
C147.8
187.8 10.02 (s)
S. aureus (WT)
S. aureus (NorA-)
S. aureus (NorA+)
372
89
>1488
45 89 89
21 45 45
NP NP NP 45
6
6
3
6
123.0
114.0
159.8
8.01 (d; 8.6)
7.00 (dd; 8.6, 2.2)
6
C146.8
a Berberine test range in the potentiation study 1.5-89 µM. NP )
no potentiation at 89 µM berberine.
5,6-OCH2O-
102.8 6.07 (s)
6-OCH3
56.2
96.6
3.88 (s)
7.18 (d; 2.2)
7
8
9
1′
94.2 7.14 (s)
150.4
119.6
110.0
160.0
56.1 3.84 (s)
100.6 6.70 (d; 2.0)
162.5
108.8 6.65 (dd; 8.4, 2.0) 108.9
133.6 7.48 (d; 8.4) 133.7
cell growth under these conditions and having no activity when
used alone were considered to exhibit MDR inhibitory activity. In
preliminary assays, compounds 1, 4, 5, and 6 showed potentiation
activity against S. aureus when used in combination with berberine,
while 2 and 3 did not. Accordingly, the active compounds were
further analyzed in a more detailed potentiation assay, and the
results are summarized in Table 2.
To test whether the MDR inhibitory effects of the active
compounds are exclusively related to the NorA pump of S. aureus,
the isogenic pair of knockout and overexpression NorA mutants
of S. aureus, containing none versus elevated levels of NorA,
respectively, were used.19-21 INF 271, a synthetic efflux pump
inhibitor, was used as a positive control.22
Berberine exhibited MICs of 372 and 89 µM against the wild-
type S. aureus and the isogenic NorA mutant, respectively. As
expected, the NorA-overexpressing S. aureus mutant was consider-
ably less susceptible to berberine (MIC > 1488 µM). Spinosan A
(1, tested at 48 µM) and its acetate (6, 42 µM) decreased the MIC
of berberine approximately 8-fold and 62-fold, respectively, against
wild-type S. aureus. Compounds 4 and 5 (at 56 and 48 µM,
respectively) potentiated the activity of berberine to a lesser degree,
each causing a 4-fold decrease in MIC. Compounds 1 (48 µM)
and 4 (56 µM) potentiated berberine against the S. aureus knockout
efflux mutant, causing 15- and 4-fold decreases in MIC, respec-
tively, whereas the effects of 5 and 6 were less. Below ∼40 µM
concentrations the inhibitory activity of all of the D. spinosa
compounds decreased steadily. Moderate potentiation (berberine
MIC ) 89 µM) was still observed, however, for 1 and 6 at
concentrations of 6.3 and 5.6 µM, respectively.
Compound 6 enhanced the antimicrobial effect of berberine
against the NorA overexpression S. aureus mutant, while also
causing a greater fold decrease in MIC in the wild-type strain
compared to the knockout mutant, suggesting NorA-associated
activity. Although the MICs of berberine against the S. aureus
knockout efflux mutant changed when combined with 1, 4, and 5,
the magnitude of these changes relative to berberine alone were
not sufficient to conclude NorA-associated activity. Additional
efflux systems are present in S. aureus that have not yet been
extensively studied or characterized,23 and the inhibitory activities
of these D. spinosa metabolites may be associated with more than
one efflux system or with an unknown mechanism. None of the
compounds showed potentiation against the other microorganisms
tested in this study.
156.3
119.3
109.9
160.1
56.2
2′
2′-OCH3
3.86 (s)
6.71 (d; 2.1)
3′
4′
5′
6′
100.6
162.6
6.66 (dd; 8.3, 2.1)
7.52 (d; 8.3)
a 75 MHz. b 400 MHz. c Assignments may be interchanged.
by an ortho-coupled aromatic proton and a methoxy group. These
data, along with HSQC and HMBC NMR correlations, and
comparison with 1, 6, and known compounds1-3 led us to elucidate
the structure of spinosan B (2) shown. NOESY correlations from
H3CO-2′ to both H-3′ and the formyl proton confirmed the relative
positions of the hydroxy and methoxy groups in this portion of the
molecule. The position of the methoxy at C-6 was further confirmed
by NOESY correlations to both H-5 and H-7. To our knowledge,
the basic structures of spinosans A and B are closely related to
only two previously reported compounds, melimessanol C1 and
maginaldehyde.3
The structures of known compounds 3 and 4 were determined
by NMR spectroscopic and mass spectrometric methods, and these
data corresponded in all essential respects with those reported.4-11
Complete NMR and other spectroscopic data for 3 are reported
here for the first time. Compound 4 was also compared to an
authentic sample, previously isolated in-house.8
Interpretation of NMR spectroscopic data and comparison of
these data to known compounds12-14 led us to elucidate the structure
of compound 5 as 6,4′-dimethoxy-7,2′-dihydroxyisoflavone.15,16 Key
HMBC correlations that established regiochemistry were those from
H-2 to C-1′, from H-6′ to C-3, and from H-5 to C-4. The relative
positions of the two methoxy groups were established by HMBC
correlations to their respective points of attachment, to which mutual
correlations from aromatic ring protons were observed. The
placement of the methoxy at C-6 was further supported by a
NOESY correlation from this group to H-5. Extensive spectroscopic
data for 5 (see Experimental Section) have not been previously
reported.
