Uncovering biosynthetic potential of plant-associated fungi: Effect of culture conditions on metabolite production by Paraphaeosphaeria quadriseptata and Chaetomium chiversii

Article abstract of DOI:10.1021/np070504b

In an attempt to uncover the biosynthetic potential of plant-associated fungi, the effect of culture conditions on metabolite production by Paraphaeosphaeria quadriseptata and Chaetomium chiversii was investigated. These studies indicated that the production of the major metabolites by P. quadriseptata differ when the water used to make the media was changed from tap water to distilled water. It resulted in the isolation of six new secondary metabolites, cytosporones F-I (1-4), quadriseptin A (5), and 5′-hydroxymonocillin III (6) together with monocillin III (7), a metabolite new to P. quadriseptata, in addition to monocillin I (8), a previously known metabolite from this organism. Aposphaerin B (9) encountered was suspected to be an artifact originating from cytosporone F (1). Incorporation of heavy metal ions to P. quadriseptata culture medium induced production of monocillin I (8) by this fungus. Cultivation of C. chiversii in liquid medium resulted in the isolation of chaetochromin A (12) as the major metabolite instead of radicicol (10), the major constituent of this organism when grown in a solid medium. Compounds 1-7 and 12 were evaluated for their potential to inhibit Hsp90 and antiproliferative activity toward the cancer cell lines NCI-H460, MCF-7, and SF-268. Only compounds 6, 7, and 8 exhibited significant activity in both assays.

Full text of DOI:10.1021/np070504b

J. Nat. Prod. 2007, 70, 1939–1945
1939
Uncovering Biosynthetic Potential of Plant-Associated Fungi: Effect of Culture Conditions on
Metabolite Production by Paraphaeosphaeria quadriseptata and Chaetomium chiWersii1
Priyani A. Paranagama,† E. M. Kithsiri Wijeratne, and A. A. Leslie Gunatilaka*
Southwest Center for Natural Products Research and Commercialization, Office of Arid Lands Studies, College of Agriculture and Life
Sciences, The UniVersity of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706-6800
ReceiVed September 19, 2007
In an attempt to uncover the biosynthetic potential of plant-associated fungi, the effect of culture conditions on metabolite
production by Paraphaeosphaeria quadriseptata and Chaetomium chiVersii was investigated. These studies indicated
that the production of the major metabolites by P. quadriseptata differ when the water used to make the media was
changed from tap water to distilled water. It resulted in the isolation of six new secondary metabolites, cytosporones
F-I (1-4), quadriseptin A (5), and 5′-hydroxymonocillin III (6) together with monocillin III (7), a metabolite new to
P. quadriseptata, in addition to monocillin I (8), a previously known metabolite from this organism. Aposphaerin B (9)
encountered was suspected to be an artifact originating from cytosporone F (1). Incorporation of heavy metal ions to P.
quadriseptata culture medium induced production of monocillin I (8) by this fungus. Cultivation of C. chiVersii in
liquid medium resulted in the isolation of chaetochromin A (12) as the major metabolite instead of radicicol (10), the
major constituent of this organism when grown in a solid medium. Compounds 1-7 and 12 were evaluated for their
potential to inhibit Hsp90 and antiproliferative activity toward the cancer cell lines NCI-H460, MCF-7, and SF-268.
Only compounds 6, 7, and 8 exhibited significant activity in both assays.
Plant-associated microorganisms represent a largely untapped
resource of small-molecule natural products, some with chemical
structures that have been optimized by coevolution for biological
and ecological relevance.2 According to Bode et al.,3 with more
than 20 000 compounds described in the literature, microorganisms
must be called metabolic artists superior to any metabolic diversity
created by man. This ability of microorganisms combined with the
potential to bring up a variety of new metabolites from a single
strain by systematic alteration of its cultivation parameters, known
as OSMAC (one strain many compounds) approach,4 and the use
of elicitors to induce or inhibit certain biosynthetic and/or signal
transduction pathways5 provide new opportunities to maximize
chemical diversity of their metabolites. The OSMAC approach has
resulted from the observation that very small changes in the
cultivation conditions can completely shift the metabolic profile of
many microorganisms. Thus, it represents a powerful tool to
elucidate the secondary metabolome (the overall number of all
secondary metabolites of one organism) of different microbes. This
approach has recently been used to release the chemical diversity
of a number of soil-borne fungi and actinomycetes6 and a fungus
of marine origin.7
etochiversins A and B.9 In the present study, we have investigated
the metabolite profiles of these organisms cultivated in PDB
medium made up separately in tap water and distilled water, and
herein we report the isolation of six new metabolites, cytosporones
F-I (1-4), quadriseptin A (5), and 5′-hydroxymonocillin III (6),
together with monocillin III (7), monocillin I (8), and aposphaerin
B (9) from P. quadriseptata and the known metabolites chaeto-
chromin A (12), eugenetin (13), and 6-methoxymethyleugenin (14)
from C. chiVersii. The octaketide metabolites structurally related
to 1-4, namely, cytosporones A-E and dothiorelone A, have
previously been encountered in Cytospora sp. CR200 and Diaporthe
sp. CR146,10 and Dothiorella sp. HTF3,11 respectively.
Results and Discussion
The rhizosphere fungus P. quadriseptata8a was cultivated for
14 days in PDB media made up separately with distilled water and
tap water, the culture supernatants were extracted with EtOAc, and
the resulting extracts were analyzed by HPLC. The HPLC profiles
of the two extracts indicated that they contained almost the same
metabolites but with significant differences in their major constitu-
ents (Figure 1). Thus, they were separately processed to isolate
and characterize the major metabolites present in these extracts.
Fractionation of the EtOAc extract of P. quadriseptata cultured in
distilled water involving reversed-phase column chromatography
and silica gel preparative TLC furnished cytosporones F (1) and G
(2), quadriseptin A (5), monocillin I (8), and aposphaerin B (9).
Cytosporone F (1) was determined to have the molecular formula
C18H22O5 by a combination of HRFABMS, 13C NMR, and HSQC
data and indicated eight degrees of unsaturation. The IR spectrum
had absorption bands due to OH (3369 cm-1), ester carbonyl (1732
cm-1), and R,ꢀ-unsaturated ketone carbonyl (1724 cm-1) groups.
