Geopyxins A-E, ent -Kaurane diterpenoids from endolichenic fungal strains geopyxis aff. majalis and Geopyxis sp. AZ0066: Structure-activity relationships of geopyxins and their analogues(1)

Article abstract of DOI:10.1021/np200769q

Four new ent-kaurane diterpenoids, geopyxins A-D (1-4), were isolated from Geopyxis aff. majalis, a fungus occurring in the lichen Pseudevernia intensa, whereas Geopyxis sp. AZ0066 inhabiting the same host afforded two new ent-kaurane diterpenoids, geopyxins E and F (5 and 6), together with 1 and 3. The structures of 1-6 were established on the basis of their spectroscopic data, while the absolute configurations were assigned using modified Mosher's ester method. Methylation of 1-3, 5, and 6 gave their corresponding methyl esters 7-11. On acetylation, 1 and 7 yielded their corresponding monoacetates 12 and 14 and diacetates 13 and 15. All compounds were evaluated for their cytotoxic and heat-shock induction activities. Compounds 2, 7-10, 12, 14, and 15 showed cytotoxic activity in the low micromolar range against all five cancer cell lines tested, but only compounds 7-9, 14, and 15 were found to activate the heat-shock response at similar concentrations. From a preliminary structure-activity perspective, the electrophilic α,β-unsaturated ketone carbonyl motif present in all compounds except 6 and 11 was found to be necessary but not sufficient for both cytotoxicity and heat-shock activation.

Full text of DOI:10.1021/np200769q

Article
pubs.acs.org/jnp
Geopyxins A−E, ent-Kaurane Diterpenoids from Endolichenic Fungal
Strains Geopyxis aff. majalis and Geopyxis sp. AZ0066: Structure−
Activity Relationships of Geopyxins and Their Analogues1
E. M. Kithsiri Wijeratne,† Bharat P. Bashyal,† Manping X. Liu,† Danilo D. Rocha,†
G. M. Kamal B. Gunaherath,† Jana M. U’Ren,‡ Malkanthi K. Gunatilaka,‡ A. Elizabeth Arnold,‡
Luke Whitesell,§ and A. A. Leslie Gunatilaka*,†
†SW Center for Natural Products Research and Commercialization, School of Natural Resources and the Environment, College of
Agriculture and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706, United States
‡Division of Plant Pathology and Microbiology, School of Plant Sciences, College of Agriculture and Life Sciences, University of
Arizona, Tucson, Arizona 85721, United States
§Whitehead Institute, 9 Cambridge Center, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Four new ent-kaurane diterpenoids, geopyxins
A−D (1−4), were isolated from Geopyxis aff. majalis, a fungus
occurring in the lichen Pseudevernia intensa, whereas Geopyxis
sp. AZ0066 inhabiting the same host afforded two new ent-
kaurane diterpenoids, geopyxins E and F (5 and 6), together
with 1 and 3. The structures of 1−6 were established on the
basis of their spectroscopic data, while the absolute
configurations were assigned using modified Mosher’s ester
method. Methylation of 1−3, 5, and 6 gave their
corresponding methyl esters 7−11. On acetylation, 1 and 7 yielded their corresponding monoacetates 12 and 14 and
diacetates 13 and 15. All compounds were evaluated for their cytotoxic and heat-shock induction activities. Compounds 2, 7−10,
12, 14, and 15 showed cytotoxic activity in the low micromolar range against all five cancer cell lines tested, but only compounds
7−9, 14, and 15 were found to activate the heat-shock response at similar concentrations. From a preliminary structure−activity
perspective, the electrophilic α,β-unsaturated ketone carbonyl motif present in all compounds except 6 and 11 was found to be
necessary but not sufficient for both cytotoxicity and heat-shock activation.
ecent studies have demonstrated that endolichenic fungi
are rich sources of structurally diverse small-molecule
ent-kaurane diterpenes geopyxins A−D (1−4) from G. aff.
majalis and geopyxins E (5) and F (6) together with geopyxins
A (1) and C (3) from Geopyxis sp. AZ0066. This constitutes
the first report of metabolites from a fungal strain of the genus
Geopyxis. The cytotoxic and heat-shock-inducing activities of
geopyxin B (2) prompted us to prepare several analogues of the
geopyxins (1−3, 5, and 6), which were available to us in
sufficient quantities, and evaluate these in a limited structure−
activity relationship study. The outcome of this study supports
our previous findings that diverse species from plants to fungi
elaborate structurally complex secondary metabolites capable of
acting on the stress responses of heterologous organisms.6,7
From a practical drug discovery perspective, it further validates
our heat-shock-guided approach to identifying natural products
and their analogues that can target the malignant phenotype by
altering protein homeostasis.
R
natural products, some with interesting biological activities.2 In
our continuing search for bioactive and/or novel metabolites of
plant- and lichen-associated fungi of the Sonoran Desert
bioregion,3 we have been particularly interested in the
identification of compounds that activate the heat-shock
response as a novel mode of anticancer activity.4 Unlike most
normal tissues, cancers must endure high levels of stress from
the profound alterations in protein homeostasis caused by
malignant transformation. Powerful innate adaptive mecha-
nisms, especially the ancient, highly conserved transcriptional
heat-shock response, are activated in cancers to enable survival
under these conditions.5 Natural products that further tax the
heat-shock response may overwhelm the ability to cope and
could provide leads for the development of new, broadly
effective anticancer drugs.6,7
Here we report the investigation of two endolichenic fungal
strains, Geopyxis aff. majalis and Geopyxis sp. AZ0066,
inhabiting the live thallus of the intense dark and light lichen
Pseudevernia intensa (Nyl.) Hale & W. L. Culb. (Parmeliaceae).