Transmembrane efflux pump mechanisms are major components
of resistance to many classes of antibiotics. Multidrug-resistance
pumps (MDRs) expel a variety of structurally diverse compounds.17
Various chemotypes have been shown to inhibit MDRs in micro-
organisms,18 including compounds reported from another Dalea sp.8
This effect enhances the utility of both conventional antibiotics and
weak plant antimicrobials. Compounds 1-6 were evaluated in
antimicrobial assays in order to determine both direct activity and
MDR inhibitory activity against a panel of pathogenic microorgan-
isms, including Gram-positive bacteria (Staphylococcus aureus,
Enterococcus faecalis), Gram-negative bacteria (Escherichia coli,
Pseudomonas aeruginosa), and yeast (Saccharomyces cereVisiae,
Candida albicans). Compounds 1-6 showed no direct antimicrobial
activity (MIC >140 µM, data not shown) against these organisms.
MDR inhibitory activities of 1-6 were evaluated by combining
each of these compounds with a subinhibitory concentration of the
relatively weak antimicrobial berberine. Compounds that inhibited
Experimental Section
General Experimental Procedures. NMR spectra were acquired
on a Varian UNITY INOVA 400 spectrometer, equipped with an
inverse detection probe, and on a Varian Unity Plus 300 spectrometer.
The NOESY NMR spectra of vanillin and m-anisaldehyde were
obtained as controls for correlations between ortho aromatic substit-
uents. Optical rotations were obtained on a JASCO model P-1010
polarimeter. IR spectra were recorded on a Nicolet Avatar 360 FT-IR
spectrophotometer, and UV spectra were acquired on a Hewlett-Packard
8453 diode array spectrophotometer. EIMS were obtained on a
Shimadzu GCMS-QP5000 equipped with a DI-50 direct sample inlet
device. ESIMS/MS and HRESIMS were obtained in positive ion mode
on a Micromass Q-TOF mass spectrometer.

Notes
Plant Material. Twigs, spines, stems, flower buds, and pieces of
Journal of Natural Products, 2006, Vol. 69, No. 2 263
MeOH in CH2Cl2 (0.5%, 1%, 1.5%, 2%, 3%, 5%, 8%, 10%, 30%; 200
mL fractions). Fraction 3 (35 mg) from this column was then purified
using a second silica gel gravity elution column (2.5 cm × 7.5 cm)
with a step gradient of 100% hexane, followed by EtOAc in hexane
(10%, 15%, 20%, 24%, 28%, 32%, 36%, 40%, 50%, 75%, 100%; 100
mL fractions). Fractions 6 and 7 from this column were combined and
evaporated (20 mg), and MeOH was added to precipitate pure spinosan
A (1, 12 mg). An additional 5 mg of 1 was later isolated from other
fractions using similar procedures.
lower bark of Dalea spinosa A. Gray (Fabaceae) “smoke tree” were
collected by one of the authors (Gil Belofsky) and Mr. Kavon Azadi
on May 31, 2002, in Coachwhip Canyon, Anza-Borrego Desert, GPS
position: N 33°17.217′, W 116°09.064′. A voucher specimen (#11563)
was authenticated by Dr. Paul Buck, Professor Emeritus, Department
of Biological Science, The University of Tulsa, and deposited in the
Barclay Herbarium at the same location. Plants were stored in a -20
°C freezer prior to extraction.
Microbial Strains, Chemicals, and Susceptibility Testing. S.
aureus strains (the wild-type 8325-4, isogenic NorA knockout/K1758,
and overexpression/K2361 mutants), E. coli (wild-type strain K12), and
P. aeruginosa (PA14) were cultured in Mueller-Hinton (MH) broth.
E. faecalis (V583) was cultured in brain heart infusion (BHI). S.
cereVisiae (BY4742) and C. albicans (F5) cells were grown in yeast
extract peptone and dextrose (YPD). INF 271 was kindly provided by
Protez Pharmaceuticals, Malvern, PA. Berberine was purchased from
Sigma Chemical Co. (St. Louis, MO). Cells (105/mL) were inoculated
into MH, BHI, or YPD and dispensed at 0.2 mL/well in 96-well
microtiter plates.