The 1H NMR spectrum exhibited signals due to a chelated OH (δ
11.51), two aromatic protons at δ 6.31, a 2H singlet at δ 3.77 due
to a CH2 group sandwiched by an aromatic ring and an ester
carbonyl, an -OCH2CH3 group [δ 4.16 (2H, q, J ) 7.1 Hz) and
1.25 (3H, t, J ) 7.1 Hz)], and four olefinic protons in a conjugated
diene system [δ 7.26 (1H, dd, J ) 15.0 and 10.1 Hz); 6.55 (1H, d,
J ) 15.0 Hz); 6.21 (2H, m)] (Table 1). The 13C NMR spectrum of
1 when analyzed with the help of HSQC data displayed signals for
18 carbon atoms and indicated the presence of a ketone carbonyl
(δ 195.1), an ester carbonyl (δ 171.1), six aromatic carbons of which
In a study to uncover the chemical diversity of plant-associated
microorganisms, we have investigated the influence of culture
conditions on metabolite production of the fungal strains
Paraphaeosphaeria quadriseptata and Chaetomium chiVersii. We
have previously reported that the rhizosphere fungal strain P.
quadriseptata, when cultivated in potato dextrose agar (PDA) and
potato dextrose broth (PDB; a medium with a similar constitution
to PDA but without any added agar) media made up in tap water,
produced the C18 polyketide monocillin I (8) as the major
metabolite,8a together with the minor isocoumarins paraphaeos-
phaerins A-C, biosynthetically related to 8, aposphaerin C (11),
eugenetin (13), 6-methoxymethyleugenin (14), and 6-hydroxym-
ethyleugenin (15).9 Similarly, the endophytic fungal strain C.
chiVersii, when cultivated in PDA medium made up in tap water,
produced the corresponding metabolites radicicol (10)8b and cha-
* To whom correspondence should be addressed. Tel: (520) 741-1691.
Fax: (520) 741-1468. E-mail: leslieg@ag.arizona.edu.
† Present address: Department of Chemistry, University of Kelaniya,
Dalugama, Sri Lanka.
10.1021/np070504b CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/06/2007

1940 Journal of Natural Products, 2007, Vol. 70, No. 12
Paranagama et al.
Figure 1. HPLC profiles of crude EtOAc extracts of P. quadrisep-
tata cultured in (A) PDB medium made up in distilled water and
(B) PDB medium made up in tap water. Peak identities (compounds
corresponding to peaks 4, 9, 10, 11, 13, and 15 have not been
isolated from P. quadriseptata, and therefore the identities of these
peaks were not determined): peak 1 ) 5′-hydroxymonocillin III
(6); 2 ) cytosporone G (2); 3 ) cytosporone H (3); 5 )
quadriseptin A (5); 6 ) monocillin III (7); 7 ) cytosporone I (4);
8 ) monocillin I (8); 12 ) aposphearin C (11); 14 ) cytosporone
F (1); 16 ) aposphearin B (10).
two are oxygenated (δ 163.6 and 160.6), four protonated olefinic
carbons, four methylene of which one is oxygenated (δ 61.4), and
data [δH 3.56 t (J ) 6.4 Hz); δC 61.6] suggested that in 2 this
carbon is oxygenated. Cytosporone G was therefore identified as
3,5-dihydroxy-2-(8′-hydroxy-1′-oxoocta-2′E,4′E-dienyl)benzeneace-
tic acid ethyl ester (2). HRFABMS, 13C NMR, and DEPT data
suggested the molecular formula C13H12O5 for quadriseptin A (5).
It exhibited a UV spectrum (λmax 322 and 215 nm) characteristic
of a 7-oxygenated coumarin.12 The IR spectrum of 5, with
absorption bands at 3571, 1770, and 1705 cm-1, indicated the
presence of OH, R,ꢀ-unsaturated lactone (of a coumarin), and ester
carbonyl groups, respectively. The 1H NMR spectrum of 5 indicated
the presence of a -CH2CO2CH2CH3 moiety [δH 3.91 (2H, s), 4.11
(2H, q, J ) 7.1 Hz), and 1.19 (3H, t, J ) 7.1 Hz)] in addition to
an OH (δ 9.43), two meta-coupled aromatic protons at δ 6.80 and
6.67 (each d, J ) 2.2 Hz), and two olefinic protons at δ 8.00 (d, J
) 9.7 Hz) and 6.17 (d, J ) 9.7 Hz). The 13C and DEPT NMR
spectra of 5 confirmed the presence of an R,ꢀ-unsaturated lactone
carbonyl (δ 161.3), an ester carbonyl (δ 170.8), six aromatic carbons
of which two are oxygenated (δ 160.7 and 157.5) and two are
protonated (δ 115.9 and 102.6 d), two protonated olefinic carbons
(δ 141.8 and 112.5), one methylene (δ 38.2) sandwiched by an
aromatic carbon and a carbonyl, one oxygenated methylene (δ 61.5),
and one methyl carbon (δ 14.4). The foregoing data suggested the
structure of quadriseptin A as 5-(ethoxycarbonylmethyl)-7-hydroxy-
2H-1-benzopyran-2-one (5). The remaining compounds present in
this extract were identified as monocillin I (8)8 and aposphaerin B
(9)13 by comparison of their spectral data with those reported in
the literature. The desethyl derivative of 9 [aposphaerin C (11)]
and monocillin I (8) have previously been encountered in P.
quadriseptata cultured in PDA and PDB made up in tap water.8,9
The occurrence of aposphaerin B and the related octaketide,
cavoxinone, as racemic mixtures has led to the proposition that
they have been formed by nonenzymatic cyclizations of the
corresponding olefinic open-chain precursors.13 The co-occurrence
of aposphaerin B (9) and cytosporone F (1) in the same extract
1
two methyl carbons (Table 2). The H-1H correlations observed
for 1 in the DQF-COSY spectrum suggested the presence of the
spin systems -OCH2CH3 (see above) and -CHdCH-CHdCH-
CH2-CH2-CH3. The cross-peaks between δH 6.55 (H-2′) and δC
129.0 (C-4′), δH 7.26 (H-3′) and δC 146.9 (C-5′), and δH 6.21 (H-
4′) and δC 35.3 (C-6′) in the HMBC spectrum (Figure 2) established
the connectivity of olefinic carbons, and the correlations between
δH 3.77 (H-7) and δC 171.1 (C-8), δH 3.77 (H-7) and δC 111.6
(C-6), and δH 4.16 (H-1′′) and δC 171.1 (C-8) suggested the
presence of a -CH2CO2CH2CH3 moiety at C-1. The HMBC
correlations between δH 6.55 (H-2′) and δC 116.9 (C-2) suggested
the attachment of the ketone carbonyl to C-2 of the aromatic ring.
The presence of two phenolic OH groups and two double bonds in
1 was confirmed by methylation with CH3I/K2CO3 to afford the
dimethyl derivative 16 and catalytic hydrogenation of 16 to yield
its tetrahydro derivative, 3,5-di-O-methylcytosporone B (17). Treat-
ment of 17 with NaBH4 afforded 3,5-dimethoxy-2-(1′-hydroxyoc-
tyl)phenethyl alcohol (19). Thus, the structure of 1 was suspected
to be related to cytosporone B (18), and this was confirmed by the
catalytic hydrogenation of 1 to yield 18 with spectroscopic data
identical with those reported for cytosporone B.10 Finally, the
treatment of 1 with NaBH4/CH3OH/NaOH afforded a product that
was identified as cytosporone C (20).10 On the basis of the foregoing
evidence, the structure of cytosporone F was elucidated as 3,5-
dihydroxy-2-(1′-oxoocta-2′E,4′E-dienyl)benzeneacetic acid ethyl
ester (1).