This work led to the isolation and characterization of the new
Special Issue: Special Issue in Honor of Gordon M. Cragg
Received: September 23, 2011
Published: January 20, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Although ent-kauranes of fungal origin are rare and reported
mainly from Gibberella fujikuroi and Phaeosphaeria sp. L487,8
this class of diterpenoids has frequently been encountered in
several plant families,9a and a recent report has suggested that
in important crop plants such as rice and maize some ent-
kauranes function as phytoalexins with potent antifungal and
antifeedant activities.9b In addition, ent-kauranes are of
considerable interest because of their cytotoxic,10−13 anti-
inflammatory,14−16 antiangiogenic,17 anti-HIV,18 antioxidant,19
anticholinesterase,19 SIRT1 (silent information regulator two
ortholog 1) inhibitory,20 antibacterial,21 NF-κB activation
inhibitory,22 and apoptosis-inducing23 activities.
RESULTS AND DISCUSSION
■
Geopyxin A (1), obtained as a white, amorphous solid, analyzed
for C20H28O5 by a combination of HRESIMS and 13C NMR
spectroscopy. The UV λmax at 238 nm indicated the presence of
an α,β-unsaturated ketone carbonyl chromophore.24 Its IR
spectrum had absorption bands at 3489, 1706, and 1641 cm−1,
suggesting the presence of OH, carboxylic acid carbonyl, and
1
α,β-unsaturated ketone carbonyl functionalities. The H and
13C NMR spectra together with HSQC data of 1 (Table 1)
showed the presence of two tertiary methyls (δH 1.22 s, 1.12 s
and δC 28.5, 11.5), seven methylenes, of which one is olefinic
(δH 5.96 s, 5.30 s; δC 115.6), five methines, of which two are
oxygenated [δH 3.84 d (J = 2.8 Hz, H-7), 3.50 dd (J = 11.4, 4.9
Hz, H-1); δC 72.1, 81.8] and one is allylic [δH 3.06 d (J = 2.3
Hz); δC 38.3], six quaternary carbons, of which one is olefinic
(δC 149.5) and two are carbonyls (δC 214.9 and 183.6), and a
set of aliphatic methylene and methine protons. Because an
exocyclic methylene and two carbonyls accounted for three
degrees of unsaturation, 1 should possess a tetracyclic
framework. The presence of three spin systems, −CH(O)-
CH2CH2−, −CHCH2CH(O)−, and −CHCH2CH2CHCH2−,
Figure 1. COSY and selected HMBC correlations for 1.
1
in 1 was evident from its H−1H COSY spectrum (Figure 1).
The connectivity of the above three spin systems, tertiary
methyls, and nonprotonated carbons was established by the
analysis of the HMBC correlations (Figure 1) to constitute an
ent-kaurane skeleton. The tertiary methyl group at δ 1.12
showed HMBC correlations with the oxygenated carbon at δ
81.8 (C-1) and methine carbons at δ 45.0 (C-5) and 47.3 (C-
9), placing it at C-10. The remaining tertiary methyl group at δ
1.22 assigned to CH3-1825 showed HMBC correlations with C-
3 (δ 35.4), C-5 (δ 45.0), and the carboxylic acid carbonyl
carbon (δ 183.6), placing methyl and carboxylic acid groups at
C-4. The oxymethine proton at δ 3.50 showed HMBC
correlations with C-5, C-9 (δ 47.3), and C-20 (δ 11.5), placing
it at C-1. The remaining oxymethine proton at δ 3.84 showed
HMBC correlations with C-5, C-9, C-14 (δ 35.3), and the
carbonyl carbon C-15 (δ 214.9), placing it at C-7. Methylation
(CH3I/K2CO3/acetone) of 1 gave its methyl ester (7), further
confirming the presence of a carboxylic acid group, while
acetylation (Ac2O/pyridine) of 1 yielded its diacetate (13),
suggesting the presence of two OH groups. The chemical shift
of the carbonyl carbon (C-15; δ 214.9)26 and its upfield shift to
δ 206.5 as a result of acetylation suggested that the OH-7 has a
β-orientation.27 This was further confirmed by the application
of modified Mosher’s ester method as described below. The
relative stereochemistry of 1 was determined with the help of
1H NMR and NOEDIFF data (Figure 2) of its methyl ester
(7). The coupling pattern of the H-1 (dd, J = 11.4, 5.3 Hz)
suggested it to have an axial orientation.28 Irradiation of this
Figure 2. Selected NOEs observed in the NOEDIFF experiment of 7.
proton led to significant enhancements of H-5 and H-9 signals,
suggesting that these were also on the same side of the
molecule and hence axially oriented. The β-orientation of the
CH3 group on C-4 was inferred from its 13C chemical shift (δ
28.5),29 which suggested that the carboxylic acid group is α-
oriented; the 13C chemical shift value for the α-CH3 group at
C-4 has been reported to be at δ 16.7.30 The NOE data for 7
(Figure 2) confirmed that both CH3-20 and H-14 at δ 2.22
have an α-orientation. The absolute stereochemistry of
positions C-1 and C-7 of geopyxin A (1) were determined by
the application of modified Mosher’s ester method.31 Reaction
of 1 with (R)- and (S)-α-methoxy-α-trifluoromethylphenylace-
tyl chloride (MTPA-Cl) afforded (S)- and (R)-bis-MTPA
esters 1a and 1b, respectively (Figure 3). Analysis of Δδ (δS −
δR) values confirmed the S absolute stereochemistry for both C-
1 and C-7. Thus, the structure of geopyxin A was established as
(1S,7S)-1,7-dihydroxy-15-oxo-ent-kaur-16-en-19-oic acid (1).
