Polyoxygenated methyl cyclohexanoids from a terrestrial ampelomyces fungus

Article abstract of DOI:10.1021/np800667e

Seven structurally related new polyoxygenated methyl cyclohexanoids, ampelomins A-G (1-7), were isolated from the mycelial solid culture of a soil-derived Ampelomyces fungus. Their structures were determined by spectroscopic and chemical means. Ampelomins

Full text of DOI:10.1021/np800667e

J. Nat. Prod. 2009, 72, 265–269
265
Polyoxygenated Methyl Cyclohexanoids from a Terrestrial Ampelomyces Fungus
Huiye Zhang,†,‡ Jinghua Xue,† Ping Wu,†,‡ Liangxiong Xu,† Haihui Xie,† and Xiaoyi Wei*,†
CAS Key Laboratory for PreserVation and Sustainable Utilization of Plant Resources, South China Botanical Garden, Chinese Academy of Sciences,
Xingke Road 723, Tianhe District, Guangzhou 510650, People’s Republic of China, and Graduate School of Chinese Academy of Sciences, Yuquanlu
19A, Beijing 100049, People’s Republic of China
ReceiVed October 22, 2008
Seven structurally related new polyoxygenated methyl cyclohexanoids, ampelomins A-G (1-7), were isolated from
the mycelial solid culture of a soil-derived Ampelomyces fungus. Their structures were determined by spectroscopic
and chemical means. Ampelomins A (1), C (3), E (5), and G (7) exhibited weak activity against R-glucosidase with
IC50 values of 1.74-5.93 mM, and ampelomin A (1) showed moderate antibacterial activity with MIC90 values ranging
from 202.4 to 1015.9 µM. A plausible polyketide biogenetic pathway is postulated for these compounds.
Glycosidases, the enzymes responsible for the hydrolysis of
glycosidic bonds in poly- and oligosaccharides or glycoconjugates,
are crucial in many metabolic pathways, including glycoprotein and
glycolipid processing and carbohydrate digestion in the intestinal
tract.1 Inhibitors of glycosidases have potential for the treatment
of various disorders and diseases such as diabetes, cancers, and
AIDS.1 Polyhydroxylated cyclohexanoids, a class of compounds
closely related to carbasugars, are known to have activity as
glycosidase inhibitors, and therefore they have received much
attention in recent years. Though a number of known compounds
have been isolated from plants and microbials and many more have
been prepared through chemical synthesis,2 the search for new
polyhydroxylated cyclohexanoids with improved biological activi-
ties remains an active area of current research in natural products.
Ampelomyces sp. SC0307 is a soil-derived fungal strain pos-
sessing strong antibacterial activity. We recently reported the
isolation of heteroatom-containing phenolics as the principle
compounds responsible for antibacterial activity.3 In continuing our
study, we investigated the other metabolites of this fungus. The
investigation led to the discovery of seven new polyoxygenated
methyl cyclohexanoids, ampelomins A-G (1-7). We herein report
the isolation, structure elucidation, biological activities, and plausible
biogenetic pathway of these compounds.
13C NMR and DEPT spectra displayed seven signals, indicating
the presence of a carbonyl group [δ 201.6 (C-1)], two olefinic
methines [δ 153.5 (C-3), 128.4 (C-2)], an oxymethine [δ 67.2 (C-
4)], an aliphatic methine [δ 40.1 (C-6)], a methylene [δ 41.2 (C-
1
5)], and a methyl group [δ 14.8 (C-7)]. The H NMR spectrum
exhibited signals at δ 1.45 (3H, d, J ) 6.5 Hz, H-7) for a methyl
group, δ 4.64 (1H, ddt, J ) 10.2, 4.8, 2.0 Hz, H-4) for an
oxymethine, and δ 2.35-2.40 (2H, m, H-5eq and H-6) and 1.72
(1H, tdd, J ) 12.2, 10.2, 2.0 Hz, H-5ax) for a methylene and a
methine. In addition, the spectrum showed signals at δ 6.85 (1H,
dt, J ) 10.0, 2.0 Hz, H-2) and 5.95 (1H, dd, J ) 10.0, 2.0 Hz,
H-3) indicating the presence of a cis double bond. Interpretation
of the COSY and HMQC spectra confirmed the presence of the
functional groups noted above and allowed assignment of the gross
structure as shown. The large proton coupling constants, J4,5ax
)
10.2 Hz and J5ax,6 ) 12.2 Hz, showed an envelope conformation
of the cyclohexenone ring in which both H-4 and H-6 were in
pseudoaxial positions.
The absolute configuration of 1 was established using the
modified Mosher’s method.4 Treatment of 1 with (R)- and (S)-R-
methoxy-R-trifluoromethylphenylacetic acids (MTPA) in the pres-
ence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-
ride (EDC-HCl) using catalytic DMAP5 afforded (R)- and (S)-
MTPA esters (1a and 1b, respectively) (see Experimental Section).