The molecular formula of cytosporone G (2) was determined as
C18H22O6 from its HRFABMS and 13C NMR data. IR absorption
bands at 3429, 1720, and 1654 cm-1 suggested the presence of
OH, and ester and ketone carbonyl groups. 1H and 13C NMR spectra
of 2 (Tables 1 and 2, respectively) resembled those of 1 except
that the signals due to the terminal CH3 group [δH 0.91 t (J ) 7.4
Hz); δC 13.7] are replaced with a CH2 group and the chemical shift

Biosynthetic Potential of Plant-Associated Fungi
Journal of Natural Products, 2007, Vol. 70, No. 12 1941
Table 1. 1H NMR Data (500 MHz) for Compounds 1–4
δH multiplicity (J in Hz)
3b
position
1a
2b
4b
4
6
7
6.31 s
6.31 s
3.77 s
6.34 d (2.3)
6.36 d (2.3)
3.75 s
6.34 d (2.2)
6.36 d (2.2)
3.70 s
6.35 d (2.1)
6.38 d (2.1)
3.93 s
2′
3′
4′
5′
6.55 d (15.0)
7.26 dd (15.0, 10.1)
6.21 m
6.21 m
2.16 m
6.61 d (15.1)
7.11 dd (15.1, 10.3)
6.30 dd (15.1, 10.3)
6.27 dt (15.1, 6.2)
2.29 m
6.61 d (15.1)
7.11 dd (15.1, 10.6)
6.29 dd (15.1, 10.6)
6.28 dt (15.1, 6.9)
2.33 m
4.87 d (6.2)
3.54 t (6.2)
5.92 m
5.72 dd (15.3, 6.2)
2.67 dd (16.4, 11.7) 2.52 dd (16.4, 3.3)
6′
7′
8′
1′′
2′′
3-OH
1.45 dq (14.7,7.4)
0.91 t (7.4)
4.16 q (7.1)
1.25 t (7.1)
11.51 (s)
1.64 p (6.4)
3.56 t (6.4)
3.84 m
1.61 m
1.14 d (6.1)
4.05 q (7.1)
1.18 t (7.1)
1.28 t (7.1)
4.04 q (7.1)
1.19 t (7.1)
4.05 q (7.1)
1.18 t (7.1)
c
c
c
-
-
-
a In CDCl3. b In acetone-d6. c Signal not observed in acetone-d6.
Table 2. 13C NMR Data (125 MHz) for Compounds 1–4
δC
position
1a c
,
2b c
,
3b c
,
4b c
,
1
136.5 C
135.3 C
137.6 C
138.2 C
2
116.9 C
119.2 C
119.3 C
114.9 C
3
163.6 C
161.3 C
161.3 C
164.3 C
4
5
102.7 CH
160.6 C
102.5 CH
161.1 C
102.5 CH
161.4 C
103.3 CH
162.0 C
6
7
8
111.6 CH
40.9 CH2
171.1 C
111.8 CH
40.4 CH2
171.2 C
111.8 CH
40.4 CH2
171.2 C
113.2 CH
41.4 CH2
172.6 C
Figure 3. ∆δ value [∆δ (in ppm) ) δS - δR] obtained for (S)-
and (R)-MTPA esters (21a and 21b, respectively) of cytosporone
H (3).
1′
2′
3′
4′
5′
6′
7′
8′
1′′
2′′
195.1 C
195.4 C
195.4 C
191.7 C
128.0 CH
144.7 CH
129.0 CH
146.9 CH
35.3 CH2
21.9 CH2
13.7 CH3
61.4 CH2
14.2 CH3
130.5 CH
143.9 CH
137.3 CH
145.6 CH
32.8 CH2
25.4 CH2
61.6 CH2
61.0 CH2
14.5 CH3
130.6 CH
142.8 CH
131.7 CH
143.8 CH
43.8 CH2
67.2 CH
23.6 CH3
61.1 CH2
14.5 CH3
62.2 CH
43.4 CH
127.8 CH
134.7 CH
31.5 CH2
28.6 CH2
14.1 CH3
61.2 CH2
14.2 CH3
13C NMR spectra of 3 (Tables 1 and 2, respectively) resembled
those of cytosporone F (1) except that in 3 signals due to the 7′-
CH2 are replaced with an oxygenated CH [δH 3.84 m; δC 67.2],
suggesting the presence of a -CH2CH(OH)CH3 moiety in 3.
Reaction of 3 with (R)- and (S)-R-methoxy-R-trifluoromethylphe-
nylacetic (MTP) acid chlorides afforded (S)- and (R)-MTP esters
21a and 21b, respectively (Figure 3). Analysis of ∆δ values
confirmed the R absolute configuration for C-7′ (Figure 3). The
structure of cytosporone H was thus established as 3,5-dihydroxy-
2-(7′R-hydroxy-1′-oxoocta-2′E,4′E-dienyl)benzeneacetic acid ethyl
ester (3). Cytosporone I (4) was determined to have the molecular
formula C18H22O6 by HRFABMS, which was consistent with its
13C NMR data and indicated eight degrees of unsaturation. It had
IR bands at 3417, 1737, and 1624 cm-1, suggesting the presence
a In CDCl3. b In acetone-d6. c Multiplicity from DEPT.
1
of OH, ester, and ketone carbonyl groups. The H and 13C NMR
data (Tables 1 and 2, respectively) showed very close resemblance
to those of cytosporones F-H (1-3). The 13C NMR spectrum of
4 analyzed with the help of the DEPT spectrum revealed the
presence of two methyl, four methylene of which one is oxygenated
(δ 61.2), six methine, and four quaternary carbons in addition to
the two carbonyl carbons. Of the six methines, two each were
aromatic (δC 103.3 and 113.2), olefinic (δC 127.8 and 134.7), and
Figure 2. Selected HMBC correlations for 1.
suggested that 9 may be an artifact originating from 1 by a Michael-
type addition of its phenolic OH to the enone moiety during the
isolation process. This was confirmed by the treatment of 1 with
p-TSA, which resulted in the formation of 9.
1
oxygenated aliphatic (δC 62.2 and 43.4) carbons. The H NMR
Fractionation of the EtOAc extract derived from P. quadriseptata
cultured in PDB made up with tap water involving reversed-phase
chromatography and silica gel preparative TLC afforded cytospor-
ones F-I (1-4), compound 6, monocillin III (7), and monocillin
I (8). Comparison (HPLC, TLC, LR-MS, and 1H NMR) with
samples obtained above allowed the identification of cytosporones
F (1) and G (2) and monocillin I (8). Monocillin III (7) was
identified by comparison of its spectroscopic (LR-MS and 1H NMR)
data with those reported in the literature.14 The molecular formula
of cytosporone H (3) was established as C18H22O6 from its
HRFABMS and 13C NMR data. Its IR absorption bands at 3283,
1712, and 1613 cm-1 suggested the presence of OH, ester, and
ketone carbonyls, respectively. The molecular formula of 3 indicated
spectrum of 4 had two olefinic protons [δH 5.92 (m) and 5.72 (dd,
J ) 15.3 and 6.2 Hz)] and two protons attached to oxygenated
carbons at δH 4.87 (d, J ) 6.2 Hz) and 3.54 (t, J ) 6.2 Hz).
