Cytotoxic and other metabolites of Aspergillus inhabiting the rhizosphere of sonoran desert plants

Article abstract of DOI:10.1021/np040139d

In a study to discover potential anticancer agents from rhizosphere fungi of Sonoran desert plants cytotoxic EtOAc extracts of four Aspergillus strains have been investigated. Two new metabolites, terrequinone A (1) and terrefuranone (2), along with N^α-acetyl aszonalemin (LL-S490β) (3) were isolated from As. terreus occurring in the rhizosphere of Ambrosia ambrosoides, whereas As. terreus inhabiting the rhizosphere of an unidentified Brickellia sp. afforded dehydrocurvularin (4), 11-methoxycurvularin (5), and 11-hydroxycurvularin (6). As. cervinus isolated from the rhizosphere of Anicasanthus thurberi contained two new compounds, 4R*,5S*- dihydroxy-3-methoxy-5-methylcyclohex-2-enone (7) and 6-methoxy-5(6)- dihydropenicillic acid (8), in addition to penicillic acid (9). Penicillic acid was also isolated from As. wentii occurring in the rhizosphere of Larrea tridentata. The structures of 1-9 were elucidated by spectroscopic methods and chemical derivatizations. Acetylation of 2 afforded 14-acetylterrefuranone (13) and 14-deoxy-13(14)-dehydroterrefuranone (14). Metabolites 1-9, the dienone 14, and 5(6)-dihydropenicillic acid (16) were evaluated for cytotoxicity in a panel of four human cancer cell lines and in normal human primary fibroblast cells. Compounds 4 and 5 displayed considerable cytotoxicity, whereas 1, 6, 9, and 14 were found to be moderately active, with 6 and 9 exhibiting selective cytotoxicity against cancer cell lines compared with the normal fibroblast cells.

Full text of DOI:10.1021/np040139d

J. Nat. Prod. 2004, 67, 1985-1991
1985
Cytotoxic and Other Metabolites of Aspergillus Inhabiting the Rhizosphere of
Sonoran Desert Plants1
Jian He,† E. M. Kithsiri Wijeratne,† Bharat P. Bashyal,† Jixun Zhan,† Christopher J. Seliga,† Manping X. Liu,†
Elizabeth E. Pierson,‡ Leland S. Pierson, III,‡ Hans D. VanEtten,‡ and A. A. Leslie Gunatilaka*,†
Southwest Center for Natural Products Research and Commercialization, Office of Arid Lands Studies, College of Agriculture
and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706-6800, and Division of Plant Pathology
and Microbiology, Department of Plant Sciences, College of Agriculture and Life Sciences, University of Arizona,
Tucson, Arizona 85721-0036
Received June 16, 2004
In a study to discover potential anticancer agents from rhizosphere fungi of Sonoran desert plants cytotoxic
EtOAc extracts of four Aspergillus strains have been investigated. Two new metabolites, terrequinone A
(1) and terrefuranone (2), along with Na-acetyl aszonalemin (LL-S490â) (3) were isolated from As. terreus
occurring in the rhizosphere of Ambrosia ambrosoides, whereas As. terreus inhabiting the rhizosphere of
an unidentified Brickellia sp. afforded dehydrocurvularin (4), 11-methoxycurvularin (5), and 11-
hydroxycurvularin (6). As. cervinus isolated from the rhizosphere of Anicasanthus thurberi contained
two new compounds, 4R*,5S*-dihydroxy-3-methoxy-5-methylcyclohex-2-enone (7) and 6-methoxy-5(6)-
dihydropenicillic acid (8), in addition to penicillic acid (9). Penicillic acid was also isolated from As. wentii
occurring in the rhizosphere of Larrea tridentata. The structures of 1-9 were elucidated by spectroscopic
methods and chemical derivatizations. Acetylation of 2 afforded 14-acetylterrefuranone (13) and 14-deoxy-
13(14)-dehydroterrefuranone (14). Metabolites 1-9, the dienone 14, and 5(6)-dihydropenicillic acid (16)
were evaluated for cytotoxicity in a panel of four human cancer cell lines and in normal human primary
fibroblast cells. Compounds 4 and 5 displayed considerable cytotoxicity, whereas 1, 6, 9, and 14 were
found to be moderately active, with 6 and 9 exhibiting selective cytotoxicity against cancer cell lines
compared with the normal fibroblast cells.
The genus Aspergillus (Moniliaceae), which contains
about 180 recognized species,2 has proved to be a rich
source of bioactive metabolites, and even after investiga-
tions spanning over two decades, the genus still continues
to provide metabolites with novel structures and interest-
ing biological activities.3 We have recently reported that
As. terreus from the rhizosphere of the staghorn cholla
(Opuntia versicolor Engelm; Cactaceae) contained a novel
cyclopentadione, asterredione, the cytotoxic sesquiterpenes
(+)-5,6-dihydro-6-methoxyterrecyclic acid A, (+)-5,6-dihy-
dro-6-hydroxyterrecyclic acid A, (+)-terrecyclic acid A, and
(-)-quadrone, and the cytotoxic quinones betulinan A,
asterriquinone C-1, and asterriquinone D.4 In continuation
of our studies to harvest bioactive secondary metabolites
from the rhizosphere of floristically diverse plant com-
munities in the Sonoran desert,1,4,5 we have investigated
As. terreus Thom. occurring in the rhizosphere of the
canyon ragweed [Ambrosia ambrosioides (Cav.) Payne;
Asteraceae] and in the rhizosphere of an unidentified
Brickellia sp. (Asteraceae), As. cervinus Massee, inhabiting
the rhizosphere of the desert honeysuckle [Anisacanthus
thurberi (Torr.) Gray; Acanthaceae], and As. wentii Weh-
mer occurring in the rhizosphere of the creosote bush
[Larrea tridentata (DC.) Coville; Zygophyllaceae]. Using a
bioassay-guided approach, we now report that the cytotoxic
EtOAc extracts of these strains of Aspergillus contain four
new metabolites, 1, 2, 7, and 8. Also isolated were a rare
fungal metabolite, 3,6 and the known compounds 4,7,8 5,8,9
6,8,9 and 9.10 All known compounds were identified by direct
comparison with authentic samples and/or by comparison
of their spectroscopic data with those reported in the
literature. Treatment of terrefuranone (2) with Ac2O/
pyridine afforded its acetate 13 and the dienone 14. To
determine the requirement of the 5(6)-unsaturation for the
cytotoxicity of penicillic acid, 9 was subjected to catalytic
hydrogenation, yielding 5(6)-dihydropenicillic acid (16).11
A previous study of the soil-borne As. cervinus has
resulted in the isolation of terremutin and 3,6-dihydroxy-
2,5-toluquinone,12 whereas As. wentii has been reported to
contain ochratoxin,13a substituted benzophenones and
xanthones,13b bianthrones and secoanthraquinones,13c
a
naphthopyran, benzopyranones and maleic anhydrides,13d
long-chain derivatives of citraconic anhydride,13e emodin,13f
and aflatoxin.13g Herein we report the isolation of 1-9,
structure elucidation of the new metabolites 1, 2, 7, and 8,
and cytotoxicities of 1-9, 14, and 16 toward a panel of four
human cancer cell lines [NCI-H460 (non-small cell lung),
MCF-7 (breast), SF-268 (CNS glioma), MIA Pa Ca-2
(pancreatic)] and normal human primary fibroblast cells
(WI-38).