Analysis of the proton chemical shift differences (∆δ ) δS - δR)
showed negative ∆δ signs for H-3 (-0.11) and H-2 (-0.03) and
positive signs for H-5ax (+0.09), H-5eq (+0.06), H-6 (+0.02), and
H3-7 (+0.02). This distribution of ∆δ signs enabled assignment of
the R-configuration to C-4.4 Accordingly, the S-configuration of
C-6 was assigned from the relative configuration. Therefore,
ampelomin A was defined as (4R,6S)-4-hydroxy-6-methylcyclohex-
2-enone (1).
Ampelomin B (2), a colorless oil, had a molecular formula of
C7H12O3, which was also determined from the HRESIMS and 13
C
NMR data. The 13C NMR (Table 2) and DEPT spectra exhibited
seven carbon resonances, indicating the presence of a methyl group,
a methylene, and five methines, of which four were oxygenated [δ
54.9 (C-2), 57.6 (C-3), 64.3 (C-1), and 68.5 (C-4)]. The upfield
shifted oxymethine signals (δ 54.9 and 57.6) were assignable to
Results and Discussion
The fermentation and extraction of the fungus have been
described in our previous report.3 The EtOAc and CHCl3 extracts
were fractionated by repeated column chromatography (CC) over
silica gel, ODS, and Sephadex LH-20, preparative TLC, and HPLC
to furnish seven new compounds, ampelomins A-G (1-7).
Ampelomin A (1), a yellowish oil, had a molecular formula of
C7H10O2 as deduced from the HRESIMS and 13C NMR data. The
1
an epoxy group. The H NMR spectrum (Table 1) confirmed the
presence of the functionalities indicated above. In addition, the
spectrum displayed two exchangeable signals at δ 3.68 (1H, d, J
) 7.6 Hz, 1-OH) and 4.18 (1H, d, J ) 4.4 Hz, 4-OH), suggesting
the presence of two hydroxy groups. Analysis of the COSY and
HMQC spectra furnished full assignments of the 1H and 13C NMR
data as shown in Tables 1 and 2 and led to basic structure 2.
Since signals H-1 and H-4 appeared as complicated multiplets
due to coupling with the protons of the geminal hydroxy group in
* To whom correspondence should be addressed. Tel: +86-20-37252538.
E-mail: wxy@scbg.ac.cn.
† South China Botanical Garden, Chinese Academy of Sciences.
‡ Graduate School of Chinese Academy of Sciences.
10.1021/np800667e CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/04/2009

266 Journal of Natural Products, 2009, Vol. 72, No. 2
Table 1. 1H NMR (400 MHz) Data for Compounds 2-7
Zhang et al.
position
1
2a
2b
3c
3b
4d
5d
6d
7e
4.05 m
4.63 td
3.39 m
4.25 td
4.42 br s
4.31 br dd
(5.8, 3.6)
4.00 ddd
4.04 ddd
(11.4, 4.2, 3.2)
4.46 br s
3.91 ddd
(11.8, 9.2, 4.6)
5.00 t (9.2)
(5.4, 5.4, 3.7)
3.77 t (3.7)
(9.2, 4.2)
4.06 td
2
3.21 t (3.7)
3.23 m
4.61 dt
(9.2, 4.2)
(10.4, 4.4)
(10.0, 5.8, 2.8)
3eq
3ax
4
3.13 dd
(3.7, 1.8)
3.71 dd
(3.7, 1.7)
1.70 dt
(12.9, 3.8)
1.44 dt
(12.9, 9.0)
3.57 m
2.52 dt
(12.7, 4.2)
2.27 dt
(12.9, 9.2)
4.15 dt
2.36 dt
(12.8, 4.4)
2.43 ddd
(12.8, 10.4, 2.8)
4.23 br s
2.62-2.46 m
2.67 dt
(13.2, 4.2)
1.86 ddd
(13.2, 10.8, 2.2)
4.16 td
(10.8, 4.2)
1.74 m
2.62-2.46 m
3.66 t (9.2)
3.89 m
4.35 dd
(4.0, 1.7)
2.34 m
3.50 td
(9.6, 5.9)
2.35 m
3.63 dd
(9.2, 5.1)
2.18 m
(9.2, 4.2)