Comparison of the 1H and 13C NMR data of 4 with those of
cytosporone F (1) suggested that in 4 the 2′(3′)-double bond is
replaced with an oxirane ring. The structure of cytosporone G was
thus established as 3,5-dihydroxy-2-(2′,3′-epoxy-1-oxoocta-4′E-
enyl)benzeneacetic acid ethyl ester (4).
Compound 6, isolated as a white, amorphous solid, was
determined to have the molecular formula C18H20O7 by a combina-
tion of HRFABMS and 13C NMR data and indicated nine degrees
of unsaturation. Its IR spectrum with absorption bands at 3400,
1718, and 1637 cm-1 suggested the presence of OH, lactone
carbonyl, and R,ꢀ-unsaturated ketone carbonyl groups. Comparison
1
that it is isomeric with cytosporone G (2). However, the H and

1942 Journal of Natural Products, 2007, Vol. 70, No. 12
Paranagama et al.
Figure 4. Selected HMBC and NOE correlations for 6.
Figure 5. Effect of heavy metal ions on the production of monocillin
I (8) by P. quadriseptata cultured in PDB medium made up in (A)
distilled water, (B) tap water, (C) 0.50 mM CuSO4 in distilled water,
(D) 0.50 mM ZnSO4 in distilled water, (E) 0.125 mM Cd(NO3)2 in
distilled water, and (F) 0.0125 mM K2Cr2O7 in distilled water.
Presented are the mean value and standard deviation (SD) of
triplicate determinations.
1
of the H NMR and 13C NMR data of 6 with those of monocillin
III (7) obtained above indicated that they are structurally related,
and this combined with the presence of an additional oxygen atom
in 6 compared with 7 as evident from their molecular formulas
suggested that 6 may be an oxygenated derivative of monocillin
III. In the 13C NMR spectrum of 6, the chemical shift of C-5′ (δ
70.3) indicated that it is oxygenated, and this was confirmed by
the presence of strong correlations between H-6′ (δ 2.53 and 1.28)
and C-5′ (δ 70.3) and between H-3′ (δ 6.10) and C-5′ (δ 70.3) in
its HMBC spectrum (Figure 4). The gross structure of 6 was
therefore suspected to be 5′-hydroxymonocillin III. The relative
Table 3. Antiproliferative Activities of 5′-Hydroxymonocillin
III (6) and Monocillin III (7) against a Panel of Three Tumor
Cell Linesa
cell lineb
1
stereochemistry of 6 was determined with the help of H NMR
compound
NCI-H460
MCF-7
SF-268
coupling constants and pulse-field gradient 1D NOE experiments
(Figure 4). The large coupling constant (16.2 Hz) observed for H-3′
and H-4′ suggested the E configuration for the 3′(4′)-double bond
as in monocillin III (7).14 The disposition of the oxirane ring was
determined as trans from the pulse-field gradient NOE experiment,
which showed an enhancement of the H signal at δ 2.62 (H-8′)
on irradiation of the signal at δ 1.28 (H-6′R), suggesting that these
protons are on the same side of the ring. Enhancement of the H
6
7
0.30
0.50
0.35
0.01
0.15
0.40
0.60
0.07
0.55
0.30
0.72
0.04
monocillin I (8)
doxorubicin
1
a Results are expressed as IC50 values in µM. b Key: NCI-H460 )
human non small cell lung cancer; MCF-7 ) human breast cancer;
SF-268 ) human CNS cancer (glioma).
1
signal at δ 4.44 (H-5′) on irradiation of the signal at δ 2.53 (H-
6′ꢀ) indicated that H-5′ and H-6′ꢀ have the same orientation,
confirming the structure of 6 as 5′-hydroxymonocillin III. Attempted
preparation of Mosher’s esters of 6 to determine its absolute
stereochemistry failed probably due to its ready dehydration under
the basic conditions used for this reaction.
analysis of these extracts indicated that this organism failed to
produce radicicol (10) in liquid culture (data not shown). Most of
the major metabolites were present in the mycelial extract of C.
chiVersii grown in both tap water and distilled water, and these
were isolated by solvent-solvent partition followed by column
chromatography to afford chaetochromin A (12) as the major
constituent together with eugenetin (13) and 6-methoxymethyleu-
genin (14). Chaetochromin A was identified by comparison of its
1H NMR, 13C NMR, and MS data with those reported,19–21 whereas
12 and 13 were identified by comparison with those obtained
previously from the same organism.9 Chaetochromin A (12) has
previously been reported from Chaetomium gracile19 and C.
Virescens.20 This constitutes the first report of its occurrence in C.
chiVersii.
We have previously reported the antiproliferative and Hsp90
inhibitory activities of monocillin I (8) and radicicol (10).8b
Compounds 1-7 and chaetochromin A (12) encountered in this
study were evaluated for their ability to induce heat shock response
and inhibit proliferation of three cancer cell lines [NCI-H460 (non
small cell lung), MCF-7 (breast), and SF-268 (CNS glioma)].
Monocillin I and/or radicicol were used in these assays as positive
controls to assess the relative activities of the compounds tested.
In the former assay, cells were exposed to serial dilutions of test
compounds for 72 h in RPMI 1640 media supplemented with 10%
fetal bovine serum, and when cell viability was evaluated by the
MTT assay,22 only 5′-hydroxymonocillin III (6) and monocillin
III (7) were found to inhibit proliferation/survival of the cell lines
used; other compounds did not show any detectable inhibition at
concentrations up to 5.0 µM. The concentrations resulting in 50%
inhibition of cell proliferation/survival as measured by the MTT
assay (IC50) were found to range between 0.15 and 0.55 µM. It is
significant that compared with monocillin I (8), 5′-hydroxymono-
cillin III (6) showed selective activity toward the breast cancer
(MCF-7) cell line (Table 3). When compounds 1-7 and chaeto-
chromin A (12) were tested in the heat shock induction assay using
the reporter 3T3-Y9/B12 cells,8b only 5′-hydroxymonocillin III (6)
Intrigued by the dependence of P. quadriseptata on the quality
of the water for the production of its major metabolites, we analyzed
the tap water used in the culture medium for metal ions, and it was
found to contain a significant amount of Cu2+ (0.15 ppm) compared
to other heavy metals (Cd2+ and Cr3+).16 Although the direct effect
of heavy metal ions in the production of secondary metabolites in
microorganisms is hitherto unknown, the influence of Cu2+ on
superoxide dismutase (SOD) activity of Aspergillus niger strain
B-77 has recently been reported.17 It has been suggested that cellular
physiology correlating with the enhancement of SOD activity in
the presence of Cu2+ is indicative of the role this enzyme plays in
the cellular adaptation to environmental (oxidative and temperature)
stress.17 We have recently reported the effect of monocillin I (8),
the major metabolite produced by P. quadriseptata when cultured
in tap water, on the stress-related protein Hsp908b and its ability to
confer thermotolerance to Arabidopsis thaliana.18 It was of interest,
therefore, to investigate the effect of Cu2+, Cd2+, and Cr3+ ions on
the production of 8 in liquid media. P. quadriseptata was thus
cultured in PDB made up in distilled water containing nontoxic
concentrations of these ions. The EtOAc extracts of the supernatants
of these cultures when analyzed by HPLC for the content of
monocillin I (8) clearly indicated that these metal ions had a
significant influence on its production (Figure 5).