Results and Discussion
The cytotoxic EtOAc extract of As. terreus, collected from
the rhizosphere of the canyon ragweed, on fractionation
involving solvent-solvent partitioning followed by Sepha-
dex LH-20 gel filtration and silica gel chromatography
afforded 1-3. Terrequinone A (1) was obtained as a purple
solid that analyzed for C32H30N2O3 by a combination of
HRFABMS and 13C NMR spectroscopy. Its IR spectrum
had absorption bands at 3410 and 1636 cm-1, suggesting
the presence of NH/OH and quinone carbonyl groups. The
1H and 13C NMR spectra of 1 (Table 1) had some similari-
ties to those of isoasterriquinone14 and suggested it to be
an asterriquinone with similar substituents but located at
different positions. NMR data also indicated that in 1 the
central quinone moiety is monohydroxylated compared
* To whom correspondence should be addressed. Tel: (520) 741-1691.
Fax: (520) 741-1468. E-mail: leslieg@ag.arizona.edu.
† Southwest Center for Natural Products Research and Commercializa-
tion.
‡ Division of Plant Pathology and Microbiology.
10.1021/np040139d CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/18/2004

1986 Journal of Natural Products, 2004, Vol. 67, No. 12
He et al.
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for
Terrequinone (1) in DMSO-d6
a
b
a
b
position
1
δH
δC
position
δH
δC
184.4 s 8′
7.03 dt (7.0,
1.0)
7.32 d (8.0)
121.8 d
2
3
4
153.8 s 9′
118.0 s 10′
188.3 s 11′
111.5 d
40.0 s
146.8 d
6.16 dd (17.5,
10.5)
5.10 br d (17.5) 111.6 t
4.99 br d (10.5)
5
6
7
147.1 s 12′
135.2 s
28.8 t
3.35 dd
13′
14′
1.52 s
27.3 qc
27.9 qc
(12.5, 6.0)
3.27 dd
1.52 s
(12.5, 6.0)
5.05 m
8
9
122.5 d 1′′
133.4 s 2′′
108.3 s
127.6 d
7.48 d (2.0)
10
11
1′
2′
1.29 s
1.57 s
17.8 q 3′′-NH 10.67 br s
25.8 q 4′′
137.3 s
128.0 s
120.8 d
120.5 d
102.7 s 5′′
143.4 s 6′′
7′′
7.39 d (8.0)
7.10 dt (8.0,
1.0)
3′-NH 10.04 br s
4′
5′
6′
7′
136.5 s 8′′
129.9 s 9′′
7.19 dt (8.0,
122.6 d
112.6 d
1.0)
7.49 d (8.0)
8.87 br s
7.21 d (7.5) 119.7 d 2-OH
6.93 dt (8.0, 119.6 d
1.0)
a Multiplicites deduced from HSQC; coupling constants (J
values in Hz) are in parentheses. Assignments are based on HSQC
and HMBC spectra. b Multiplicites deduced from DEPT. Assign-
ments are based on HSQC and HMBC spectra. c Signals may be
interchanged.
with all known asterriquinones, which contain dioxygen-
ated quinone moieties. This was further supported by the
presence of a 1H broad singlet at δ 8.87, which disappeared
on methylation, in addition to two 1H broad singlets at δ
1
10.67 and 10.04 due to indole NH groups. The H NMR
spectrum of terrequinone A also suggested the presence of
two indole residues, one of which is 3-substituted and the
other 2,3-disubstituted, two 3H singlets at δ 1.29 and 1.57,
and a 6H singlet at δ 1.52 in addition to two independent
spin systems. On the basis of their chemical shifts, these
3H and 6H singlets were assigned to four CH3 groups on
quaternary aliphatic/olefinic carbons. The 1H-1H COSY
spectrum of 1 indicated that one of the spin systems
consisted of two 1H double doublets at δ 3.35 and 3.27 (J
) 12.5 and 6.0 Hz) and a 1H multiplet at δ 5.05. In the
HMBC spectrum of 1 (Figure 1) the protons at δ 3.35 and
3.27 showed strong correlations with carbons at δ 122.5
(to which the proton at δ 5.05 is attached as determined
by the HSQC spectrum), 188.3 (quinone carbonyl), 147.1
(C-5 of quinone), and 133.4 (olefinic quaternary carbon).
The strong HMBC correlations observed between the 3H
singlets at δ 1.57 and 1.29 with the olefinic carbon signals
at δ 133.4 (C-9) and 122.5 (C-8) together with the above
observations confirmed the presence of a dimethylallyl
group attached to C-5 of the quinone moiety of 1. The
remaining spin system of terrequinone A consisted of two
1H doublets [δ 5.10 (J ) 17.5 Hz) and 4.99 (J ) 10.5 Hz)]
and a 1H double doublet [δ 6.16 (J ) 17.5 and 10.5 Hz)],
suggesting the presence of a vinyl group on a quaternary
carbon. In the HMBC spectrum, the protons at δ 5.10 and
4.99 showed strong correlations with the quaternary carbon
signal at δ 40.0 (C-10′), which also had cross-peaks with
the 6H singlet at δ 1.52, and the olefinic signal at δΗ 6.16
(H-11′) with the CH3 signals at δC 27.9 (C-13′/C-14′) and
27.3 (C-13′/C-14′). The foregoing suggested the presence of
a 1,1-dimethyprop-2-enyl (inverted γ,γ-dimethylallyl) group
in 1 at C-2′ of the 2,3-disubstituted indole residue (see
above). This was confirmed by the presence of a strong
ROESY correlation between the 6H singlet at δ 1.52 and
Figure 1. HMBC and ROESY correlations for 1 and NOE correlations
for 10.