Growth inhibition was determined by serial 2-fold dilution of test
compounds, starting at 50 µg/mL (42-50 µM), combined with 30 µg/
mL (89 µM) of berberine against Gram-positive bacteria, 10 µg/mL
(13.6 µM) erythromycin for Gram-negative bacteria, and 1 µg/mL (3.3
µM) fluconazole for yeast. An MDR inhibitor was defined as a
compound that completely prevented cell growth in the presence of
subinhibitory concentrations of an antibiotic during an 18-h incubation
at 37 °C for S. aureus, E. faecalis, E. coli, and P. aeruginosa and a
24-h incubation at 30 °C for S. cereVisiae and C. albicans. All tests
were done in triplicate by following National Center for Clinical
Laboratory Standards (NCCLS) recommendations.24 Growth was as-
sayed with a microtiter plate reader (Spectramax PLUS384, Molecular
Devices) by absorption at 600 nm.
Berberine Potentiation Assay. A checkerboard assay was conducted
to specify the degree of potentiation of berberine by 1, 4, 5, and 6 and
to determine the specificity of these compounds for the NorA efflux
pump. Serial 2-fold dilutions of berberine and a test compound were
mixed in each well of a 96-well microtiter plate so that each row (and
column) contained a fixed amount of one agent and increasing amounts
of the second agent. The resulting plate presents a pattern in which
every well contains a unique combination of concentrations between
the two molecules. The concentrations of berberine (row) ranged from
30 to 0.5 µg/mL (89-1.5 µM), while plant compound (column)
concentrations ranged from 15 to 0.015 µg/mL (corresponding to molar
concentrations in the ranges 56-42 µM to 0.06-0.04 µM). Each plate
also contained a row and column in which a serial dilution of each
agent was present alone. The >89 µM (>30 µg/mL) MIC values for
berberine alone, against S. aureus wild-type and NorA overexpression
strains (Table 2), were determined in a separate assay. Cells were added
to each well at a final concentration of 5 × 106 CFU/mL, and plates
incubated at 37 °C for 24 h. Growth was assayed by absorption at 600
nm with a microtiter plate reader (Spectramax PLUS384, Molecular
Devices). An OD less than 0.04 was considered to reveal no bacterial
growth.
Extraction and Isolation. Finely divided D. spinosa bark (220 g)
was extracted with MeOH (1.5 L, 24 h) to provide, after evaporation,
4.1 g of crude extract. This material was preadsorbed in a CH2Cl2-
MeOH solution onto ∼10 g of silica gel, the solvent removed under
vacuum, and the resulting powder subjected to vacuum liquid chro-
matography (VLC) over a prepacked column bed, 10 cm (i.d.) × 3.5
cm (h), of TLC-grade silica gel (Selecto Scientific). The column was
eluted using a stepwise gradient of solvents (500 mL each), beginning
with hexane and continuing with mixtures of EtOAc in hexane (20%,
40%, 60%, 80%, 100%), followed by mixtures of MeOH in CH2Cl2,
up to 30%. The five fractions that eluted with 20-100% EtOAc were
combined on the basis of TLC analysis (EM Science, silica gel 60,
Fraction 9 (86 mg) from the Sephadex column was further purified
using column chromatographic procedures nearly identical to that
described above, resulting in a 23 mg fraction that, following addition
of MeOH, precipitated pure spinosan B (2, 13 mg).
Fractions 8 (62 mg) and 10 (56 mg) from the Sephadex column
were purified over silica gel using a sequence similar to that described
above (step gradients of MeOH in CH2Cl2, followed by EtOAc in
hexane), resulting in, respectively, compound 3 (9 mg) and, with one
additional EtOAc-hexane silica gel column procedure, compound 4
(8 mg).
Fraction 12 (86 mg) from the Sephadex column was subjected to
three successive silica gel chromatography procedures (step gradients
in MeOH-CH2Cl2, EtOAc-hexane, and MeOH-CH2Cl2) to provide
a 22 mg fraction of interest (primarily a purple spot with vanillin/H2SO4
spray reagent on TLC, Rf ) 0.6 in 9:1 CH2Cl2-MeOH). This fraction
was purified over silica gel (2.5 cm × 7 cm; Davison 100-200 mesh)
using a linear gradient of MeOH (0 f 2%) in CH2Cl2, at a flow rate
of ∼20 mL/min, to afford compound 5 (6 mg).