C. chiVersii, an endophytic fungal strain known to produce the
Hsp90 inhibitor radicicol (10) as the major metabolite when cultured
in PDA,8b was also investigated for the effect of culture conditions
on the production of 10 and other metabolites. The organism was
cultured for 7 days separately in PDB made up with distilled water
and tap water and filtered, and the resulting culture supernatants
and mycelia were separately extracted with EtOAc. TLC and HPLC

Biosynthetic Potential of Plant-Associated Fungi
Journal of Natural Products, 2007, Vol. 70, No. 12 1943
water. Fifty-two fractions were collected and combined on the basis
of their TLC profiles to yield 11 fractions [A (4.8 mg), B (7.4 mg), C
(4.6 mg), D (3.4 mg), E (29.3 mg), F (12.8 mg), G (2.6 mg), H (1.6
mg), I (14.0 mg), J (1.8 mg), and K (12.0 mg)]. Of these, fraction E
(29.0 mg) was purified by silica gel preparative TLC (CH2Cl2/MeOH,
95:5) to afford 1 (25.4 mg). Fractions A and F were purified separately
by reversed-phase preparative TLC on RP-18 (MeOH/H2O, 75:25) to
obtain 5 (3.0 mg) and 2 (9.8 mg), respectively. The combined fractions
B, C, and D (15.4 mg) on further purification by preparative silica gel
TLC (CH2Cl2/MeOH, 97:3) yielded 8 (3.0 mg) and 9 (11.3 mg). A
portion (380 mg) of the EtOAc extract obtained from the liquid culture
prepared with tap water was subjected to reversed-phase column
chromatography on a column of LRP-2 (12.0 g) using a gradient of
MeOH in water. Seventy-five fractions were collected and combined
on the basis of their TLC profiles to yield four fractions [L (81.0 mg),
M (76.2 mg), N (141.0 mg), and O (55.2 mg)]. Of these, fraction M
was purified by preparative silica gel TLC (CH2Cl2/MeOH, 90:10)
followed by reversed-phase (RP-18) preparative TLC (MeOH/H2O,
60:40) to furnish 2 (2.9 mg), 3 (3.0 mg), 4 (0.9 mg), 6 (3.5 mg), and
7 (1.5 mg). A portion (20.0 mg) of fraction N was purified by
preparative silica gel TLC (Et2O/MeOH, 99:1) to obtain 7 (1.4 mg)
and 8 (6.7 mg). A portion (10.0 mg) of the combined fraction O when
purified by preparative silica gel TLC (CH2Cl2/MeOH, 95:5) afforded
a further quantity of 8 (1.0 mg).
Figure 6. Cell-based heat shock induction assay (HSIA) of
monocillin I (8), 5′-hydroxymonocillin III (6), and monocillin III
(7). Radicicol (10) and DMSO were used as positive and negative
control, respectively. All samples were tested at 1.0 µM concentra-
tion. The mean value and standard deviation (SD) of triplicate
determinations are presented, expressed as a percentage of the
negative control.
Cytosporone F (1): pale brown oil; UV (EtOH) λmax (log ε) 288
and monocillin III (7) showed activity at a concentration less than
5.0 µM. The ability of the monocillin analogues 6 and 7 to induce
heat shock response was found to be slightly less than that observed
for monocillin I (8) and radicicol (10) in this assay (Figure 6).
Further studies are in progress to understand the effect of heavy
metal ions in the production of metabolites by these and other plant-
associated fungal strains.
(7.47), 208 (8.32) nm; IR (KBr) νmax 3369, 2960, 1732, 1724 1614,
1
1593, 1461, 1319, 1267, 1161, 1020, 1002 cm-1; H and 13C NMR
data, see Tables 1 and 2, respectively; HRFABMS m/z 319.1552 [M
+ 1]+ (calcd for C18H23O5, 319.1546).
Cytosporone G (2): pale brown oil; UV (EtOH) λmax (log ε) 287
1
(7.58), 206 (7.44) nm; IR νmax 3429, 1720, 1654, 1029, 856 cm-1; H
and 13C NMR data, see Tables 1 and 2, respectively; HRFABMS m/z
335.1502 [M + 1]+ (calcd for C18H23O6, 335.1495).
25
Cytosporone H (3): yellow oil; [R]D -13.04 (c 0.13, MeOH);
Experimental Section
UV (EtOH) λmax (log ε) 305 (5.55), 298 (5.09), 204 (5.23) nm, IR νmax
3283, 2922, 1712, 1613 cm-1; 1H and 13C NMR data, see Tables 1 and
2, respectively; HRFABMS m/z 335.1497 [M + 1]+ (calcd for
C18H23O6, 335.1495).
General Experimental Procedures. Optical rotations were mea-
sured with a Jasco DIP-370 digital polarimeter using MeOH as solvent.
IR spectra were recorded on a Shimadzu FTIR-8300 spectrometer in
KBr disks, and UV spectra in MeOH on a Shimadzu UV-1601
spectrometer. Analytical HPLC was performed on a Hitachi instrument
equipped with an L-6200A intelligent pump, an L-4500 diode array
detector, and a P-6000 interface utilizing Hitachi model D-7000
chromatography data station software using a Kromasil 5 µm C-18
column (4.6 mm × 250 mm); injections were made with an AS-4000
intelligent autosampler, and the mobile phase consisted of a gradient
of MeOH (60–100%) in water at a flow rate of 0.4 mL min-1. 1D and
2D NMR spectra were recorded in CDCl3, acetone-d6, and pyridine-d5
using residual solvents as internal standards on a Bruker DRX-500
instrument at 500 MHz for 1H NMR and 125 MHz for 13C NMR. The
chemical shift values (δ) are given in parts per million (ppm), and the
coupling constants are in Hz. LR-MS and HR-MS were recorded on
Shimadzu LCMS QP8000R and JEOL HX110A spectrometers,
respectively.