the NH signal at δH 10.04 of this indole ring, and the
resistance of this NH to undergo acetylation on treatment
of 1 with Ac2O/pyridine at room temperature. The remain-
ing NH signal of 1 at δH 10.67, which underwent ready
acetylation under the above conditions, probably due to the
absence of steric crowding at C-2′′ of this indole ring,
showed strong ROESY correlations with CH3 signals at δ
1.57 and 1.29, suggesting the 3-substituted indole residue
and the dimethylallyl group to be on the same side of the
central quinone core of terrequinone A (1). Methylation of
1 with CH3I/K2CO3 in acetone at room temperature af-
forded the monomethyl derivative 10. In the NOE spectrum
of 10, irradiation of the signal due to 6′-H (at δH 7.21) of
the disubstituted indole moiety induced an enhancement
of the 3H singlet at δ 3.72 of the newly introduced Me
group, indicating that the OH group and the 2,3-disubsti-
tuted indole residue of 1 are on the same side of the
quinone core. Acetylation of 1 afforded two products, which
were identified as its monoacetate (11) and the diacetate
(12). The 1H NMR spectrum of 12 indicated that, in
addition to the OH of the quinone system, the NH of the
monosubstituted indole ring has undergone acetylation as

Cytotoxic Metabolites of Aspergillus
Journal of Natural Products, 2004, Vol. 67, No. 12 1987
evident from the disappearance of the signal at δH 10.67
and significant downfield shifts of 2′′-H and 9′′-H of 12
compared with those of 1. On the basis of the foregoing
evidence the structure of terrequinone A was elucidated
as 2-hydroxy-3-[2′-(1,1-dimethylprop-2-en)indol-3′-yl)]-5-(3-
methylbut-2-enyl)-6-(indol-3′-yl)cyclohex-2,5-diene-1,4-di-
one (1).
The molecular formula of terrefuranone (2) was deter-
mined as C14H20O3 from its HRFABMS and 13C NMR data
and indicated five degrees of unsaturation. The IR absorp-
tion bands at 3425 and 1697 cm-1 and the 13C NMR signals
at δ 65.6 and 204.7 suggested the presence of OH and R,â-
1
unsaturated ketone carbonyl groups. The H NMR spec-
trum of 2 consisted of one 3H singlet at δ 1.47 assigned to
a CH3 group on a quaternary carbon (C-5), one olefinic
singlet at δ 5.43, and two independent spin systems. The
1H-1H COSY spectrum of 2 indicated that one of the spin
systems consisted of a 3H doublet (J ) 6.5 Hz) at δ 0.97
attributable to a CH3 attached to an oxygenated methine
at δ 4.21 (1H, m), which in turn is attached to a methylene
group at δ 2.69 (2H, m). The chemical shift of this CH2
indicated that it is attached to an olefinic carbon, suggest-
ing the presence of the fragment CH3CH(OH)CH2CdC in
2. This was further confirmed by the treatment of 2 with
Ac2O/pyridine, which resulted in the formation of its
acetate 13 and the corresponding dienone, 13-deoxy-12(13)-
Figure 2. Selected HMBC correlations for 2 and 8.
configuration at C-5 and C-14, is known to occur in As.
niveus,16 its spectroscopic data have not been reported in
the literature.
Bioassay-guided fractionation of the cytotoxic EtOAc
extract of As. terreus, isolated from the rhizosphere of an
unidentified Brickellia sp., involving solvent-solvent parti-
tion, followed by Sephadex LH-20 gel filtration and re-
peated silica gel and reversed-phase chromatography fur-
nished three cytotoxic compounds, 4-6. These were
identified as dehydrocurvularin (4), 11-methoxycurvularin
(5), and 11-hydroxycurvularin (6) by comparison of their
spectroscopic data with those reported in the literature7,9
and by direct comparison with authentic samples.8 Al-
though 5 and 6 have been reported as metabolites of several
fungi,7-9 isolation of each of these as a mixture of 11R- and
11â-epimers suggests their possible artifactual origin,
probably by a Michael-type addition of MeOH and H2O to
the enone system of dehydrocurvularin (4) during the
processing of these microorganisms.
The EtOAc extract of As. cervinus, isolated from the
rhizosphere of A. thurberi, was selected for detailed inves-
tigation on the basis of its cytotoxicity and because of the
absence of any reported biological activity of the metabo-
lites previously encountered in this fungal species.12 Frac-
tionation of the EtOAc extract as above furnished 7-9. The
molecular formula of compound 7, isolated as an optically
active colorless semisolid, was deduced as C8H12O4 from
its HRFABMS and 13C NMR spectra and indicated three
degrees of unsaturation. IR absorption bands at 3402 and
1620 cm-1 suggested the presence of OH and R,â-unsatu-
rated ketone carbonyl groups. The 1H NMR spectrum of 7
indicated the presence of two 3H singlets at δ 1.34 and 3.76
due to CH3 and OCH3, respectively, two 1H doublets (J )
16.5 Hz) at δ 2.68 and 2.50, and two 1H singlets at δ 4.32
and 5.35, accounting for all but two protons which were
suspected to belong to two OH groups. The 13C NMR
spectrum, assigned on the basis of DEPT, HSQC, and
HMBC spectra, showed the presence of a ketone carbonyl
(δ 196.1), two olefinic carbons (δ 101.5 and 174.3), of which
one was oxygenated (δ 174.3), two oxygenated sp3 carbons
(δ 74.5 and 73.7), a methoxy carbon (δ 56.6), and a methyl
carbon (δ 22.5). The UV spectrum of 7 had an absorption
band at 250.5 nm, indicating the presence of a cyclic
â-oxygenated-enone structure. The chemical shifts and
coupling constants of the 1H doublets at δH 2.68 and 2.50
suggested that they belong to a COCH2 moiety of a cyclic
system with its CH2 group attached to a quaternary carbon.
In the HMBC spectrum the signal at δH 2.68 had cross-
peaks with signals at δC 196.1, 101.5, and 73.7, and the
signal at δH 4.32 showed correlations with those at δC 174.3
1
dehydroterrefuranone (14). The H NMR spectrum of 13
showed downfield shifts of all the protons of this fragment,
especially the shift from δ 4.21 to 5.25 of the signal assigned
to the proton geminal to the OH group. The 1H-1H COSY
spectrum indicated that the remaining spin system of 2
consisted of a 3H triplet (J ) 6.5 Hz) at δ 0.97 attributable
to a CH3 attached to a methylene at δ 2.08 (m) and that
the latter is attached to a methine group at δ 5.78 (m),
which is a part of a conjugated diene [δ 5.96 (dd, J ) 15.0
and 9.5 Hz), 6.26 (dd, J ) 15.5 and 4.5 Hz), 5.52 (d, J )
15.5 Hz)] attached to a quaternary carbon. The large
coupling constants of the olefinic protons of this spin system
together with these 1H NMR data identified this fragment
as E,E-hexa-1,3-diene. These two spin systems and the CH3
group on C-5 (see above) accounted for C10H19O and 2 units
of unsaturation. Thus, the remaining fragment of 2 has
the partial formula C4HO2 with 3 units of unsaturation and
a carbonyl function, which suggested this to be a trisub-
stituted dihydrofuran-4-one. The chemical shift (δH 5.43)
of the olefinic singlet and the ready dehydration of 2 on
treatment with Ac2O/pyridine yielding the corresponding
dienone 14 indicated the attachment of the CH3CH(OH)-
CH2 fragment (see above) to C-2 of the dihydrofuran-4-one.