2.36 m
5
1.89 m
1.85 m
2.39 m
6eq
1.18 ddd
1.71 ddd
1.59 ddd
2.15 dt
1.98 dt
2.14 dt
1.98 dt
1.91 ddd
(13.8, 4.3, 3.2)
(14.0, 5.4, 3.1) (13.4, 5.9, 4.2) (13.4, 4.2)
(13.6, 4.0)
(14.0, 3.6)
(12.2, 4.2)
(13.4, 4.6, 3.0)
6ax
7
1.57 ddd
2.02 ddd 1.23 ddd 1.82 ddd
2.08 ddd
1.28 ddd
2.10 td
1.67 ddd
(13.4, 11.8, 4.6)
1.03 d (7.4)
(13.8, 10.3, 5.8) (14.0, 9.9, 5.4) (13.4, 9.0, 4.2) (13.4, 9.2, 4.2) (13.6, 11.2, 2.4) (14.0, 12.2, 2.4) (12.2, 11.4)
0.86 d (7.1) 1.14 d (7.1) 0.83 d (7.1) 1.20 d (7.1) 1.17 d (6.8) 1.23 d (6.6) 1.27 d (6.4)
a Measured in acetone-d6. Data for exchangeable protons: δ 3.68 (1H, d, J ) 7.6 Hz, 1-OH) and 4.18 (1H, d, J ) 4.4 Hz, 4-OH). b Measured in
C5D5N-D2O (5:1). c Measured in DMSO-d6. Data for exchangeable protons: δ 4.46 (1H, d, J ) 3.6 Hz, 1-OH), 4.67 (1H, d, J ) 4.8 Hz, 2-OH), and
4.47 (1H, d, J ) 5.0 Hz, 4-OH). d Measured in C5D5N. e Measured in CD3OD. Data for 6-MSA moiety: δ 6.74 (2H, d, J ) 8.0 Hz, H-3′ and H-5′),
7.23 (1H, t, J ) 8.0 Hz, H-4′), and 2.53 (s, 6′-Me).
Table 2. 13C NMR (100 MHz) Data for Compounds 2-7a
position
2
3
4
5
6
7b
1
2
3
4
5
6
7
64.3
54.9
57.6
68.5
28.3
32.8
15.9
69.3
71.8
35.1
69.0
32.4
34.1
14.0
70.0
68.7
37.7
70.4
30.6
34.8
17.9
69.6
71.3
39.5
74.0
33.9
37.9
18.8
71.8
71.2
41.6
70.4
39.2
36.9
19.4
68.2
82.2
72.6
75.3
33.5
36.8
13.2
Figure 1. Key HMBC (arrows) and NOESY (curves) correlations
of 7.
a Solvents: acetone-d6 for 2, DMSO-d6 for 3, C5D5N for 4, 5, and 6,
CD3OD for 7. b Data for 6-MSA moiety: δ 117.1 (C-1′), 160.9 (C-2′),
115.6 (C-3′), 133.9 (C-4′), 123.5 (C-5′), 141.4 (C-6′), 171.7 (C-7′), and
22.7 (6′-Me).
a chair conformation for the cyclohexane ring in which the three
hydroxy groups were in equatorial positions, while 5-Me was in
the axial position. In the 1H NMR spectrum of 4, Ja,a values between
H-2/H-3ax (10.4 Hz) and H-5/H-6ax (11.2 Hz) and Ja,e/e,e values
(2.4-4.4 Hz) for other couplings were observed, indicating a chair
form for the six-membered ring with 1-OH and 4-OH equatorial
and 2-OH and 5-Me axial. For 5, Ja,a values (9.6-12.2 Hz) between
H-2/H-3ax, H-3ax/H-4, H-4/H-5, and H-5/H-6ax and Ja,e/e,e values
(2.4-5.8 Hz) for other couplings were found, in accord with a chair
form for the six-membered ring with 1-OH in the axial orientation
and other substituents in equatorial orientations. For 6, the Ja,a values
(10.8-12.2 Hz) found between H-1/H-6ax, H-3ax/H-4, H-4/H-5,
and H-5/H-6ax and Ja,e/e,e values (2.2-4.2 Hz) for other coupling
constants showed that the cyclohexane ring adopted a chair
conformation with 2-OH in the axial position and other substituents
in equatorial positions. Therefore, the structures of ampelomins
C-F were elucidated as (1S*,2S*,4R*,5S*)-5-methylcyclohexane-
1,2,4-triol (3), (1S*,2R*,4R*,5S*)-5-methylcyclohexane-1,2,4-triol
(4), (1S*,2R*,4S*,5S*)-5-methylcyclohexane-1,2,4-triol (5), and
(1R*,2S*,4S*,5S*)-5-methylcyclohexane-1,2,4-triol (6), respectively.
Ampelomin G (7), a white solid, had a molecular formula of
C15H20O6 as determined from the HRESIMS and 13C NMR data.