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with 3. The 1H NMR coupling pattern (brs) of the oxymethine
proton at δ 3.88 suggested the presence of a 7β-OH group (see
above) in 3. Thus, geopyxin C was identified as (7S)-7-
hydroxy-1,15-dioxo-ent-kaur-16-en-19-oic acid (3).
Geopyxin D (4), obtained as a white, amorphous powder,
analyzed for C20H28O5 by a combination of HRESIMS and 13
C
NMR spectroscopy. The H and 13C NMR spectroscopic data
of 4 (Table 1) closely resembled those of 2. The only difference
was the presence of an oxygenated methine group [δH 4.10 d (J
= 4.2 Hz); δC 66.1] in 4 instead of a methylene group in 2,
suggesting that 4 is an oxygenated derivative of 2. The
difference in molecular formulas between these compounds
confirmed that 4 contains an additional oxygen atom. The
oxymethine proton at δ 4.12 showed HMBC correlations with
C-13 (δC 36.9) and C-8 (δC 51.1) (Supporting Information,
S1a) placing this additional OH group at C-11. Irradiation of
CH3-20 of 4 during the NOEDIFF experiment caused
enhancement of H-11 (δ 4.10), H-14 (δ 2.27), and H-6 (δ
2.04), suggesting that they were α-oriented and hence 11-OH
should be β-oriented (Supporting Information, S1b). Because
compounds 1 and 4 presumably share a common biosynthetic
origin, it is proposed that 4 should have the same configuration
at C-7 as 1. On the basis of this and the above observed NOE
correlations the absolute configuration at C-11 of 4 was
determined as S. Thus, geopyxin D was identified as (7S,11S)-
7,11-dihydroxy-15-oxo-ent-kaur-16-en-19-oic acid (4).
Repeated chromatographic separation of the EtOAc extract
of Geopyxis sp. AZ0066 over silica gel resulted in the isolation
of two new compounds, geopyxins E and F (5 and 6), together
with geopyxins A (1) and C (3). The molecular formula,
C20H28O6, was assigned to geopyxin E (5) on the basis of its
HRESIMS and NMR data and indicated the presence of one
more oxygen atom than geopyxin A (1). Comparison of 1H and
13C NMR data of 5 (Table 1) with those of 1 showed them to
be similar with some minor differences. The significant
difference was the presence of an oxygenated methine group
(δC 68.7) in 5 replacing a methylene (δC 30.2) group in 1. This
was placed at C-2, due to the presence of the spin system
−CH(O)CH(O)CH2− identified from its 1H−1H COSY
spectrum. In the NOEDIFF experiment (Supporting Informa-
tion, S2), irradiation of H-1 (δ 3.06) exhibited enhancement of
H-2, indicating both protons were β-oriented and hence the
OH group at C-2 should be α. Application of modified
Mosher’s ester method31 for the determination of absolute
stereochemistry at C-1 and C-2 of geopyxin E was not
attempted, as related 1,2-bis-MTPA esters are reported to show
interactions in ΔδS−R effects among the modification sites due
to their close proximity.32 However, biosynthetically it could
share a common origin with geopyxin A (1). This together with
the NOE correlations observed above led to the identification
of geopyxin E as (1R,2S,7S)-1,2,7-trihydroxy-15-oxo-ent-kaur-
16-en-19-oic acid (5).
1
Figure 3. Δδ values [(Δδ in ppm)= δS − δR ] obtained for (S)- and
(R)-bis MTPA esters (1a and 1b, respectively) of geopyxin A (1).
Geopyxin B (2), obtained as a white, amorphous solid,
analyzed for C20H28O4 by a combination of HRESIMS and
1
NMR data. The H and 13C NMR data of 2 (Table 1) were
similar to those of 1, and the molecular weight difference of 16
Da between these suggested that 1 was an oxygenated analogue
of 2. The major difference in NMR data was found to be the
absence of the oxymethine group at δC 81.8, which was
assigned to C-1 in 1; instead 2 showed the presence of a
methylene group (δC 39.8), which exhibited an HMBC
correlation to CH3-20 (δ 1.02). This was further confirmed
by the upfield chemical shift of the two carbons (C-2 and C-10)
adjacent to C-1 in 2 when compared with those of 1 (Table 1).
The planar structure of 2 was thus determined to be 7-hydroxy-
15-oxo-ent-kaur-16-en-19-oic acid. The absolute stereochemis-
try of C-7 was determined by the application of modified
Mosher’s ester method.31 Reaction of geopyxin B with (R)- and
(S)-MTPA-Cl afforded (S)- and (R)-MTPA esters 2a and 2b,
respectively. Analysis of Δδ (δS − δR) values (Figure 4)
Figure 4. Δδ values [(Δδ in ppm) = δS−δR] obtained for (S)- and (R)-
MTPA esters (2a and 2b, respectively) of geopyxin B (2).
confirmed the S absolute stereochemistry for C-7. On the basis
of the above data, the structure of geopyxin B was established as
(7S)-7-hydroxy-15-oxo-ent-kaur-16-en-19-oic acid (2).