HSQC and HMBC data further confirmed the presence of
these three fragments, and HMBC correlations (Figure 2)
were used to establish their connectivities, leading to the
planar structure 2 proposed for terrefuranone. It remained
to determine the stereochemical dispositions of the groups
at C-5 and C-14 in 2. The configuration at C-5 was assigned
using CD data for the dienone 14 derived from 2. The CD
spectrum of 14 consisted of a curve (R band) with a strong
negative first Cotton effect (306 nm) and a weak positive
second Cotton effect (275 nm), indicating negative (S)
chirality at C-5.15 Attempts to prepare the Mosher ester of
2 resulted in the formation of the dienone 14 with no trace
of the desired ester, and this precluded the determination
of the configuration at C-14. On the basis of the foregoing
evidence, the structure of terrefuranone was elucidated as
5â-(1E,3E)-1,3-hexadienyl-4,5-dihydro-2-(2-hydroxypropyl)-
5R-methylfuran-4-one (2). Although the related anti-HIV
active carboxylic acid, nivefuranone C (15), with undefined

1988 Journal of Natural Products, 2004, Vol. 67, No. 12
He et al.
Table 2. Cytotoxicities of Compounds 1-9, 14, and 16 against
a Panel of Four Tumor Cell Lines and Normal Human Primary
Fibroblast Cellsa
and 73.7. These HMBC data suggested that the CH2 of the
COCH2 moiety is attached to the oxygenated quaternary
carbon at δ 73.7 and that the oxygenated carbon at δ 74.5
(δH 4.32) is attached to the oxygenated carbon of the olefin
of the enone system. In the NOESY spectrum 7 had strong
spatial correlations between δH 2.68 and 4.32 and between
δH 2.50 and 1.34, indicating a trans relationship between
the vicinal OH groups. On the basis of the above data, the
structure of 7 was elucidated as 4R*,5S*-dihydroxy-3-
methoxy-5-methylcyclohex-2-enone.
cell lineb
compound NCI-H460 MCF-7 SF-268 MIA Pa Ca-2 WI-38
1
5.60
1.10
0.90
2.10
5.80
5.50
0.01
6.80
1.30
13.90
2.50
0.90
4.10
20.00
6.80
0.04
5.40
1.90
1.20
3.30
8.00
6.30
0.05
NTc
3.60
1.70
11.60
57.50
NTc
4
5
0.60
6
2.00
9
12.80
11.70
0.07
14
Compound 8 was obtained as an optically active colorless
oil. Its molecular formula, C9H14O5, determined by a
combination of HRFABMS and 13C NMR spectroscopy
indicated three degrees of unsaturation. It showed IR
absorption bands at 3356, 1751, and 1643 cm-1, suggesting
the presence of OH and R,â-unsaturated lactone groups.
The UV spectrum of 8 showed very close resemblance to
that of 7 with an absorption band at 249.5 nm, indicating
the presence of a cyclic â-oxygenated-enone structure. Its
1H NMR spectrum had three singlets, one due to an olefinic
proton at δ 5.02 and two due to OCH3 groups at δ 3.87
and 3.38, a D2O exchangeable proton at δ 6.70, and signals
assignable to the -CH(CH3)CH2O- spin system. The 13C
NMR spectrum of 8 when analyzed with the help of HSQC
showed the presence of one CH3, two OCH3, one CH2
bearing an oxygen atom, an olefinic CH, and three qua-
ternary carbons. The above data indicated 8 to be a furan-
2-enone bearing -OCH3, -OH, and -CH(CH3)CH2OCH3
groups. The points of attachments of these groups to the
furan-2-enone ring system were determined with the help
of HMBC correlations (Figure 2). On the basis of the
foregoing evidence the structure of 8 was elucidated as
6-methoxy-5(6)-dihydropenicillic acid. Analysis of NMR
coupling constants, NOE data, and the CD spectrum did
not permit definitive assignment of the configuration at
C-4 and C-5 of 8. Compound 9 was identified as penicillic
acid by comparison of its 1D and 2D NMR spectral data
with those reported in the literature.10 The presence of
penicillic acid (9) in the same extract and the use of MeOH
during the extraction of the fungus suggested possible
artifactual origin of 8 from 9. However, 9 failed to react
with MeOH to produce 8 under a variety of conditions
including such harsh conditions as refluxing MeOH in the
presence of p-toluenesulfonic acid (p-TSA), indicating that
8 is a genuine natural product. Penicillic acid (9) has
previously been reported from several Aspergillus and
Penicillium species.10 Co-occurrence of 7 and 9 in As.
cervinus is noteworthy, as compounds structurally related
to 7 (e.g., 6-methoxy-2-methyl-1,4-benzoquinone) have been
implicated as biosynthetic precursors of penicillic acid (9).17
Bioactivity-guided fractionation of the cytotoxic EtOAc
extract of As. wentii, isolated from the rhizosphere of L.
tridentata, afforded penicillic acid (9) as the only cytotoxic
constituent of this extract.
doxorubicin
0.30
a Results are expressed as IC50 values in µM; compounds 2, 3,
7, 8, and 16 were found to be inactive in all cell lines at 10.0 µg/
mL. b Key: NCI-H460 ) human non-small cell lung cancer; MCF-7
) human breast cancer; SF-268 ) human CNS cancer (glioma);
MIA Pa Ca-2 ) human pancreatic cancer; WI-38 ) normal human
primary fibroblast cells. c NT ) not tested.
0.90 to 57.50 µM. The most cytotoxic are the curvularins
4-6. It is significant that penicillic acid (9) is selectively
cytotoxic against non-small cell lung and pancreatic cancer
cell lines used in this study. Compounds 4-6 have been
reported to inhibit sea urchin embryogenesis by acting on
components of the mitotic apparatus,19 and a recent patent
application suggests their weak interaction with heat shock
protein 90 (Hsp90),20 a promising target for anticancer drug
discovery.21 Penicillic acid (9) has been reported to be
cytotoxic to A2780 human ovarian carcinoma,22a Chinese
hamster ovary cells,22b and HeLa cells22c and was found to
inhibit the growth of sarcoma originally induced by it.22d
The mechanism of cytotoxicity of 9 has been determined
to be due to its ability to bind to SH groups in macromole-
cules,22e induction of DNA single-strand breaks,22b and
inhibition of DNA synthesis.22c It has recently been dem-
onstrated that penicillic acid (9) inhibits Fas ligand-induced
apoptosis by targeting self-processing of caspase-8.22f The
absence of any cytotoxic activity of its derivatives 8 and
16 suggests the requirement of the isopropenyl moiety for
the cytotoxicity of 9. Studies to elucidate the molecular
mechanism(s) of action of the most potent cytotoxins 4 and
5 and animal studies to evaluate their antitumor potential
are currently in progress.