The 1H (Table 1), 13C (Table 2), and DEPT NMR spectra showed
the presence of a secondary methyl group, an aromatic methyl group
[δH 2.53 (3H, s, 6′-Me); δC 22.7 (6′-Me)], a methylene, an aliphatic
methine, four oxymethines, and an ester carbonyl group [δC 171.7
(C-7′)], as well as a 1,2,3-trisubstituted benzene ring [δH 7.23 (1H,
t, J ) 8.0 Hz, H-4′) and 6.74 (2H, d, J ) 8.0 Hz, H-3′ and H-5′);
δC 160.9 (C-2′), 141.4 (C-6′), 133.9 (C-4′), 123.5 (C-5′), 117.1 (C-
1′), and 115.6 (C-3′)]. Interpretation of the COSY and HMQC
spectra led to a partial structure, 5-methylcyclohexane-1,2,3,4-tetrol.
The other functional groups were assembled into another partial
structure, 6-methylsalicylic acid (6-MSA), from the HMBC spec-
trum (key correlations depicted in Figure 1), in which correlations
were observed from the aromatic methyl protons at δ 2.53 to C-1′,
C-5′, and C-6′ and from H-4′ to C-2′ and C-6′. The presence of
the 6-methylsalicylic acid moiety was confirmed by alkaline
1
the H NMR spectrum of 2 in acetone-d6, it was quite difficult to
define their coupling patterns. The H NMR spectrum of 2 was
1
remeasured in a mixture of C5D5N and D2O (5:1) (Table 1). The
remeasured 1H NMR spectrum revealed the coupling constants, J1,2
) J2,3 ) 3.7 Hz, J3,4 ) 1.7 Hz, J4,5 ) 4.0 Hz, J5,6eq ) 3.1 Hz, J5,6ax
) 9.9 Hz, and J1,6eq ) J1,6ax ) 5.4 Hz. In the NOESY spectrum of
2, measured in the same mixture, key NOE interactions were
observed between H-1/H-6ax, H-1/H-6eq, H-1/H-2, H-2/H-3, H-3/
H-4, H-4/H-5, and H-4/H3-7, while no correlations were found
between H-5/H-6ax, H-5/H-1, and H-4/H-6ax. These findings
showed a half-chair conformation for the cyclohexane ring in which
1-OH and 4-OH were pseudoaxial, 5-Me was equatorial, and H-1,
H-2, and H-3 had a mutual cis relationship and a trans relationship
to H-4.6 Therefore, 2 was deduced to be (1S*,2S*,3R*,4S*,5S*)-
2,3-epoxy-5-methylcyclohexane-1,4-diol (2).
Ampelomins C-F (3-6), all obtained as white powders, had
the same molecular formula, C7H14O3, as determined from the
HRESIMS and 13C NMR data. The 1H and 13C NMR spectra
(Tables 1 and 2) showed that they all comprised a secondary methyl
group, two methylenes, and four methines, of which three were
oxygenated. By interpretation of COSY and HMQC, their basic
structures were all derived to be 5-methylcyclohexane-1,2,4-triol.
The structural differences were derived from configurational
diversity, which was assigned by examination of the vicinal proton
coupling constants in the 1H NMR spectra. In the 1H NMR spectrum
of 3 in DMSO-d6, the signals of H-1, H-2, and H-4 appeared as
complicated multiplets. In order to provide coupling patterns of
1
these signals, the H NMR spectrum of 3 was remeasured in the
mixture of C5D5N and D2O (5:1). The remeasured 1H NMR
spectrum of 3 revealed axial-axial coupling constants (Ja,a ) 9.2
Hz) between H-1/H-2, H-1/H-6ax, H-2/H-3ax, and H-3ax/H-4 and
axial-equatorial or equatorial-equatorial coupling constants (Ja,e/
e,e ) 4.2 Hz) between H-1/H-6eq, H-2/H-3eq, H-3eq/H-4, H-4/H-
5, H-5/H-6ax, and H-5/H-6eq. These values were consistent with

Polyoxygenated Methyl Cyclohexanoids from Ampelomyces
Journal of Natural Products, 2009, Vol. 72, No. 2 267
Scheme 1. Plausible Biogenetic Pathway for 1-7
Table 3. Antibacterial Activities of 1, Determined by MABA
(µM)
1
ceftazidime
bacteria
MIC50 ( SD MIC90 ( SD MIC50 ( SD MIC90 ( SD
S. aureus
E. coli
P. Vulgaris
54.9 ( 5.4
89.9 ( 4.8
51.6 ( 4.8
234.2 ( 8.7 3.11 ( 0.16 14.65 ( 0.37
265.1 ( 16.3 0.37 ( 0.15 0.46 ( 0.09
202.4 ( 4.8 0.18 ( 0.11 0.21 ( 0.01
P. aeruginosa 63.5 ( 0.8 1015.9 ( 19.8 0.73 ( 0.07 3.94 ( 0.16
routes, is that p-toluquinone may undergo partial hydrogenation,
leading to (S)-5-methylcyclohex-2-ene-1,4-dione, the key precursor
for 1-7, instead of a reaction involving epoxide precursors.8-10
The closely related carbasugar structures of these compounds,
among which 3-6 could be regarded as 2,6-deoxy-5a-carbasugars
and 2 and 7 as 6-deoxy-5a-carbasugars, inspired us to evaluate their
glycosidase inhibitory activities. In the evaluation 1, 3, 5, and 7
exhibited activity against R-glucosidase with IC50 values of
1.74-5.93 mM, much weaker than acarbose (IC50 ) 0.03 mM),
while 2, 4, and 6 were not active toward R-glucosidase. All
compounds were inactive against ꢀ-glucosidase and R-rhamnosidase
at concentrations of 10 mM and lower. It was interesting that the
active methylcyclohexanes (3, 5, and 7) all possess an equatorial
2-O-substituent, differing from the inactive compounds (2, 4, and
6), which all have an axial or pseudoaxial 2-O-substituent. At
present, there is no explanation for such an SAR regarding
R-glucosidase inhibition. The antibacterial activity of these com-
pounds against Staphylococcus aureus, Escherichia coli, Proteus
Vulgaris, and Pseudomonas aeruginosa was also evaluated, and
only 1 showed activity, with MIC50 values of 51.6-89.7 µM and
MIC90 values ranging from 202.4 to 1015.9 µM, less potent than
ceftazidime (Table 3).