Geopyxin F (6), obtained as a white, amorphous powder,
analyzed for C20H30O6 by a combination of HRESIMS and 13
C
1
NMR data. The H and 13C NMR data of 6 (Table 1) were
similar to those of 1, except for the absence of the exo-cyclic
double bond in 6. It showed the presence of an oxygenated
methylene group, suggesting that it contained a −CHCH2OH
group instead of a −CCH2 group in 1. In the HMBC
spectrum, oxygenated methylene protons of 6 showed
correlations with the ketone carbonyl at δ 229.1 (C−15) and
methine carbons at δ 32.6 (C-13) and 56.0 (C-16), confirming
the above observation (Supporting Information, S3). The
Geopyxin C (3), obtained as a white, amorphous powder,
analyzed for C20H26O5 by a combination of HRESIMS and
1
NMR data. The H and 13C NMR data of 3 (Table 1) were
very similar to those of 1 except for the presence of an
additional ketone carbonyl (δC 213.6) in 3 instead of an
oxymethine group [δH 3.50 dd (J = 11.4, 4.9 Hz); δC 81.8] in 1.
Oxidation of 1 with pyridinium chlorochromate afforded a
monoketo compound as the only product, which was identical
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a
Table 2. Cytotoxic Activity of Compounds 2, 7−10, 12, 14, and 15 against a Panel of Five Cancer Cell Lines
b
cell line
compound
NCI-H460
SF-268
MCF-7
PC-3M
MDA-MB-231
2
2.25 0.15
1.08 0.15
0.92 0.01
2.28 0.10
2.27 0.17
3.52 0.25
0.82 0.05
1.26 0.05
0.42 0.30
2.35 0.31
0.72 0.00
0.69 0.06
1.56 0.16
1.02 0.03
2.37 0.14
0.66 0.02
1.18 0.07
0.62 0.07
4.32 1.55
0.79 0.04
0.63 0.12
1.67 0.45
2.08 0.20
3.48 0.20
0.53 0.14
1.18 0.07
0.42 0.13
5.41 0.91
2.27 0.07
1.48 0.11
3.63 0.09
3.99 0.34
3.88 0.21
1.54 0.04
2.31 0.37
0.24 0.09
3.31 0.44
0.73 0.14
0.91 0.10
3.38 0.37
3.81 0.48
4.11 0.15
1.15 0.07
1.93 0.10
0.58 0.23
7
8
9
10
12
14
15
doxorubicin
a
Results are expressed as IC50 values standard deviation in μM. Doxorubicin and DMSO were used as positive and negative controls. Compounds
b
1, 3−6, 11, and 13 were found to be inactive at 5.0 μM. Key: NCI-H460 = human non-small-cell lung cancer; SF-268 = human CNS cancer
(glioma); MCF-7 = human breast cancer; PC-3M = metastatic human prostate adenocarcinoma; MDA-MB-231= human breast adenocarcinoma.
treatment of 6 with Ac2O/pyridine gave the diacetate 13, which
was previously obtained by the acetylation of 1 (see above),
suggesting that it was formed as a result of dehydration and
acetylation of 6. The upfield shift of both C-12 (δC 25.5) and
the oxygenated C-17 (δC 59.1) due to a γ-steric compression
effect between 12β-H and 16-CH2OH as observed before for
related ent-kauranes bearing a 16-CH2OH33 suggested the β-
orientation of the hydroxymethylene group at C-16 of 6.
Although it is possible that 6 was formed from 1 as a result of
the addition of a molecule of water during the workup, the
absence of the C-16 epimer and the previous reports33,34 of the
natural occurrence of related ent-kauranes bearing 16α-CH2OH
suggested 6 to be a genuine natural product. Since acetylation
of both geopyxin A (1) and geopyxin F (6) afforded the same
product and 1 was determined to have an S configuration at C-
1 and C-7, geopyxin F should also have the same absolute
configuration at these two positions. Thus, geopyxin F was
identified as (1S,7S)-1,7,17-trihydroxy-15-oxo-ent-kauran-19-oic
acid (6).
Compounds 1−15 were evaluated for in vitro inhibition of
cell proliferation/survival using a panel of five cancer cell lines,
NCI-H460 (human nonsmall cell lung cancer), SF-268 (human
CNS glioma), MCF-7 (human breast cancer), PC-3 M (human
metastatic prostate adenocarcinoma), and MDA-MB-231
(human metastatic breast adenocarcinoma). Cells were exposed
to serial dilutions of test compounds for 72 h in RPMI-1640
media supplemented with 10% fetal bovine serum, and relative
viable cell number was measured by standard dye reduction
assay using resazurin (AlamarBlue).35 Among the natural ent-
kauranes, only geopyxin B (2) was found to be cytotoxic (Table
2 and Supporting Information S4). In contrast, all methyl ester
analogues except 11 showed cytotoxic activity at low micro-
molar concentrations, and among these 1-O-acetylmethylgeo-
pyxin A (14) was the most active. However, no evidence for
significant cell-type selectivity was observed. Compounds 1−15
were also evaluated for heat-shock-inducing activity over a
range of concentrations using a heat-shock reporter cell line.4,6
As demonstrated for a concentration of 2.5 μM, the natural ent-
kaurane geopyxin B (2) showed weak activity, whereas its
methyl ester 8 and 1-O-acetylmethylgeopyxin A (14) induced
the greatest response (Figure 5). Heat-shock activation
occurred in the same concentration range at which these
compounds demonstrated cytotoxic activity against human
cancer lines, but not all cytotoxic compounds induced a heat-
shock response. This structure−activity relationship is con-
sistent with our previous finding that heat-shock activation is
Figure 5. Cell-based heat-shock induction assay. The materials tested
were DMSO (negative control), monocillin I (MON, positive
control), and compounds 1−15. All samples were tested at 2.5 μM
except for MON, which was tested at 0.5 μM. The mean and standard
deviation of triplicate determinations are presented, expressed as a
percentage of the negative control; results are representative of three
independent experiments.