Experimental Section
General Experimental Procedures. Melting points were
determined on an Electrothermal melting point apparatus and
are uncorrected. Optical rotations were measured with a
JASCO Dip-370 polarimeter using CHCl3 as solvent. CD
spectrum was obtained using a JASCO J-805 spectropolarim-
eter. IR spectra for KBr disks were recorded on a Shimadzu
FTIR-8300 spectrometer. 1D and 2D NMR spectra were
recorded in CDCl3, acetone-d6, and DMSO-d6 and using
residual solvents as internal standards with a Bruker DRX-
500 instrument at 500 MHz for 1H NMR and 125 MHz for 13
C
1
NMR and a Bruker DRX-600 instrument at 600 MHz for H
NMR and 150 MHz for 13C NMR. The chemical shift values
(δ) are given in parts per million (ppm), and the coupling
constants are in Hz. Low-resolution and high-resolution MS
were recorded respectively on Shimadzu LCMS QP8000R and
JEOL HX110A spectrometers.
Compounds 1-9, 14, and 16 were evaluated for in vitro
cytotoxicity against a panel of four cancer cell lines (NCI-
H460, MCF-7, SF-268, and MIA Pa Ca-2) and normal
human primary fibroblast cells (WI-38). Cells were treated
with test compounds for 72 h in RPMI-1640 media supple-
mented with 10% fetal bovine serum, and cell viability was
evaluated by the MTT assay.18 The concentrations resulting
in 50% inhibition of cell proliferation/survival (IC50) as
measured by this assay are given in Table 2. Of those
tested, terrequinone A (1), dehydrocurvularin (4), 11-
methoxycurvularin (5), 11-hydroxycurvularin (6), penicillic
acid (9), and 13-deoxy-12(13)-dehydroterrefuranone (14)
were found to be cytotoxic, with IC50 values ranging from
Cytotoxicity Bioassays. The tetrazolium-based colorimet-
ric assay (MTT assay)18 was used for the in vitro assay of
cytotoxicity to human non-small cell lung carcinoma (NCI-
H460), human breast carcinoma (MCF-7), human glioma (SF-
268), human pancreatic cancer (MIA Pa Ca-2) cell lines, and
normal human primary fibroblast (WI-38) cells as previously
reported.4 All samples for cytotoxicity assays were dissolved
in DMSO. During bioassay-guided fractionation, cytotoxicity
of fractions was monitored using the NCI-H460 cell line.

Cytotoxic Metabolites of Aspergillus
Journal of Natural Products, 2004, Vol. 67, No. 12 1989
Fungal Isolations. The fungal strains were isolated from
the rhizospheres of Ambrosia ambrosoides (collected from
Tucson Mountains, AZ; herbarium sample accession No. AH-
00-21), Brickellia sp. (from Sycamore Springs in Greasewood
Mountains, AZ; accession No. AH-02-30), Anicasanthus thurb-
eri (from Santa Rita Mountains near Sonoita, AZ; accession
No. AH-00-70), and Larrea tridentata (from Puerto Blanco
Mountains, AZ; accession No. AH-00-115). All plant species
were identified by Dr. Annita Harlan of the University of
Arizona. Identification of the isolated fungal strains was made
by Ms. Donna Bigelow, Ms. Jun Zhang, and Dr. Elizabeth
Pierson (all of Department of Plant Sciences, University of
Arizona) by the analysis of the ITS regions of the ribosomal
DNA as described previously.4 Excised roots of each plant (1
cm long sections; ca. 5 g) were separately placed in 5 mL of
phosphate-buffered saline (PBS, 0.1M, pH ) 7.4), and micro-
organisms were detached from the roots by vortexing and
sonication. Serial dilutions of the resulting suspensions were
placed on Petri dishes containing potato dextrose agar (PDA,
Difco, Plymouth, MN) supplemented with chloramphenicol and
streptomycin. After 4 days of incubation at 25 °C, single
colonies from each Petri dish were transferred to water agar
Petri dishes containing the same antibiotics, and after 3 days
pure fungal cultures were obtained by hyphal tipping. Each
fungal strain is deposited in the Division of Plant Pathology
and Microbiology, Department of Plant Sciences, and South-
west Center for Natural Products Research and Commercial-
ization of the University of Arizona microbial culture collec-
tions under the following accession numbers: As. terreus from
A. ambrosoides, AH-00-21-F12; As. terreus from Brickellia sp.,
AH-02-30-F7; As. cervinus from A. thurberi, AH-00-70-F11; and
As. wentii from L. tridentata, AH-00-115-F15. Each organism
was subcultured on PDA, and for long-term storage isolates
were subcultured on PDA slants, overlaid with 40% glycerol,
and stored at -80 °C.
Cultivation and Isolation of Metabolites of As. terreus
from the Rhizosphere of A. ambrosoides. For isolation of
secondary metabolites, the fungus was cultured in 40 T-flasks
(800 mL), each containing 135 mL of PDA coated on five sides
of the flasks, maximizing the surface area for fungal growth
(total surface area/flask ca. 460 cm2). After incubation for 28
days at 27 °C, MeOH (200 mL/T-flask) was added to all 40
T-flasks, which were sonicated and shaken in a rotary shaker
for 12 h at room temperature, and the resulting extract was
filtered through Whatman No. 1 filter paper and a layer of
Celite 545. The filtrate was concentrated to one-fourth of its
original volume and extracted with EtOAc (5 × 300 mL).
Combined EtOAc extracts were evaporated under reduced
pressure to afford a brown semisolid (657.4 mg), a portion
(477.0 mg) of which was partitioned between hexane and 80%
aqueous MeOH. The cytotoxic 80% aqueous MeOH fraction
was diluted to 50% aqueous MeOH with H2O and extracted
with CHCl3. Evaporation of CHCl3 under reduced pressure
yielded a brown semisolid (184.0 mg), which was subjected to
gel permeation chromatography on a column of Sephadex LH-
20 (2.0 g) in hexane/CH2Cl2 (1:4) and eluted with hexane/
CH2Cl2 (1:4) (50 mL), CH2Cl2/acetone (3:2) (50 mL), and
CH2Cl2/MeOH (1:1) (50 mL) to furnish six fractions, 1-6 (25
mL each), of which fraction 2 (90.4 mg) was found to be the
most cytotoxic. Column chromatography of this fraction (90.0
mg) on silica gel (1.5 g) and elution with CH2Cl2 (50.0 mL)
and CH2Cl2/MeOH (50:1) (50 mL) afforded 1 (6.0 mg) and three
subfractions, A-C. Chromatography of fraction A (50.9 mg)
on silica gel (0.3 g) by elution with hexane/EtOAc (1.5:1)
afforded 2 (5.0 mg) and 3 (30.0 mg).