hydrolysis of 7, which afforded 6-MSA, a metabolite previously
obtained from this fungus.3 The attachment of the 6-MSA moiety
to C-2 was indicated by the downfield shifts of H-2 (δ 5.00) and
C-2 (δ 82.2) as well as the presence of an HMBC correlation from
H-2 to C-7′. The relative configuration was determined from the
Experimental Section
General Experimental Procedures. Optical rotations were obtained
1
on a Perkin-Elmer 343 spectropolarimeter. H NMR (400 MHz), 13C
1
vicinal proton coupling constants in the H NMR spectrum. The
NMR (100 MHz), and 2D NMR spectra were recorded on a Bruker
DRX-400 instrument with the residual solvent peak as reference.
HRESIMS data were obtained on a Bruker Bio TOF IIIQ mass
spectrometer. ESIMS data were collected on a MDS SCIEX API 2000
LC/MS/MS instrument. Preparative HPLC was run with a Shimazu
LC-6AD pump and a Shimazu RID-10A refractive index detector using
a XTerra prep MS C18 column (10 µm, 300 × 19 mm). Preparative
TLC was performed on precoated silica gel plates (GF254, Qingdao
Marine Chemical Ltd., Qingdao, China, 0.25 mm thickness) with
detection under fluorescent light (λ ) 254 nm). For column chroma-
tography, silica gel 60 (100-200 mesh, Qingdao Marine Chemical Ltd.,
Qingdao, China), Develosil ODS (10 µm, Nomura Chemical Co. Ltd.,
Japan), and Sephadex LH-20 were used.
Producing Fungus and Fermentation. The producing fungus,
Ampelomyces sp. SC0307, was isolated from a soil sample collected
in the Dinghu Mountain Biosphere Reserve, Guangdong, China, in
January 2002.3 The fermentation process was described in our previous
report.3 Briefly, the seed culture (3000 mL) was prepared by growing
the fungus in YMG broth on a rotary shaker for 3 days in the dark at
28 °C, 120 rpm. The culture were transferred into 30 5000 mL
Erlenmeyer flasks containing 1000 mL of YMG medium and 550 g of
wheat grains, and the flasks were incubated for 7 days in the stationary
phase in the dark at 28 °C.
Extraction and Isolation. The obtained mycelial solid culture (the
medium upon which the mycelia were grown) was extracted with 95%
EtOH, and the resultant extract was partitioned using sequential
extraction with petroleum ether, CHCl3, EtOAc, and n-BuOH.3 The
EtOAc-soluble extract (36 g) was separated into 10 fractions by Si gel
column chromatography (CC) using CHCl3-MeOH mixtures of
increasing polarity (100:0 to 60:40). Fraction 5, obtained on elution
with CHCl3-MeOH (95:5), was further chromatographed on a Si gel
column using CHCl3-MeOH (95:5 to 80:20) and resulted in the
collection of 40 subfractions of 35 mL each. Subfractions 11-14,
obtained on elution with CHCl3-MeOH (95:5), were combined on the
basis of TLC analysis and purified by Sephadex LH-20 CC using MeOH
Ja,a values H-1/H-2 (9.2 Hz), H-2/H-3 (9.2 Hz), H-3/H-4 (9.2 Hz),
and H-6ax/H-1 (11.8 Hz) were observed, and the Ja,e/e,e values H-4/
H-5 (5.1), H-5/H-6eq (3.0), H-5/H-6ax (4.6 Hz), and H-1/H-6eq
(4.6 Hz) were also observed, indicative of a chair conformation
for the cyclohexane ring in which only the 5-Me substituent was
in an axial position, while the other substituents were all in
equatorial positions. The configuation was further supported by
correlations in the NOESY spectrum (Figure 1). Thus, the structure
of 7 was deduced as (1R*,2R*,3S*,4S*,6S*)-2,3,6-trihydroxy-4-
methylcyclohexyl 2-hydroxy-6-methylbenzoate (7).