not a general consequence of toxicity.6 For example, conven-
tional cytotoxic chemotherapeutics such as doxorubicin and
cisplatin do not induce the response.36 Instead, activation of the
heat-shock response represents a specific response to
disturbances of protein homeostasis within the cell. Consistent
with this concept, we found that the α,β-unsaturated ketone
carbonyl motif present in all compounds except 6 and 11 was
necessary but not sufficient for both tumor cell growth
inhibition and heat-shock activation. This electrophilic moiety
possesses strong potential for Michael addition of the reactive
cysteine residues exposed on many key regulatory proteins in
cells. Rather than forming adducts with proteins indiscrimin-
ately, however, it is likely that these compounds selectively add
nucleophilic residues, which are poised to react by virtue of
local electronic and structural constraints as well as the
particular pH maintained in specific intracellular compart-
ments.37 Some of these proteins are perhaps the fundamental
electrophile sensors in cells that are among the first-line
defenses for launching adaptive transcriptional and post-
transcriptional responses.38,39
CONCLUSIONS
■
Here and in our previous studies we have found that the α,β-
unsaturated ketone carbonyl moiety in these ent-kauranes and
other small-molecule natural products is necessary, but not
sufficient, for their biological activities. A number of
compounds with this moiety were completely inactive in our
assays. A plausible explanation may be that these molecules
contain a structurally complex targeting moiety that confers
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described species of Pyronemataceae (Ascomycota) chosen on the
basis of highest-affinity BLASTn matches to the GenBank non-
redundant (nr) database in NCBI and recent phylogenetic analyses
within the family.44,45 Taxon sampling included seven strains of
Geopyxis spp. corresponding to all available sequences in GenBank for
the relevant loci (partial LSU rDNA), as well as nine representative
genera within the family. Phylogenetic analyses using maximum
likelihood were conducted in GARLI46 using the GTR+I+G model of
evolution as determined by ModelTest,47 followed by 1000 bootstrap
replicates to assess support for the resulting topology. The analysis
placed AZ0484 and AZ0066 with strong support within Geopyxis (data
not shown). The alignment then was trimmed to exclude (1) all
redundant sequences of Geopyxis and (2) all terminal taxa placed
outside of Geopyxis, except for three strains of Trichophaea and one of
Pulvinula, used as outgroups or to clarify the boundaries of Geopyxis in
a second analysis. Analysis of 38 strains as described above placed
AZ0484 with strong support as sister to Geopyxis majalis; thus it was
determined here as Geopyxis aff. majalis AZ0484. AZ0066 was placed
with strong support within Geopyxis, albeit with unknown affinity at
the species level. Thus it was determined here as Geopyxis sp. AZ0066
(Supporting Information, S5).
Cytotoxicity Assay. Relative cell growth and survival were
measured in 96-well microplate format using the fluorescent detection
of resazurin (AlamarBlue) dye reduction as an end point. Compounds
were tested against human non-small-cell lung cancer (NCI-H460),
CNS glioma (SF-268), breast cancer (MCF-7), metastatic prostate
adenocarcinoma (PC-3M), and human metastatic breast adenocarci-
noma (MDA-MB-231). Serial dilutions of test compounds or vehicle
control (DMSO) were added to triplicate wells. After 72 h incubation,
dye solution was added to each well (1:10 dilution). After brief
agitation, incubation was continued for 4 h at 37 °C before data
acquisition using a microplate fluorometer (Ex/Em: 560/590). Mean
fluorescence intensity per well as a measure of relative viable cell
number in compound-treated wells was compared to that of DMSO-
treated wells. The conventional chemotherapeutic drug doxorubicin
served as a positive control.
Heat-Shock Induction Assay (HSIA). Mouse fibroblasts stably
transduced with a reporter construct encoding enhanced green
fluorescent protein under the transcriptional control of a minimal
consensus heat-shock element were used to measure the heat-shock-
inducing activity of extracts and pure compounds as previously
described.25 Reporter cells were seeded into flat-bottom 96-well plates
(Falcon; Becton Dickinson, Lincoln Park, NJ) at a density of 20 000
cells/well and allowed to adhere overnight (one column of wells was
left empty to serve as a blank control). The following day, serial 2-fold
dilutions of pure compounds or a single concentration of extracts and
fractions was added to triplicate wells. DMSO vehicle alone (volume
not to exceed 0.1%) served as a negative control. Cells were incubated
overnight, the medium was removed, and wells were rinsed once with
PBS, followed by addition of 150 μL of PBS to each well. Fluorescence
was determined on an Analyst AD (LJL Biosystems) plate reader
equipped with filter sets for excitation at 485 nm and emission at 525
nm.