5.96 (1H, dd, J ) 15.0, 9.5 Hz, H-8), 5.78 (1H, m, H-9), 5.52
(1H, d, J ) 15.5 Hz, H-6), 5.43 (1H, s, H-3), 4.21 (1H, m, H-14),
2.69 (2H, m, CH2-13), 2.08 (2H, m, CH2-10), 1.47 (3H, s, CH3-
12), 1.30 (3H, d, J ) 6.5 Hz, CH3-15), 0.97 (3H, t, J ) 6.5 Hz,
CH3-11); 13C NMR (125 MHz, CDCl3) δ 204.7 (C, C-4), 188.6
(C, C-2), 138.8 (CH, C-9), 131.1 (CH, C-7), 127.9 (CH, C-8),
126.5 (CH, C-6), 90.2 (C, C-5), 65.6 (CH, C-14), 40.4 (CH2,
C-13), 25.7 (CH2, C-10), 23.4 (CH3, C-15), 22.3 (CH3, C-12),
13.3 (CH3, C-11); APCIMS (+)-ve mode m/z 237 [M + 1]+;
APCIMS (-)-ve mode m/z 235 [M - 1]+; HRFABMS m/z
237.1490 [M + 1]+ (calcd for C14H21O3, 237.1491).
Na-Acetylaszonalemin (3): colorless amorphous solid; 1H
and 13C NMR and MS data were consistent with those reported
in the literature.6b
Methylation of Terrequinone A. Methyl iodide (0.2 mL)
and K2CO3 (10.0 mg) were added to a stirred solution of 1 (1.2
mg) in acetone (0.2 mL) at 0 °C. After 5 min at 0 °C, the ice
bath was removed and the reaction mixture was stirred at 25
°C until the starting material disappeared (TLC control). It
was then filtered, solvent was removed under reduced pres-
sure, and the crude product was purified by preparative TLC
(silica gel) using 1% MeOH in CH2Cl2 as eluant to give 10 (1.2
mg).
Terrequinone A monomethyl ether (10): dark brown
1
solid; H NMR (600 MHz, acetone-d6) δ 10.68 (1H, brs, NH),
10.15 (1H, brs, NH), 7.51 (1H, d, J ) 7.8 Hz, H-6′′), 7.49 (1H,
d, J ) 2.4 Hz, H-2′′), 7.38 (1H, d, J ) 7.8 Hz, H-9′′), 7.35 (1H,
d, J ) 8.0 Hz, H-6′), 7.25 (1H, d, J ) 8.0 Hz, H-9′), 7.18 (1H,
t, J ) 7.8 Hz, H-7′′), 7.09 (1H, t, J ) 8.0 Hz, H-7′), 7.07 (1H,
t, J ) 7.8 Hz, H-8′′), 6.98 (1H, t, J ) 8.0 Hz, H-8′), 6.14 (1H,
dd, J ) 17.5, 10.6 Hz, H-11′), 5.09 (1H, dd, J ) 17.4, 0.8 Hz,
H-12′a), 5.05 (1H, m, H-8), 5.02 (1H, dd, J ) 10.6, 0.9 Hz,
H-12′b), 3.72 (3H, s, OCH
3), 3.32 (1H, dd, J ) 12.9, 7.4 Hz,
H-7a), 3.21 (1H, dd, J ) 13.5, 6.5 Hz, H-7b), 1.55 (3H, s, CH
3),
1.51 (3H, s, CH3), 1.50 (3H, s, CH3), 1.29 (3H, s, CH3); APCIMS
(-)-ve mode m/z 503 [M - H]+; HRFABMS m/z 504.2448 [M]+
(calcd for C33H32N2O3, 504.2413).
Acetylation of Terrequinone A. Acetic anhydride (0.2
mL) was added to a solution of terrequinone A (1) (2.0 mg) in
pyridine (0.1 mL) and stirred at 25 °C until the starting
material disappeared (TLC control). Pyridine and excess Ac2O
were removed under reduced pressure, and the products were
separated by preparative TLC (silica gel) using 1% methanol
in CH2Cl2 as eluant to give 11 (0.9 mg) and 12 (0.9 mg).
Terrequinone A monoacetate (11): dark brown solid; 1H
NMR (600 MHz, acetone-d6) δ 10.74 (1H, brs, NH), 10.30 (1H,
brs, NH), 7.51 (1H, d, J ) 2.6 Hz, H-2′′), 7.52 (1H, d, J ) 7.0
Hz, H-6′′), 7.39 (1H, d, J ) 7.0 Hz, H-9′′), 7.35 (1H, d, J ) 7.8
Hz, H-6′), 7.24 (1H, d, J ) 7.8 Hz, H-9′), 7.19 (1H, dt, J ) 7.0,
0.9 Hz, H-7′′), 7.10 (1H, dt, J ) 7.8, 0.9 Hz, H-7′), 7.08 (1H,
dt, J ) 7.0, 0.9 Hz, H-8′′), 6.98 (1H, dt, J ) 7.8, 0.9 Hz, H-8′),
6.16 (1H, dd, J ) 17.4, 10.6 Hz, H-11′), 5.08 (1H, dd, J ) 17.4,
0.8 Hz, H-12′a), 5.07 (1H, m, H-8), 5.04 (1H, dd, J ) 10.6, 1.2
Hz, H-12′b), 3.39 (1H, dd, J ) 13.5, 7.4 Hz, H-7a), 3.27 (1H,
dd, J ) 13.5, 6.7 Hz, H-7b), 1.96 (3H, s, OAc), 1.57 (3H, s,
CH3), 1.51 (3H, s, CH3), 1.50 (3H, s, CH3), 1.29 (3H, s, CH3);
APCIMS (-)-ve mode m/z 531 [M - H]+.
Terrequinone A diacetate (12): dark brown solid; 1H
NMR (600 MHz, acetone-d6) δ 10.36 (1H, brs, NH), 7.91 (1H,
s, H-2′′), 8.47 (1H, d, J ) 8.3 Hz, H-6′′), 7.41-7.38 (3H, m,
H-7′′, H-8′′, H-9′′), 7.35 (1H, d, J ) 7.8 Hz, H-6′), 7.24 (1H, d,
J ) 7.8 Hz, H-9′), 7.09 (1H, dt, J ) 7.8, 0.7 Hz, H-7′), 6.98
(1H, dt, J ) 7.8, 0.7 Hz, H-8′), 6.17 (1H, dd, J ) 17.4, 10.7 Hz,
H-11′), 5.07 (1H, d, J ) 17.4 Hz, H-12′a), 5.06 (1H, d, J ) 10.6
Hz, H-12′b), 5.02 (1H, brt, H-8), 3.36 (1H, dd, J ) 14.4, 6.9
Hz, H-7a), 3.27 (1H, dd, J ) 14.4, 5.2 Hz, H-7b), 1.96 (3H, s,
OAc), 1.55 (3H, s, CH3), 1.51 (3H, s, CH3), 1.50 (3H, s, CH3),
1.29 (3H, s, CH3); APCIMS (-)-ve mode m/z 573 [M - H]+.