Cultivation, Extraction, and Isolation of Metabolites of P.
quadriseptata. The rhizosphere fungus P. quadriseptata isolated from
the Christmas cactus, Opuntia leptocaulis DC. (Cactaceae), as described
previously,8a was grown on PDA for 14 days and used for inoculation
of the liquid cultures. Mycelia were scraped out and mixed with sterile
PDB (150 mL) and filtered through a 100 µm filter to separate spores
from mycelia. The optical density of the filtrate containing fungal spores
was adjusted to a value between 0.5 and 0.3 with sterile PDB, and
this spore suspension (50 mL) was used to inoculate two Erlenmeyer
flasks (2.0 L), each containing 1.0 L of PDB prepared with distilled
water and PDB prepared with tap water. Flasks were shaken on a rotary
shaker at 28 °C and 160 rpm. On day 14, mycelia of each culture were
separated from the supernatant by filtration using Whatman No. 1 filter
paper. The mycelia were dried by vacuum filtration, frozen at -80 °C,
and lyophilized for 24 h. The supernatants from each experiment were
neutralized to pH 7 and extracted separately with EtOAc (8 × 500
mL), and the EtOAc extracts were evaporated under reduced pressure
to afford dark brown semisolids (208 and 388 mg, respectively, from
culture media prepared with distilled water and tap water). A portion
(120 mg) of the EtOAC extract obtained from the liquid culture prepared
with distilled water was subjected to reversed-phase column chroma-
tography on a column of LRP-2 (4.0 g) using a gradient of MeOH in
Cytosporone I (4): colorless, amorphous solid; UV (EtOH) λmax
(log ε) 309 (5.39), 277 (4.33), 217 (4.22), 201 (5.29) nm; IR νmax 3417,
1
2922, 1737, 1624, 1577, 1542, 1463, 1425 cm-1; H and 13C NMR
data, see Tables 1 and 2, respectively; HRFABMS m/z 335.1509 [M
+ 1]+ (calcd for C18H23O6, 335.1495).
Quadriseptin A (5): colorless, amorphous solid; UV (EtOH) λmax
(log ε) 322 (6.60), 258 (6.50), 215 (6.42) nm; IR νmax 3571, 1770,
1705, 1670, 829 cm-1; 1H NMR (500 MHz, acetone-d6) δ 9.43 (1H, s,
OH), 8.00 (1H, d, J ) 9.7 Hz, H-4), 6.80 (1H, d, J ) 2.2 Hz, H-6),
6.67 (1H, d, J ) 2.2 Hz, H-8), 6.17 (1H, d, J ) 9.7, H-3), 4.11 (2H,
q, J ) 7.1 Hz, H-1′′), 3.91 (2H, s, H-1′), 1.19 (3H, t, J ) 7.1 Hz,
H-2′′); 13C NMR (125 MHz, acetone-d6) δ 170.8 (C, C-2′), 161.3 (C,
C-2), 160.7 (C, C-7), 157.5 (C, C-9), 141.8 (CH, C-4), 135.7 (C, C-5),
115.9 (CH, C-6), 112.5 (CH, C-3), 111.9 (C, C-10), 102.6 (CH, C-8),
61.5 (CH2, C-1′′), 38.2 (CH2, C-1′), 14.4 (CH3, C-2′′); HRFABMS m/z
249.0773 [M + 1]+ (calcd for C13H13O5, 249.0763).
25
5′-Hydroxymonocillin III (6): white, amorphous solid; [R]D
+40.98 (c 0.18, MeOH); UV (EtOH) λmax (log ε) 304 (5.40), 264 (4.84),
216 (4.75), 198 (5.25) nm; IR νmax 3400, 1718, 1637, 1579 cm-1; H
1
NMR (500 MHz, acetone-d6) δ 11.93 (1H, s, OH), 6.82 (1H, dd, J )
16.2 and 8.0 Hz, H-4′), 6.32 (2H, s, H-3 and H-5), 6.10 (1H, d, J )
16.2 Hz, H-3′), 5.25 (1H, m, H-10′), 4.72 (1H, d, J ) 17.6 Hz, H-1′a),
4.44 (1H, m, H-5′), 3.75 (1H, d, J ) 17.6 Hz, H-1′b), 2.89 (1H, m,
H-7′), 2.62 (1H, dt, J ) 10.0 and 2.9 Hz, H-8′), 2.53 (1H, dt, J ) 12.5
and 3.9 Hz, H-6′ꢀ), 2.05 (1H, m, H-9′a), 1.72 (1H, dt, J ) 15.9 and
4.9 Hz, H-9′b), 1.43 (3H, d, J ) 6.4 Hz, H-11′), 1.28 (1H, m, H-6′R);
13C NMR (125 MHz, acetone-d6) δ 197.2 (C, C-2′), 172.0 (C, C-1′′),
167.0 (C, C-4), 163.3 (C, C-6), 150.2 (CH, C-4′), 141.0 (C, C-2), 129.2
(CH, C-3′), 113.7 (CH, C-3), 105.8 (C, C-1), 102.8 (CH, C-5), 72.4
(CH, C-10′), 70.3 (CH, C-5′), 55.9 (CH, C-7′), 54.6 (CH, C-8′), 48.4
(CH2, C-1′), 41.4 (CH2, C-6′), 36.9 (CH2, C-9′), 18.1 (CH3, C-11′);
HRFABMS m/z 349.1285 [M + 1]+ (calcd for C18H21O7 349.1287).
Methylation of Cytosporone F. Methyl iodide (0.5 mL) and K2CO3
(10.0 mg) were added to a stirred solution of 1 (10.0 mg) in acetone
(0.5 mL) at 0 °C. After 5 min at 0 °C, the ice bath was removed and
the reaction mixture was stirred at room temperature until the starting
material disappeared (TLC control). It was then filtered, the solvent

1944 Journal of Natural Products, 2007, Vol. 70, No. 12
Paranagama et al.
was removed under reduced pressure, and the crude product was purified
by preparative TLC (silica gel) using CH2Cl2/MeOH (95:5) as eluant
to give 3,5-di-O-methylcytosporone F (8.6 mg).
under reduced pressure to afford cytosporone C (3.0 mg). Its 1H NMR
and MS data were consistent with those reported in the literature.10
Conversion of Cytosporone F (1) to Aposphaerin B (9).
p-Toluenesulfonic acid (0.1 mg) was added to a stirred solution of 1
(2.0 mg) in dry toluene (0.5 mL), and the reaction mixture was heated
at 65 °C for 2 h (TLC control). Toluene was removed under reduced
pressure, and the crude mixture was dissolved in CH2Cl2/MeOH
(90:10). The solution was passed through a short bed of silica gel using
CH2Cl2/MeOH (9:1) (10 mL). The solvents were evaporated under
3,5-Di-O-methylcytosporone F (16): pale brown oil; UV (EtOH)
max (log ε) 279 (5.68), 213 (5.57) nm; IR νmax 2925, 1735, 1602, 1460,
λ
1157 cm-1; 1H NMR (500 MHz, CDCl3) δ 6.94 (1H, dd, J ) 15.4 and
10.8 Hz, H-3′), 6.41 (1H, d, J ) 2.2 Hz, H-6), 6.38 (1H, d, J ) 2.2
Hz, H-4), 6.35 (1H, d, J ) 15.4 Hz, H-2′), 6.21 (1H, dd, J ) 15.1 and
10.8 Hz, H-4′), 6.12 (1H, dt, J ) 15.1 and 6.9 Hz, H-5′), 4.06 (2H, q,
J ) 7.1 Hz, H-1′′), 3.80 (3H, s, OCH3), 3.74 (3H, s, OCH3), 3.55 (2H,
s, H-7), 2.12 (2H, m, H-6′), 1.41 (2H, dq, J ) 14.8 and 7.4 Hz, H-7′),
1.18 (3H, t, J ) 7.1 Hz, H-2′′), 0.89 (3H, t, J ) 7.4 Hz, H-8′); 13C
NMR (125 MHz, CDCl3) δ 196.2 (C, C-1′), 171.1 (C, C-8), 161.2 (C,
C-3), 158.6 (C, C-5), 145.4 (CH, C-5′), 143.4 (CH, C-3′), 134.6 (C,
C-1), 130.3 (CH, C-2′), 129.3 (CH, C-4′), 113.9 (C, C-2), 107.3 (CH,
C-6), 97.6 (CH, C-4), 60.8 (CH2, C-1′′), 55.8 (CH3, OCH3), 55.4 (CH3,
OCH3), 38.7 (CH2, C-7), 35.2 (CH2, C-6′), 21.9 (CH2, C-7′), 14.2 (CH3,
C-2′′), 13.6 (C, C-8′); HRFABMS m/z 347.1860 [M + 1]+ (calcd for
C20H27O5, 347.1858).