Spinosan A (1): pale yellow solid; UV(MeOH) λmax (log ꢀ) 211
(4.32), 248 (4.03), 296 (3.72), 347 (3.88) nm; IR νmax (CHCl3) 3300
(br OH), 3019, 1664, 1616, 1458, 1310, 1125, 1034 cm-1; 1H and 13
C
NMR data, see Table 1; HMBC correlations (acetone-d6) CHO f C-2,
3, 9; H-4 f C-3, 5, 6, 7*, 8; OCH2O f C-5, 6; H-7 f C-4*, 5, 6, 8,
9; H3CO f C-2′; H-3′ f C-1′, 2′, 4′, 5′; H-5′ f C-1′, 2′*, 3′, 4′, 6′;
H-6′ f C-2, 2′, 3′*, 4′ (*indicates weak four-bond correlation);
HRESIMS found m/z 313.0670 (M + H)+, calcd for C17H13O6 313.0712.
Spinosan B (2): pale yellow solid; UV(MeOH) λmax (log ꢀ) 207
(4.42), 244 (4.28), 279 (3.80), 343 (3.98) nm; IR νmax (film on NaCl)
3323 (br OH), 2905, 1654, 1618, 1589, 1498, 1315, 1266, 1202, 1143,
1
1062 cm-1; H and 13C NMR data, see Table 1; HMBC correlations
(acetone-d6) CHO f C-2, 3; H-4 f C-3, 5, 6, 7*, 8; H-5 f C-6, 7, 9;
H3CO-6 f C-6; H-7 f C-5, 6, 8, 9; H3CO-2′ f C-2′; H-3′ f C-1′,
2′, 4′ 5′; H-5’ f C-1′, 2′*, 3′, 4′; H-6′ f C-2′, 3′*, 4′ (*indicates
weak four-bond correlation); HRESIMS found m/z 299.0894 (M + H)+,
calcd for C17H15O5 299.0919.
(+)-Melilotocarpan A (3): yellow oil; [R]D +21.0 (c 0.20, CHCl3);
UV(MeOH) λmax (log ꢀ) 208 (4.67), 228sh (4.11), 283 (3.75) nm; IR
νmax (CHCl3) 3528 (br OH), 3020, 2938, 1623, 1605, 1497, 1477, 1278,
1147, 1090 cm-1; 1H NMR (CDCl3) δ 7.19 (1H, d, J ) 8.4, H-5), 7.15
(1H, d, J ) 8.8, H-6′), 6.71 (1H, d, J ) 8.4, H-6), 6.47 (1H, d, J )
2.2, H-3′), 6.47 (1H, dd, J ) 8.8, 2.2, H-5′), 5.51 (1H, d, J ) 6.5,
H-4), 4.36 (1H, dd, J ) 10.5, 4.8, H-2â), 3.92 (3H, s, H3CO-7), 3.78
(3H, s, H3CO-4′), 3.64 (1H, d, J ) 10.5, H-2R), 3.57 (1H, dd, J ) 6.5,
4.8, H-3); 13C NMR (CDCl3) δ 161.4 (C-4′), 160.9 (C-2′), 149.9 (C-
8), 148.7 (C-9), 135.1 (C-7), 126.3 (C-5), 125.0 (C-6′), 119.1 (C-1′),
113.4 (C-10), 109.2 (C-6), 106.7 (C-3′), 97.1 (C-5′), 78.7 (C-4), 66.9
(C-2), 61.4 (OCH3-7), 55.7 (OCH3-4′), 39.6 (C-3); HMBC correlations
(CDCl3) H2-2 f C-3, 4, 9, 1′; H-3 fC-2, 1′, 2′; H-4 f C-2, 3, 5, 9,
10; H-5 f C-4, 7, 9; H-6 f C-7, 8, 10; H3CO-7 f C-7; H-3′ f C-1′,
2′, 5′; H3CO-4′ f C-4′; H-5′ f C-1′, 3′; H-6′ f C-3, 2′, 4′, 5′;
HRESIMS found m/z 301.1053 (M + H)+, calcd for C17H17O5 301.1076.
(+)-Medicarpin (4): yellow oil; [R]D +16.7 (c 0.20, CHCl3); UV,
1H NMR, and 13C NMR spectra were consistent with an authentic
sample;8 the structure of 4 was also confirmed by HSQC and HMBC
NMR spectroscopy; EIMS m/z 271 (M + H+, rel int 100), 256 (36),
242 (47), 213 (7), 185 (5), 175 (8), 162 (33), 148 (29), 135 (24).