The absolute configuration of 2-7 was not assigned in the present
study. However, in the postulated biogenetic pathway (see next
paragraph), 1-7 are derived from a common precursor, (S)-5-
methylcyclohex-2-ene-1,4-dione. In consideration of this, the
absolute configuration of 2-7 could be tentatively assigned as
shown.
Polyoxygenated methyl cyclohexanoids of fungal origin,7 such
as theobroxide,8 epoformin,8 and terremutin,9 have been proven to
be biosynthetically derived from a polyketide pathway via a
tetraketide intermediate, 6-MSA.8,10 The co-occurrence of 1-7 and
6-MSA in this Ampelomyces sp. strongly suggests that these
metabolites are produced via a similar biosynthetic pathway. By
inspection of the structures of 1-7, it is clear that these metabolites
are chemically related (Scheme 1); that is, 7 might be derived from
2 through reaction with 6-MSA via an SN2 mechanism, 2 might be
generated from the 4-epimer of 1 by epoxidation and reduction,
and 3-6 could be transformed from 1 or its 4-epimer via hydration
in a mechanism similar to that for R,ꢀ-unsaturated ketones11
followed by reduction. Thus, a plausible but speculative biogenetic
pathway for 1-7 is postulated as shown in Scheme 1. The
uniqueness of this pathway, compared with the previously proposed

268 Journal of Natural Products, 2009, Vol. 72, No. 2
Zhang et al.
to afford compound 2 (20 mg). Fraction 9, obtained on elution with
CHCl3-MeOH (9:1), was subjected to Si gel CC using petroleum
ether-acetone (6:4-4:6) and resulted in the collection of 27 subfrac-
tions of 100 mL each. Subfractions 10 and 11, obtained on elution
with petroleum ether-acetone (55:45), were combined and applied to
Sephadex LH-20 CC using MeOH, followed by preparative HPLC using
5% aqueous MeOH to afford 3 (15 mg) (tR ) 19.98 min) and 4 (50
mg) (tR ) 24.13 min). Fraction 10, obtained on elution with
CHCl3-MeOH (9:1), was subjected to Si gel CC using petroleum
ether-acetone (6:4-4:6) and resulted in the collection of 27 subfrac-
tions of 10 mL each. Subfraction 8, obtained on elution with petroleum
ether-acetone (55:45), was purified by preparative HPLC using 5%
aqueous MeOH to afford 6 (9 mg) (tR ) 20.30 min).
The CHCl3-soluble extract (28 g) was subjected to passage over a
Si gel column, eluted with petroleum ether-acetone mixtures in a step
gradient of increasing polarity (95:5 to 50:50) to obtain 10 fractions.
Fractions 4, 7, and 10, obtained by elution with petroleum ether-acetone
(9:1, 6:4, and 5:5, respectively), were applied to further separation.
Fraction 4 was chromatographed on an ODS column using 60% aqueous
MeOH (6:4), followed by purification with Sephadex LH-20 CC using
MeOH to afford 1 (20 mg). Fraction 7 was chromatographed on an
ODS column and eluted with 30% MeOH to obtain 59 subfractions of
10 mL each. Subfractions 22-30 were combined and purified by
Sephadex LH-20 CC using MeOH, followed by preparative TLC
(CHCl3-MeOH, 9:1, Rf ) 0.55), to afford 7 (12 mg). Fraction 10 was
chromatographed on a Si gel column using CHCl3-MeOH mixtures
of increasing polarity (90:10 to 60:40) to obtain 30 subfractions of 35
mL each. Subfraction 4 was purified by Sephadex LH-20 CC using
MeOH, followed by HPLC using 5% MeOH, to afford 5 (20 mg) (tR
)33.25 min).
similar procedure, the (S)-MTPA ester (0.6 mg) was prepared from 1
(3.0 mg) by the use of (S)-MTPA (28.1 mg), EDC-HCl (23.0 mg), and
4-DMAP (14.6 mg).
1
(R)-MTPA Ester of 1 (1a): H NMR (400 MHz, CDCl3) δ 7.510
(2H, m, Ar-H), 7.410 (3H, m, Ar-H), 6.770 (1H, dd, J ) 10.2, 2.0
Hz, H-2), 6.066 (1H, dd, J ) 10.2, 2.0 Hz, H-3), 5.891 (1H, ddd, J )
7.6, 5.2, 2.8 Hz, H-4), 3.550 (3H, s, OCH3), 2.480 (1H, m, H-6),
2.440(1H, m, H-5eq), 1.837 (1H, dddd, J ) 12.2, 7.6, 5.2, 2.8 Hz,
H-5ax), 1.151 (3H, d, J ) 6.8 Hz, H3-7).