1
reduced pressure to afford aphosphaerin B (2.0 mg). Its H NMR and
MS data were consistent with those reported in the literature.13
Monocillin III (7): white solid; 1H NMR, 13C NMR, and MS data
were consistent with those reported in the literature.14
Preparation of the (R)- and (S)-MTPA Ester Derivatives of
Cytosporone H (3) by a Convenient Mosher Ester Procedure.15
Compound 3 (0.5 mg) was transferred into a clean NMR tube and was
dried under vacuum. Pyridine-d5 (0.6 mL) and (R)-(-)-R-methoxy-R-
(trifluoromethyl)phenylacetyl chloride (5 µL) were added into the NMR
tube immediately under a stream of dry N2, and then the NMR tube
was shaken carefully to mix the sample and MTPA chloride evenly.
The reaction mixture in the NMR tube was allowed to stand at room
temperature, and the 1H NMR was recorded after 6 h. 1H NMR data of
the (S)-MTPA ester derivative (21a) of 3 (500 MHz, pyridine-d5): δ
6.459 (1H, d, J ) 15.1 Hz, H-2′), 6.316 (1H, m, H-4′), 6.127 (1H, m,
H-5′), 5.277 (1H, m, H-7′), 4.040 (2H, q, J ) 7.1 Hz, H-1′′), 3.905
(2H, s, H-7), 2.450 (2H, m, H-6′), 1.178 (3H, d, J ) 6.2 Hz, H-8′),
1.051 (3H, t, J ) 7.1 Hz, H-2′′). In the manner described for 21a,
another portion of 3 (0.5 mg) was reacted in a second NMR tube with
(S)-(-)-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride (5 µL) at
room temperature for 6 h using pyridine-d5 (0.5 mL) as solvent to afford
Catalytic Hydrogenation of 3,5-Di-O-methylcytosporone F (16).
A solution of 16 (8.0 mg) in EtOH (0.5 mL) containing 10% Pd on
carbon (1 mg) was stirred in an atmosphere of H2 for 3 h (TLC control).
The solution was filtered through a plug of cotton, and the solvent was
evaporated under reduced pressure. The crude product was purified by
preparative TLC (silica gel) using CH2Cl2/i-PrOH (99:1) as eluant to
give 3,5-di-O-methylcytosporone B (17) (5.3 mg).
3,5-Di-O-methylcytosporone B (17): colorless oil; UV (EtOH) λmax
(log ε) 283 (6.00), 226 (5.97) nm; IR νmax 2927, 1735, 1681, 1317,
1
1155 cm-1; H NMR (500 MHz, CDCl3) δ 6.37 (1H, d, J ) 2.3 Hz,
H-6), 6.36 (1H, d, J ) 2.3 Hz, H-4), 4.12 (2H, q, J ) 7.1 Hz, H-1′′),
3.79 (3H, s, OCH3), 3.78 (3H, s, OCH3), 3.59 (2H, s, H-7), 2.80 (2H,
t, J ) 7.4 Hz, H-2′), 1.62 (2H, m, H-3′), 1.26 (8H, m, H-4′-H-7′),
1.23 (3H, t, J ) 7.1 Hz, H-2′′), 0.86 (3H, t, J ) 7.4 Hz, H-8′); 13C
NMR (125 MHz, CDCl3) δ 206.9 (C, C-1′), 171.2 (C, C-8), 161.2 (C,
C-3), 158.7 (C, C-5), 134.4 (C, C-1), 124.3 (C, C-2), 107.7 (CH, C-6),
97.5 (CH, C-4), 60.9 (CH2, C-1′′), 55.6 (CH3, OCH3), 55.4 (CH3,
OCH3), 44.5 (CH2, C-2′), 38.9 (CH2, C-7), 31.6 (CH2, C-6′), 29.3 (CH2,
C-4′), 29.1 (CH2, C-5′), 24.1 (CH2, C-3′), 22.6 (CH2, C-7′), 14.2 (CH3,
C-2′′), 14.1 (CH3, C-8′); HRFABMS m/z 351.2186 [M + 1]+ (calcd
for C20H31O5, 351.2171).
1
the (R)-MTPA ester derivative (21b) of 3. H NMR data of 21b (500
MHz, pyridine-d5): δ 6.426 (1H, d, J ) 15.1 Hz, H-2′), 6.204 (1H, m,
H-4′), 5.917 (1H, m, H-5′), 5.288 (1H, m, H-7′), 4.042 (2H, q, J ) 7.1
Hz, H-1′′), 3.905 (2H, s, H-7), 2.386 (2H, m, H-6′), 1.255 (3H, d, J )
6.2 Hz, H-8′), 1.053 (3H, t, J ) 7.1 Hz, H-2′′).
Effect of Metal Ions on the Production of Monocillin I (8). PDB
(50 mL) was prepared with distilled water into each 250 mL conical
flask, and appropriate weights of CuSO4, ZnSO4, Cd(NO3)2, and
K2Cr2O7, were added into each flask separately to obtain the final
concentrations of 0.5 mM CuSO4, 0.5 mM ZnSO4, 0.125 mM Cd(NO3)2,
and 0.0125 mM K2Cr2O7. The spore solutions (1 mL) prepared as above
were added into each flask and shaken on a rotary shaker at 160 rpm
and 28 °C. The experiment with each metal ion concentration was
carried out in triplicate. After two weeks the supernatant was extracted
into EtOAc (3 × 50 mL) and concentrated to dryness. Each EtOAc
extract was analyzed using reversed-phase HPLC, and the percentage
of monocillin I produced by each culture was estimated.