6,4′-Dimethoxy-7,2′-dihydroxyisoflavone (5): pale yellow solid;
mp 198-200 °C (lit. 195-198 °C);16 UV(MeOH) λmax (log ꢀ) 204
(4.46), 216 (4.38), 256 (4.15), 288 (3.97), 320 (3.92), 359 (3.46) nm;
IR νmax (film on NaCl) 3393 (br OH), 1616, 1576, 1506, 1480, 1437,
F254, with vanillin/concentrated H2SO4 spray reagent, 1% w/v), and the
solvents were evaporated. The residue (1.5 g) was further fractionated
by Sephadex LH-20 (Sigma) column chromatography (2.5 cm × 58
cm) eluting with 1 L of 3:1:1 hexane-toluene-MeOH at a flow rate
of 0.5 mL/min, collecting ∼5 mL fractions. Fractions of similar
composition as determined by TLC were pooled, resulting in 15
fractions. Fraction 11 (81 mg) from this column was further purified
over silica gel (2.5 cm × 9 cm, Davison Chemical 100-200 mesh) by
gravity elution using a step-gradient of 100% CH2Cl2, followed by
1
1285, 1201, 1160 cm-1; H NMR (CDCl3) δ 8.08 (1H, s, H-2), 7.69
(1H, s, H-5), 7.09 (1H, d, J ) 8.6, H-6′), 7.07 (1H, s, H-8), 6.67 (1H,
d, J ) 2.6, H-3′), 6.56 (1H, dd, J ) 8.6, 2.6, H-5′), 4.06 (3H, s, OCH3-
6), 3.84 (3H, s, OCH3-4′); 13C NMR (CDCl3) δ 178.6 (CdO), 162.2
(C-4′), 158.2 (C-2′), 154.7 (C-2), 152.9 (C-9), 152.5 (C-7), 146.2 (C-
6), 130.5 (C-6′), 124.4 (C-3), 116.9 (C-10), 113.3 (C-1′), 107.9 (C-5′),

264 Journal of Natural Products, 2006, Vol. 69, No. 2
Notes
104.64 (C-5), 104.55 (C-3′), 102.6 (C-8), 56.9 (OCH3-6), 55.6 (OCH3-
4′); HMBC correlations (CDCl3) H-2 f C-3, 4, 9, 1′; H-5 f C-4, 6,
7, 8*, 9, 10; H3CO-6 f C-6; H-8 f C-4*, 6, 7, 9, 10; H-3′ f C-1′,
2′, 4’, 5′; H3CO-4′ f C-4′; H-5′ f C-1′, 3′, 4′; H-6′ f C-3, 2′, 3′*,
4′; *indicates weak four-bond correlation; EIMS m/z 314 (M+, rel int
40), 297 (11), 282 (2), 271 (2), 175 (3), 166 (13), 157 (9), 148 (100),
133 (16).
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Spinosan A Acetate (6). To 9 mg of 1 were added 2 mL of
triethylamine and 1 mL of Ac2O. The reaction mixture was stirred at
RT for 1.5 h and evaporated under dry N2. To the residue was added
3 mL of H2O, which was then extracted twice in succession with 3
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°C; UV(MeOH) λmax (log ꢀ) 209 (4.44), 244 (4.11), 289 (3.86), 341
(3.96) nm; IR νmax (CHCl3) 3018, 1766, 1670, 1614, 1591, 1500, 1462,
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6.04 (2H, s, OCH2O), 3.86 (3H, s, OCH3), 2.36 (3H, s, OCOCH3); 13
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Acknowledgment. Funding for G.B. was generously provided by
The Research Corporation. Funding for K.L. was provided by NIH
grant R21AI059483. The authors would like to thank Dr. M. Eastman
and Ms. G. Bell-Eunice of the Oklahoma State University, Department
of Chemistry, Statewide Shared NMR Facility, funds for which were
provided by the National Science Foundation (BIR-9512269), the
Oklahoma State Regents for Higher Education, the W.M. Keck
Foundation, and Conoco, Inc. Additional thanks to Dr. L. Zhang of
the University of Oklahoma, Department of Chemistry and Biochem-
istry, Mass Spectrometry Facilities. K.L. would like to thank G. Kaatz
of Wayne State University School of Medicine, Michigan, for S. aureus
strains K1758 and K2361, and F. Ausubel, Department of Molecular
Biology, Massachusetts General Hospital, for P. aeruginosa and E.
faecalis. Special thanks to Mr. T. Allport and Ms. F. Galvani for lodging
and plant collection tools.
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NP058057S