Acetylation of Terrefuranone (2). Compound 2 (1.0 mg)
was acetylated with Ac2O (0.5 mL) in pyridine (0.5 mL) with
stirring at room temperature for 12 h. The reaction mixture
was purified on preparative silica gel eluting with hexane/
EtOAc (3:1) to yield 14-acetyl terrefuranone (13) (0.3 mg) and
14-deoxy-13(14)-dehydroterrefuranone (14) (0.6 mg). Com-
pound 13: colorless oil; 1H NMR (600 MHz, CDCl3) δ 6.26 (1H,
Terrequinone A (1): purple powder; mp 160-165 °C; UV
(MeOH) λmax (log ꢀ) 223.0 (5.63), 274.5 (5.18), 360.0 (4.24) nm;
IR (KBr) νmax 3410, 2932, 1636, 1435, 1096, 741 cm-1; 1H and
13C NMR data, see Table 1; APCIMS (+)-ve mode m/z 491 [M
+ 1]+; APCIMS (-)-ve mode m/z 489 [M - 1]+; HRFABMS
m/z 490.2260 [M]+ (calcd for C32H30N2O3, 490.2256).
Terrefuranone (2): colorless oil; [R]20 +16.8 (c 0.13
D
MeOH); UV (MeOH) λmax (log ꢀ) 233 (4.34), 265 (3.94) nm; IR
(KBr) νmax 3425, 2924, 2855, 1697, 1589, 1373, 1111 cm-1; 1H
NMR (500 MHz, CDCl3) δ 6.26 (1H, dt, J ) 15.5, 4.5 Hz, H-7),

1990 Journal of Natural Products, 2004, Vol. 67, No. 12
He et al.
m, H-2′′), 5.95 (1H, m, H-3′′), 5.79 (1H, m, H-4′′), 5.50 (1H,
dd, J ) 15.5, 2.5 Hz, H-1′′), 5.39 (1H, s, H-4), 5.25 (1H, m,
H-2′), 2.78 (2H, m, H-1′), 2.07 (2H, m, H-5′′), 1.99 (3H, s,
CH3CO), 1.45 (3H, s, H-1′′′), 1.32 (3H, d, J ) 6.5 Hz, H-3′),
0.97 (3H, t, J ) 7.5 Hz, H-6′′); APCIMS (+)-ve mode m/z 279
[M + 1]+, 320 [M + CH3CN]+, 260 [M + H - HOAc]+, 260 [M
+ CH3CN -HOAc]+. Compound 14: colorless oil; UV (MeOH)
λmax (log ꢀ) 235 (3.36), 276 (3.36), 306 (3.00) nm; CD λmax (4.23
× 10-4 M EtOH) nm 306 ([θ] -37770); 1H NMR (600 MHz,
CDCl3) δ 6.83 (1H, dq, J ) 15.5, 7.0 Hz, H-2′), 6.26 (1H, m,
H-2′′), 6.24 (1H, dd, J ) 15.5, 1.5 Hz, H-1′), 5.96 (1H, dd, J )
15.0, 10.2 Hz, H-3′′), 5.77 (1H, m, H-4′′), 5.56 (1H, d, J ) 15.2
Hz, H-1′′), 5.36 (1H, s, H-4), 2.07 (2H, m, H-5′′), 1.95 (3H, dd,
J ) 7.0, 1.5 Hz, H-3′), 1.48 (3H, s, H-1′′′), 0.97 (3H, t, J ) 7.3
Hz, H-6′′); APCIMS (+)-ve mode m/z 219 [M + 1]+.
A-E, of which fraction C was found to be the most cytotoxic.
Chromatography of fraction A (92.0 mg) on silica gel (2.5 g)
by elution with CH2Cl2/MeOH (100:1) afforded 8 (54.0 mg).
Fraction C (25.0 mg) was further purified on silica gel (1.2 g)
chromatography by elution with CH2Cl2/MeOH (20:1) to yield
9 (18.0 mg). Chromatography of fraction D (15.0 mg) on silica
gel (0.3 g) by elution with hexane/acetone (1:1) afforded 7 (4.5
mg).
4R*,5S*-Dihydroxy-3-methoxy-5-methylcyclohex-2-
enone (7): colorless semisolid; [R]20D +160.3° (c 0.01, CHCl3);
UV (MeOH) λmax (log ꢀ) 250.5 (4.24) nm; IR (KBr) νmax 3402,
2924, 2855, 1620, 1458, 1373, 1227, 1057 cm-1; 1H NMR (500
MHz, CDCl3) δ 5.35 (1H, s, H-2), 4.32 (1H, s, H-4), 3.76 (3H,
s, OCH3-3), 2.68 (1H, d, J ) 16.5 Hz, H-6a), 2.50 (1H, d, J )
16.5 Hz, H-6b), 1.34 (3H, s, CH3-5); 13C NMR (125 MHz, CDCl3)
δ 196.1 (C, C-1), 174.3 (C, C-3), 101.5 (CH, C-2), 74.5 (CH,
C-4), 73.7 (C, C-5), 56.6 (CH3, OCH3-3), 48.6 (CH2, C-6), 22.5
(CH3, CH3-5); APCIMS (+)-ve mode m/z 173 [M + 1]+; APCIMS
(-)-ve mode m/z 171 [M - 1]+; HRFABMS: m/z 173.0811 [M
+ 1]+ (calcd for C8H13O4, 173.0814).
Cultivation and Isolation of Metabolites of As. terreus
from the Rhizosphere of Brickellia sp. For isolation of
secondary metabolites, the fungus was cultured on PDA in 60
T-flasks for 30 days at 27 °C. Extraction and liquid-liquid
fractionation as for As. terreus from A. ambrosoides (see above)
afforded the cytotoxic CHCl3 fraction as a dark brown solid
(486.1 mg). A portion (300.0 mg) of this was subjected to gel
permeation chromatography on a column of Sephadex LH-20
(10.0 g) in hexane/CH2Cl2 (1:4) and eluted with hexane/CH2-
Cl2 (1:4) (75 mL), CH2Cl2/acetone (3:2) (100 mL), and CH2Cl2/
acetone (1:4) (50 mL). Seventeen fractions (ca. 15 mL each)
were collected and combined on the basis of their TLC patterns
to yield three cytotoxic fractions, A-C. Column chromatogra-
phy of fraction A (91.5 mg) on reversed-phase silica gel (4.0 g)
and elution with MeCN/H2O (32:68) afforded a cytotoxic
subfraction (40.9 mg), a portion (17.0 mg) of which was further
purified by preparative TLC on silica gel (CH2Cl2/2-PrOH, 95:
5) to afford 4 (4.6 mg). Fraction B (118.0 mg) was subfraction-
ated on silica gel (5.0 g) using a gradient of EtOAc in hexane
to afford a cytotoxic subfraction (62.4 mg). Further purification
of this by column chromatography on reversed-phase silica gel
(3.0 g) and elution with increasing amounts of MeCN in H2O
yielded a crude fraction (11.0 mg), which on further purifica-
tion by preparative TLC on silica gel (CH2Cl2/2-PrOH, 95:5)
afforded pure 5 (9.0 mg). Evaporation of the cytotoxic 50%
aqueous MeOH fraction obtained during liquid-liquid frac-
tionation yielded a dark brown solid (176.2 mg). A portion
(100.0 mg) of this was subjected to gel permeation chroma-
tography on a column of Sephadex LH-20 (4.0 g) in hexane/
CH2Cl2 (1:4) and eluted with hexane/CH2Cl2 (1:4) (40 mL),
CH2Cl2/acetone (3:2) (80 mL), and CH2Cl2/acetone (1:4) (40
mL). Twenty fractions (ca. 10 mL each) were collected and
combined on the basis of their TLC patterns to yield two
cytotoxic fractions, C (37.1 mg) and D (39.3 mg). Chromatog-
raphy of fraction C (27.0 mg) on reversed-phase silica gel (0.5
g) and elution with increasing amounts of MeCN in H2O
afforded 6 (6.9 mg) and a further quantity of 5 (8.7 mg).