Cultivation and Isolation of Metabolites of Geopyxis aff.
majalis. A seed culture of G. aff. majalis (AZ0484) grown on PDA for
two weeks was used for inoculation. Mycelia were scraped out, mixed
with sterile water, and filtered through a 100 μm filter to separate
spores from the mycelia. Absorbance of the spore solution was
measured (at 600 nm) and adjusted to between 0.3 and 0.5. This spore
solution was used to inoculate 4 × 2.0 L Erlenmeyer flasks, each
holding 1.0 L of the medium (PDB) containing 0.25 mM CuSO4 and
incubated at 160 rpm and 28 °C. The glucose level in the medium was
monitored using glucose strips (URISCAN glucose strips). On day 66,
the strip gave a green color for the glucose test, indicating the absence
of glucose in the medium. Mycelia were then separated by filtration,
and the filtrate was extracted with EtOAc (3 × 2 L). The combined
EtOAc layer was washed with H2O, dried over anhydrous Na2SO4, and
evaporated under reduced pressure to give crude EtOAc extract (1.05
g), which showed activity in HSIA. A portion (938.0 mg) of this
extract was partitioned between hexane and 80% aqueous MeOH. The
target selectivity and a more generic effector motif that confers
thiol reactivity via Michael addition chemistry. We have seen
similar structure−activity scenarios for several other heat-shock-
active natural products encountered during our screening
efforts.6 The chemical reactivity of such heat-shock-inducing
compounds makes elucidating their mechanisms of action
challenging. While targets for some thiol-reactive molecules
have been proposed, it is still unclear if these are the most
biologically meaningful interactions. At high concentrations of
compound, targeting may be swamped, and more promiscuous
adduct formation can occur, leading to less relevant effects.40,41
These natural products have been honed by nature over an
evolutionary time scale to modulate the biology of diverse
species by engaging the heat-shock response and perhaps other
transcriptional responses that act system-wide to impact their
survival. In this regard, they could prove very useful in probing
the mechanisms responsible for maintaining protein homeo-
stasis in the cell and perhaps serve as leads for the development
of broadly effective anticancer drugs with novel modes of
action.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured in CHCl3 with a JASCO Dip-370 digital polarimeter. UV
spectra were recorded on a Shimadzu UV-1601 UV−vis spectropho-
tometer. IR spectra were recorded on a Shimadzu FTIR-8300
spectrometer using KBr disks. 1D and 2D NMR spectra were
recorded in CDCl3 with a Bruker Avance III 400 spectrometer at 400
MHz for 1H NMR and 100 MHz for 13C NMR using residual CHCl3
as an internal reference. Low-resolution and high-resolution MS were
recorded on Shimadzu LCMS-QP8000α and JEOL HX110A
spectrometers, respectively. Analytical and preparative thin layer
chromatography (TLC) were performed on precoated 0.25 mm
thick plates of silica gel 60 F254; spraying with a solution of
anisaldehyde in EtOH followed by heating was used to visualize
compounds on analytical TLC. Preparative HPLC was performed on a
Waters Delta Prep 4000 preparative chromatography system equipped
with a Waters 996 photodiode array detector and a Waters Prep LC
controller utilizing Empower Pro software and using a RP column
(Phenomenex Luna 5 μm, C18, 100 Å, 250 × 10 mm) with a flow rate
of 3.0 mL/min; chromatograms were acquired at 254 and 270 nm.
Fungal Isolation and Identification. In June 2007, a healthy
thallus of Pseudevernia intensa was collected from the lower branches of
a mature individual of Pseudotsuga menziesii in a mixed coniferous
forest ca. 9.5 km northwest of the Southwestern Research Station in
the eastern Chiricahua Mountains of southeastern Arizona, USA (2100
m.a.s.l.; 31°53′00″ N, 109°12′18″ W).42 The thallus was cut into 2 × 2
cm segments, washed in tap water, surface-sterilized by agitating
sequentially in 95% EtOH for 30 s, 0.5% NaOCl for 2 min, and 70%
EtOH for 2 min following previous procedures,42 and then surface-
dried under sterile conditions before cutting into 2 × 2 mm pieces.