Cultivation, Extraction, and Isolation of Metabolites of Chaeto-
mium chiWersii. A culture of C. chiVersii grown on PDA for one week
was used for inoculation. Mycelia were scraped out and mixed with
sterile PDB (150 mL) and filtered through a 100 µm filter to separate
spores from the mycelia. Absorbance of the spore solution was
measured and adjusted to between 0.5 and 0.3. This spore solution (50
mL) was used to inoculate 2 L Erlenmeyer flasks containing 1.0 L of
PDB prepared with filtered water. Flasks were shaken on a rotary shaker
at 28 °C and 160 rpm for one week. On day 7, mycelia were separated
from the supernatant by filtering through Whatman No. 1 filter paper.
The supernatant was neutralized to pH 7 and extracted with EtOAc (8
× 500 mL). The lyophilized mycelia (20.0 g) were extracted with
EtOAc (8 × 500 mL) and the EtOAc extracts evaporated under reduced
pressure to afford a dark green semisolid (2.1 g). A portion (1.8 g) of
this extract was partitioned between hexane and 80% aqueous MeOH.
Evaporation of solvents under reduced pressure yielded hexane (1.02
g) and 80% aqueous MeOH (0.77 g) fractions. The 80% aqueous MeOH
fraction was diluted with water to 50% aqueous MeOH and extracted
with CHCl3. Evaporation of CHCl3 under reduced pressure yielded a
brown semisolid (0.71 g), which was subjected to column chromatog-
raphy on a column of LiChroprep DIOL (22 g, 25–40 µm) made up in
hexane (100 mL) and eluted with hexane and mixtures of hexane/
CH2Cl2 of increasing polarity, CH2Cl2, mixtures of CH2Cl2/MeOH of
increasing polarity, and finally with MeOH. A total of 55 fractions
(7.5 mL each) were collected, and fractions having similar TLC
behavior were combined to give 17 subfractions. Of these, the
subfraction 12 afforded chaetochromin A (12) (157.9 mg), the major
Reduction of 3,5-Di-O-methylcytosporone F (17) with NaBH4.
NaBH4 (5 mg) was added to a stirred solution of 17 (5.0 mg) in
anhydrous MeOH (0.5 mL) at 0 °C. After 5 min, the ice bath was
removed and the reaction mixture was stirred at room temperature until
the starting material disappeared (TLC control). It was then filtered,
the solvent was removed under reduced pressure, and the crude product
was purified by preparative TLC (silica gel) using CH2Cl2/i-PrOH
(99:1) as eluant to give 3,5-dimethoxy-2-(1′-hydroxyoctyl)phenethyl
alcohol (19) (3.9 mg) as a colorless oil: UV (EtOH) λmax (log ε) 283
(5.64), 227 (5.55), 214 (5.52) nm; IR νmax 3427, 2925, 1602, 1460,
1147 cm-1; 1H NMR (500 MHz, CDCl3) δ 6.37 (1H, d, J ) 2.4, H-6),
6.32 (1H, d, J ) 2.4 Hz, H-4), 4.83 (1H, dd, J ) 8.6 and 5.5 Hz,
H-1′), 3.83 (2H, t, J ) 6.4 Hz, H-8), 3.82 (3H, s, OCH3), 3.72 (3H, s,
OCH3), 2.89 (2H, t, J ) 6.4 Hz, H-7), 1.95 (1H, m, H-2′a), 1.67 (1H,
m, H-2′b), 1.26 (10H, m, H-3′-H-7′), 0.85 (3H, t, J ) 7.4 Hz, H-8′);
13C NMR (125 MHz, CDCl3) δ 159.2 (C, C-3), 158.9 (C, C-5), 137.6
(C, C-1), 123.5 (C, C-2), 106.9 (CH, C-6), 97.7 (CH, C-4), 70.5 (CH2,
C-8), 63.5 (CH, C-1′), 55.4 (CH3, OCH3), 55.3 (CH3, OCH3), 37.8 (CH2,
C-7), 36.8 (CH2, C-2′), 31.9 (CH2, C-6′), 29.6 (CH2, C-4′), 29.3 (CH2,
C-5′), 26.3 (CH2, C-3′), 22.7 (CH2, C-7′), 14.1 (CH3, C-8′); HRFABMS
m/z 293.2125 [MH - H2O]+ (calcd for C18H29O3, 293.2117).
Conversion of Cytosporone F (1) to Cytosporone B (18).
A
solution of 1 (3.0 mg) in EtOH (0.5 mL) containing Pd on carbon (10%,
1 mg) was stirred in an atmosphere of H2 for 2 h (TLC control). The
solution was filtered through a plug of cotton, and the solvent was
evaporated under reduced pressure to afford cytosporone B (18) as a
white solid (3.0 mg). Its 1H NMR, 13C NMR, and MS data were
consistent with those reported in the literature.10
Conversion of Cytosporone F (1) to Cytosporone C (20).
A
solution of 1 (3.0 mg) in 0.2 N NaOH (0.5 mL) was stirred with NaBH4
(3.0 mg) at room temperature for 1 h (TLC control). The reaction
mixture was acidified by adding an appropriate amount of 2 N HCl at
0 °C. After 5 min the ice bath was removed and the aqueous solution
was extracted with EtOAc (2 × 10 mL). The solvent was evaporated

Biosynthetic Potential of Plant-Associated Fungi
Journal of Natural Products, 2007, Vol. 70, No. 12 1945
H. D.; Whitesell, L.; Bolzani, V.; Da, S.; Gunatilaka, A. A. L. J.
Antibiot. 2004, 57, 541–546. (b) Turbyville, T. J.; Wijeratne, E. M. K.;
Liu, M. X.; Burns, A. M.; Seliga, C. J.; Luevano, L. A.; David, C. L.;
Feath, S. H.; Whitesell, L.; Gunatilaka, A. A. L. J. Nat. Prod. 2006,
69, 178–184.
constituent of this extract. Subfractions 5 and 6 were purified by
preparative silica gel TLC with CH2Cl2/i-PrOH (99:1) and CH2Cl2/
MeOH (95:5) separately to give eugenetin (13) (20.2 mg) and
6-methoxymethyleugenin (14) (5.2 mg), respectively.
1
Chaetochromin A (12): yellow solid; MS and H and 13C NMR
(9) Wijeratne, E. M. K.; Paranagama, P. A.; Gunatilaka, A. A. L.
data were consistent with those reported in the literature.21
Cytotoxicity and Heat Shock Inhibition Assays. The tetrazolium-
based cytotoxicity and heat shock induction assays were carried out as
described previously.8b
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Acknowledgment. Financial support for this work was provided by
Grant R01 CA 90265 funded by the National Cancer Institute. P.A.P.
thanks the Council for International Exchange of Scholars for a
Fulbright Fellowship. We thank Drs. H. D. VanEtten, L. S. Pierson,
and E. E. Pierson (University of Arizona) and Dr. S. H. Faeth (Arizona
State University) for providing fungal strains used in this study, Ms.
A. Ray-Maitra (Environmental Research Laboratory, Department of
Soil Water and Environmental Science, University of Arizona) for
providing analytical data for the tap water sample used in this study,
and Ms. A. M. Burns and Ms. M. X. Liu for their assistance with
biological assays.
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optical emission spectrometry (ICP-OES; Perkin Elmer model 5300DV)
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NP070504B