(S)-MTPA Ester 1 (1b): 1H NMR (400 MHz, CDCl3) δ 7.510 (2H,
m, Ar-H), 7.420 (3H, m, Ar-H), 6.663 (1H, dd, J ) 10.2, 2.0 Hz,
H-2), 6.034 (1H, dd, J ) 10.2, 2.0 Hz, H-3), 5.900 (1H, ddd, J ) 7.6,
5.2, 2.8 Hz, H-4), 3.550 (3H, s, OCH3), 2.501 (2H, m, H-5eq and H-6),
1.925 (1H, dddd, J ) 7.6, 5.2, 2.8 Hz, H-5ax), 1.175 (3H, d, J ) 6.8
Hz, H3-7).
Glycosidase Inhibition Assay. The enzymes of R-glucosidase from
baker’s yeast, ꢀ-glucosidase from almonds, and naringinase from
Penicillium decumbens, as well as the substrates of p-nitrophenyl R-D-
glucopyranoside, p-nitrophenyl ꢀ-D-glucopyranoside, and p-nitrophenyl
R-L-rhamnopyranoside, were purchased from Sigma Chemical Com-
pany. Other chemicals were purchased from native companies. The
glycosidase inhibition assay was performed according to the reported
methods12-14 with slight modification. As for the R-glucosidase
inhibition assay, 50 µL of 0.2 U/mL enzyme in 0.2 M phosphate buffer
(pH 6.8) and 50 µL of compound solution in the same buffer were
incubated in 96-well plates at 37 °C for 30 min. Then, 50 µL of 2.5
mM p-nitrophenyl R-D-glucopyranoside in 0.2 M phosphate buffer was
added, and the plate was incubated at 37 °C for another 5 min. The
reaction was quenched by the addition of 0.1 M Na2CO3 solution (0.1
mL). Acarbose was tested as a positive control. Before and after
reaction, absorbance readings were recorded at 405 nm by microplate
reader (Genios) and compared to a negative control, which had 50 µL
of buffer solution in place of compound solution to calculate percentage
inhibition. This procedure was also used for ꢀ-glucosidase and
R-rhamnosidase inhibition assays. ꢀ-Glucosidase inhibition was evalu-
ated using p-nitrophenyl ꢀ-D-glucopyranoside as a substrate, citrate-
phosphate buffer (pH 5.0) as a reaction buffer, and borate buffer (pH
9.0) as an alkali solution to stop the reaction. R-Rhamnosidase inhibition
was assayed using naringinase as an enzyme, p-nitrophenyl R-L-
rhamnopyranoside as a substrate, citrate-phosphate buffer (pH 5.0) as
a reaction buffer, and 0.4 M NaOH as an alkali solution to stop the
reaction. Glycosidase inhibitory activity was expressed as IC50 values.
The IC50 values (IC50 ( SD, mM) against R-glucosidase were
determined as 3.59 ( 0.008 (1), 6.12 ( 0.006 (3), 5.93 ( 0.002 (5),
1.74 ( 0.004 (7), >10 (2, 4, and 6), and 0.03 ( 0.003 (acarbose). The
values of all test compounds against ꢀ-glucosidase and R-rhamnosidase
were more than 10 mM.
Antibacterial Activity. The antibacterial activity against S. aureus,
E. coli, P. Vulgaris, and P. aeruginosa was evaluated by microplate
Alamar Blue assay as described in our previous report.3 Briefly, serial
2-fold dilutions of compounds 1-7 and ceftazidime were made in
DMSO, and the test bacteria at a concentration of 2 × 105 cfu/mL
were prepared in MHB medium supplemented with 8% Alamar Blue.
In a 96-well plate, 78 µL of each test organism and 2 µL each of the
compound dilutions were mixed and incubated at 32 °C until growth
control wells developed the growth (pink color). The fluorescence
intensity was then measured using a plate reader (Synergy HT, Bio-
Tek, Winooski, VT) with excitation at 530 nm and emission at 590
nm. The percentage inhibition of each compound dilution was
calculated. All tests were run in triplicate and averaged. MIC50 and
MIC90 values (Table 3), defined as the concentrations required to reduce
bacterial growth by 50% and 90%, respectively, were obtained by
interpolation of concentration-inhibition curves.