Pieces were plated on 2% malt extract agar (MEA) in a Petri plate. The
plate was sealed with Parafilm and incubated under ambient light/dark
conditions at room temperature (ca. 21.5 °C). Two of the emergent
fungi were isolated into pure culture on 2% MEA, vouchered in sterile
water, and deposited as living vouchers at the Robert L. Gilbertson
Mycological Herbarium at the University of Arizona under accession
nos. ARIZ0484 and ARIZ0066 (AZ0484, AZ0066). Total genomic
DNA was isolated from fresh mycelium of each strain,42 and the
nuclear ribosomal internal transcribed spacers and 5.8s gene (ITS
rDNA; ca. 600 bp) and an adjacent portion of the nuclear ribosomal
large subunit (LSU rDNA; ca. 500 bp) were amplified as a single
fragment by PCR. Positive amplicons were sequenced bidirectionally
as described previously.42
Sequence data for these strains were aligned manually in Mesquite43
with a core alignment containing 148 closely related endophytic,
endolichenic, and saprotrophic fungi from the same site42 and 19
366
dx.doi.org/10.1021/np200769q | J. Nat. Prod. 2012, 75, 361−369

Journal of Natural Products
Article
HSIA-active 80% aqueous MeOH fraction was diluted to 50% aqueous
MeOH by addition of water and extracted with CHCl3. The CHCl3
fraction (795.0 mg) was then subjected to gel permeation
chromatography over a column of Sephadex LH-20 (30 g) made up
in hexanes/CH2Cl2 (1:4) and eluted with hexanes/CH2Cl2 (1:4, 500
mL), CH2Cl2/acetone (3:2, 400 mL), CH2Cl2/acetone (1:4, 400 mL),
CH2Cl2/MeOH (1:1, 100 mL), and finally MeOH (100 mL). Of the
78 fractions (20 mL each) collected, those having similar TLC patterns
were combined to give 18 major fractions [A (20.4 mg), B (28.6 mg),
C (16.3 mg), D (12.4 mg), E (14.7 mg), F (15.4 mg), G (10.7 mg), H
(47.5 mg), I (23.5 mg), J (20.9 mg), K (20.9 mg), L (135.7 mg), M
(217.6 mg), N (24.9 mg), O (56.6 mg), P (21.1 mg), Q (15.9 mg), R
(96.1 mg), and S (42.1 mg)]. Fraction M (100.0 mg) was
chromatographed over a column of silica gel (4.0 g) made up in
hexanes/EtOAc (4:1) and eluted with hexanes containing increasing
amounts of EtOAc followed by EtOAc. The fractions eluted with
hexanes/EtOAc (7:3) were combined to give 1 (55.2 mg). Fraction L
(135.0 mg) was further fractionated on a column of silica gel (5.0 g)
by elution with hexanes/Et2O (1:1), hexanes/Et2O (3:7), and Et2O to
give 55 fractions (6 mL each), and those having similar TLC patterns
were combined to give 12 subfractions (F1−F12). Fraction F2 (35.9
mg) was rechromatographed over a column of silica gel (1.0 g) made
up in CH2Cl2/MeOH (100:2) and eluted with CH2Cl2/MeOH
(100:2) followed by CH2Cl2/MeOH (100:4) to give seven
subfractions (F2A−F2G). Subfractions F2B−F2D were combined (20.5
mg) and purified by RP-HPLC using 60% aqueous CH3CN containing
0.25% HCOOH to give 2 (13.2 mg, tR = 24 min). Subfraction F4 on
evaporation gave an additional quantity of 1 (29.9 mg). Further
separation of subfraction F5, (9.5 mg) by RP-HPLC using 65%
aqueous CH3CN containing 0.25% HCOOH gave 1 (1.5 mg, tR = 9
min) and 4 (1.7 mg, tR = 14 min). Fraction O was chromatographed
over a column of silica gel (3.5 g) made up in CH2Cl2/MeOH (100:2)
and eluted with CH2Cl2/MeOH (100:2) and CH2Cl2/MeOH (100:4)
followed by MeOH to afford 40 (6 mL) fractions, and those having
similar TLC patterns were combined to give 11 subfractions (F1−F11).
Of these, F2 and F3 were combined (36.7 mg) and subjected to RP-
HPLC using 50% aqueous CH3CN containing 0.25% HCOOH to give
3 (21.2 mg, tR = 17.5 min).
Mycelia were then separated by filtration, and the filtrate was extracted
with EtOAc (3 × 1.2 L). The combined EtOAc layer was washed with
H2O, dried over anhydrous Na2SO4, and evaporated under reduced
pressure to give a crude EtOAc extract (2.42 g), which showed activity
in HSIA. A portion (2.10 g) of this extract was chromatographed over
a column of silica gel (50.0 g) made up in hexanes/EtOAc (7:3) and
eluted with hexanes/EtOAc (7:3), hexanes/EtOAc (6:4), EtOAc, and
EtOAc/MeOH (99:1) to afford 30 (150 mL) fractions (F1−F30).
Fractions F6−F17 (1.30 g) were found to contain geopyxin A (1) as the
major compound (>95%). Chromatographic separation of fraction F5
gave geopyxin C (3, 12.0 mg). Fraction F23 (220.0 mg) was further
fractionated by column chromatography over silica gel (10.0 g) made
up in CH2Cl2 and eluted with CH2Cl2 containing increasing amounts
of MeOH to give 120 (8 mL) fractions, and those having similar TLC
patterns were combined to give 14 subfractions (A−N). Subfraction E
(30.0 mg) was purified by column chromatography over silica gel (1.0
g) using CH2Cl2/MeOH (98:2) as eluant to give 5 (25.9 mg).
Fraction, F24 (195.0 mg) was subjected to column chromatography
over silica gel (4.0 g) made up in CH2Cl2 and eluted with CH2Cl2
containing increasing amounts of MeOH to give 6 (161.9 mg).
Geopyxin E (5): white, amorphous solid; [α]25 − 93.5 (c 0.13,
D
CH3OH); UV (EtOH) λmax (log ε) 239 (2.84) nm; IR (KBr) νmax
3323, 2933, 2868, 1703, 1641, 1473, 1444, 1406, 1259, 1201, 1076,
1049, 983 945, 905, 733 cm−1; for 1H and 13C NMR data, see Table 1;
HRESIMS m/z 363.1812 [M − H]− (calcd for C20H27O6, 363.1813).