Separation of fraction D (39.0 mg) as for fraction C afforded 6
(21.3 mg).
Dehydrocurvularin (4): pale yellow amorphous solid;
comparison (TLC, 1H NMR, 13C NMR, and MS) with an
authentic sample8 confirmed its identity.
11-Methoxycurvularin (5): yellow oil, the identity of
which was confirmed by comparison (TLC, 1H NMR, 13C NMR,
and MS) with an authentic sample.8
11-Hydroxycurvularin (6): yellow oil; comparison (TLC,
1H NMR, 13C NMR, and MS) with an authentic sample8
confirmed its identity.
Cultivation and Isolation of Metabolites of As. cervi-
nus from the Rhizosphere of A. thurberi. For isolation of
secondary metabolites, the fungus was cultured on PDA in 60
T-flasks for 28 days at 27 °C and processed as for As. terreus
(see above) to yield the cytotoxic EtOAc extract (1.06 g). A
portion (1.05 g) of this extract was suspended in 80% aqueous
MeOH (100 mL) and extracted with hexane (3 × 200 mL). The
aqueous MeOH layer was concentrated, and the cytotoxic
residue (696 mg) was subjected to gel filtration over Sephadex
LH-20 (5.0 g) and eluted with CH2Cl2/acetone (3:2) (200 mL)
followed by MeOH (300 mL). Fractions were combined on the
basis of their TLC patterns to furnish five combined fractions,
6-Methoxy-5(6)-dihydropenicillic acid (8): colorless oil;
[R]20 +19.8° (c 0.19 CHCl3); UV (MeOH) λmax (log ꢀ) 249.5
D
(4.46) nm; IR (KBr) νmax 3356, 3132, 2932, 1751, 1643, 1342,
1219, 1034, 926 cm-1; 1H NMR (500 MHz, CDCl3) δ 6.70 (1H,
s, OH-4), 5.02 (1H, s, H-2), 3.87 (3H, s, OCH3-3), 3.75 (1H, t,
J ) 10.0 Hz, H-6a), 3.49 (1H, d, J ) 10.0 Hz, H-6b), 3.38 (3H,
s, OCH3-6), 2.46 (1H, m, H-5), 0.68 (3H, d, J ) 7.0 Hz, CH3-7);
13C NMR (125 MHz, CDCl3) 177.8 (C, C-3), 170.3 (C, C-1), 105.6
(C, C-4), 89.6 (CH, C-2), 75.0 (CH2, C-6), 59.5 (CH3, OCH3-3),
59.2 (CH3, OCH3-6), 36.5 (CH, C-5), 11.1 (CH3, C-7); APCIMS
(+)-ve mode m/z 203 [M + 1]+; HRFABMS m/z 203.0919 [M +
1]+ (calcd for C9H15O5, 203.0919).
Penicillic Acid (9): colorless crystalline solid; mp 79-80
1
°C (lit.10c 82-85 °C); IR, H and 13C NMR, and FABMS data
consistent with literature values.10
Cultivation and Isolation of Metabolites of As. wentii
from the Rhizosphere of L. tridentata. For isolation of
bioactive compounds, the fungus was cultured in 20 T-flasks
(800 mL) each containing 135 mL of PDA, for 28 days at 27
°C. Extraction of the culture medium with EtOAc and liquid-
liquid fractionation of a portion (1.0 g) of the resulting extract
(1.122 g) as for As. terreus (see above) afforded the cytotoxic
CHCl3 fraction (673 mg). A portion (630 mg) of this was
subjected to gel permeation chromatography on a column of
Sephadex LH-20 (15.0 g) in hexane/CH2Cl2 (1:4) and eluted
with hexane/CH2Cl2 (1:4) (350 mL), CH2Cl2/acetone (3:2) (200
mL), CH2Cl2/acetone (1:4) (100 mL), CH2Cl2/MeOH (1:1) (100
mL), and MeOH (100 mL). Seventeen fractions (20 mL each)
were collected while the column was eluted with hexane/
CH2Cl2 (1:4). The last two fractions (fractions 16 and 17) eluted
with hexane/CH2Cl2 (1:4) (182.0 mg) and the fractions eluted
with CH2Cl2/acetone (3:2) (204.1 mg) were found to be cyto-
toxic. These fractions were combined, and a portion (30.0 mg)
of it was purified by preparative TLC on silica gel (diethyl
ether, double elution) to obtain 9 (22.0 mg), identical with the
above obtained sample of penicillic acid.
Catalytic Hydrogenation of 9. A solution of 9 (6.0 mg)
in EtOH (0.5 mL) containing Pd on carbon (10%, 1.5 mg) was
stirred in an atmosphere of H2 for 5 min (TLC control). The
solution was filtered through a plug of cotton, and the solvent
was evaporated under reduced pressure to afford 5(6)-dihy-
1
dropenicillic acid (16) as a white solid (6.0 mg). Its H NMR,
13C NMR, and MS data were consistent with those reported
in the literature.13
Acknowledgment. This work was supported by grants
from the Arizona Disease Control Research Commission
(ADCRC), and this support is gratefully acknowledged. We
thank Dr. A. Harlan for identification of the plant species from
which the rhizosphere fungi were obtained, and Ms. D. Bigelow
and Ms. J. Zhang for their assistance in the identification of
some fungal strains. The NMR spectrometer used in this
research was funded by a grant from the National Science
Foundation (Grant No. 9729350).

Cytotoxic Metabolites of Aspergillus
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(11) Sassa, T.; Hayakaru, S.; Ikeda, M.; Miura, Y. Agric. Biol. Chem. 1971,
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(12) Elsohly, H. N.; Slatkin, D. J.; Schiff, P. L. Jr.; Knapp, J. E. J. Pharm.
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(13) (a) Varga, J.; Kevei, E.; Rinyu, E.; Teren, J.; Kozakiewicz, Z. Appl.
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NP040139D