Ampelomin A (1): yellowish oil; [R]2D0 +50.1 (c 1.6, MeOH);
positive ESIMS m/z 253 [2 M + H]+, 165 [M + K]+, 149 [M + Na]+,
127 [M + H]+; 1H NMR (400 MHz, CDCl3): δ 6.85 (1H, dt, J ) 10.0,
2.0 Hz, H-2), 5.95 (1H, dd, J ) 10.0, 2.0 Hz, H-3), 4.64 (1H, ddt, J )
10.2, 4.8, 2.0 Hz, H-4), 2.35-2.40 (2H, m, H-5eq and H-6), 1.72 (1H,
tdd, J ) 12.2, 10.2, 2.0 Hz, H-5ax), 1.45 (3H, d, J ) 6.5 Hz, H3-7);
13C NMR (100 MHz, CDCl3) δ 201.6 (C-1), 128.4 (C-2), 153.5 (C-3),
67.2 (C-4), 41.2 (C-5), 40.1 (C-6), 14.8 (C-7); HRESIMS m/z 127.0758
[M + H]+ (calcd for C7H11O2, 127.0754).
Ampelomin B (2): colorless oil; [R]2D0 +90.6 (c 0.415, MeOH);
positive ESIMS m/z 311 [2 M + Na]+, 183 [M + K]+, 167 [M +
1
Na]+; H NMR (400 MHz) see Table 1; 13C NMR (100 MHz) see
Table 2; HRESIMS m/z 167.0676 [M + Na]+ (calcd for C7H12O3Na,
167.0679).
Ampelomin C (3): white solid; [R]2D0 -15.3 (c 0.74, MeOH); positive
ESIMS m/z 315 [2 M + Na]+, 293 [2 M + H]+, 169 [M + Na]+, 147
1
[M + H]+; H NMR (400 MHz) see Table 1; 13C NMR (100 MHz)
see Table 2; HRESIMS m/z 169.0848 [M + Na]+ (calcd for C7H14O3Na,
169.0835).
Ampelomin D (4): white solid; [R]2D0 -5.9 (c 0.22, MeOH); positive
1
ESIMS m/z 315 [2 M + Na]+, 169 [M + Na]+; H NMR (400 MHz)
see Table 1; 13C NMR (100 MHz) see Table 2; HRESIMS m/z 169.0841
[M + Na]+ (calcd for C7H14O3Na, 169.0835).
Ampelomin E (5): white solid; [R]2D0 -46.7 (c 0.67, MeOH); positive
1
ESIMS m/z 315 [2 M + Na]+, 169 [M + Na]+; H NMR (400 MHz)
see Table 1; 13C NMR (100 MHz) see Table 2; HRESIMS m/z 169.0820
[M + Na]+ (calcd for C7H14O3Na, 169.0835).
Ampelomin F (6): white solid; [R]2D0 +37.6 (c 0.5, MeOH); positive
1
ESIMS m/z 315 [2 M + Na]+, 169 [M + Na]+; H NMR (400 MHz)
see Table 1; 13C NMR (100 MHz) see Table 2; HRESIMS m/z 145.0864
[M - H]- (calcd for C7H13O3, 145.0870).
Ampelomin G (7): white solid; [R]2D0 -17.0 (c 0.73, MeOH); positive
1
ESIMS m/z 319 [M + Na]+; H NMR (400 MHz) see Table 1; 13C
NMR (100 MHz) see Table 2; HRESIMS m/z 319.1149 [M + Na]+
(calcd for C15H20O6Na, 319.1152).
Preparation of the (R)-MTPA and (S)-MPTA Esters of 1. A
solution of 1 (3.0 mg, 0.024 mmol) in CH2Cl2 (2.0 mL) was treated
with (R)-MTPA (28.1 mg, 0.12 mmol) in the presence of EDC-HCl
(23.0 mg, 0.12 mmol) and 4-DMAP (14.6 mg, 0.12 mmol), and the
mixture was stirred at room temperature (25 °C) for 7 days. The reaction
mixture was poured into ice-water, and the mixture was extracted with
CHCl3. The CHCl3 extract was successively washed with 5% HCl,
saturated NaHCO3, and brine, dried over MgSO4, and filtered. Evapora-
tion of the solvent furnished a residue, which was purified by Si gel
CC (1 g, CHCl3) to give the (R)-MTPA ester (0.6 mg). Through a
Acknowledgment. We thank Prof. R. Chen, Guangzhou Institute
of Chemistry, Chinese Academy of Sciences, for 1D and 2D NMR
spectroscopic measurements, Dr. D. Fang, Chengdu Institute of Biology,
Chinese Academy of Sciences, for HRESIMS data, and Prof. T. Li,
Guangdong Institute of Microbiology for microbiological authentication.
This work was supported by an NSFC grant (No. 20672114) and the
Knowledge Innovation Program of CAS, Grant No. KSCX2-YW-N-
036.

Polyoxygenated Methyl Cyclohexanoids from Ampelomyces
Journal of Natural Products, 2009, Vol. 72, No. 2 269
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