Geopyxin F (6): white, amorphous solid; [α]25 −63.3(c 0.10,
D
CH3OH); IR (KBr) νmax 3423, 2941, 2872, 1709, 1641, 1445, 1412,
1
1250, 1213, 1161, 1070, 1022, 984, 735 cm−1; for H and 13C NMR
data, see Table 1; HRESIMS m/z 365.1967 [M − H]− (calcd for
C20H29O6, 365.1970).
Preparation of Methyl Ester Derivatives. To a solution of 1
(10.0 mg) in acetone (0.5 mL) were added K2CO3 (20.0 mg) and
CH3I (0.2 mL), and the mixture was stirred at 25 °C. After 1 h the
reaction mixture was filtered, and the filtrate was evaporated under
reduced pressure to give methylgeopyxin A (7, 10.3 mg). Four other
methyl ester derivatives, methylgeopyxin B (8), methylgeopyxin C (9),
methylgeopyxin E (10), and methylgeopyxin F (11), were prepared
from 2, 3, 5, and 6, respectively, in the same way as described above
for the preparation of 7.
Geopyxin A (1): white, amorphous solid; [α]25 −79.9 (c 0.11,
D
Methylgeopyxin A (7): white, amorphous solid; IR (KBr) νmax 3489,
3442, 2945, 2874, 1726, 1711, 1641, 1465, 1423, 1257, 1238, 1190,
1149, 1080, 1033, 975 cm−1; 1H NMR data, see Supporting
Information S6; 13C NMR data, see Supporting Information S7;
APCIMS (−) mode m/z 361 [M − H]−.
CH3OH); UV (EtOH) λmax (log ε) 238 (3.52) nm; IR (KBr) νmax
3489, 3157, 2939, 1755, 1706, 1641, 1473, 1442, 1409, 1263, 1244,
1199, 1157, 1078, 1037, 987, 941 cm−1; for 1H and 13C NMR data, see
Table 1; HRESIMS m/z 347.1862 [M − H ]− (calcd for C20H27O5,
347.1864).
1
Geopyxin B (2): white, amorphous solid; [α]25 −118.8 (c 0.11,
Methylgeopyxin B (8): white, amorphous solid; H NMR data, see
D
Supporting Information S6; 13C NMR data, see Supporting
CH3OH); UV (EtOH) λmax (log ε) 237 (3.07) nm; IR (KBr) νmax
3467, 2931, 2864, 1708, 1641, 1462, 1441, 1263, 1238, 1164, 1078,
1045, 980, 941 cm−1; for 1H and 13C NMR data, see Table 1;
HRESIMS m/z 331.1913 [M − H ]− (calcd for C20H27O4, 331.1915).
Information S7; APCIMS (−) mode m/z 345 [M − H]−.
1
Methylgeopyxin C (9): white, amorphous solid; H NMR data, see
Supporting Information S6; 13C NMR data, see Supporting
Information S7; APCIMS (+) mode m/z 343 [M + H − H2O]+.
Methylgeopyxin E (10): white, amorphous solid; 1H NMR data, see
Supporting Information S6; 13C NMR data, see Supporting
Information S7; APCIMS (+) mode m/z 379 [M + H]+, 361 [M +
H − H2O]+, 343 [M + H − 2H2O]+, 325 [M + H − 3H2O]+.
Methylgeopyxin F (11): white, amorphous solid; 1H NMR data, see
Supporting Information S6; 13C NMR data, see Supporting
Information S7; APCIMS (+) mode m/z 363 [M + H − H2O]+,
345 [M + H − 2H2O]+, 327 [M + H − 3H2O]+.
Geopyxin C (3): white, amorphous solid; [α]25 −112.7 (c 0.18,
D
CH3OH); UV (EtOH) λmax (log ε) 238 (2.94) nm; IR (KBr) νmax
3363, 2929, 2866, 1726, 1703, 1639, 1471, 1443, 1410, 1280, 1281,
1045, 982 cm−1; for 1H and 13C NMR data, see Table 1; HRESIMS m/
z 347.1857 [M + H]+ (calcd for C20H27O5, 347.1853).
Geopyxin D (4): white, amorphous solid; [α]25 −49.2 (c 0.31,
D
CH3OH); UV (EtOH) λmax (log ε) 236 (3.13) nm; IR (KBr) νmax
3462, 2938, 1756, 1705, 1639, 1474, 1443, 1410, 1283, 1281, 1045,
1
982 cm−1; for H and 13C NMR data, see Table 1; HRESIMS m/z
347.1864 [M − H]− (calcd for C20H27O5, 347.1864).
Acetylation of Geopyxin A. To a solution of 1 (30.0 mg) in
pyridine (0.5 mL) was added Ac2O (0.5 mL), and the mixture was
stirred at 25 °C. After 48 h pyridine and excess Ac2O were evaporated
under reduced pressure and by adding EtOH, and the residue was
chromatographed over a column of silica gel (1.0 g) made up in
CH2Cl2 and eluted with CH2Cl2 containing increasing amounts of
MeOH to give 12 (22.3 mg) and 13 (7.8 mg).
Cultivation and Isolation of Metabolites of Geopyxis sp.
AZ0066. A seed culture of Geopyxis sp. AZ0066 grown on PDA for
two weeks was used for inoculation. Mycelia were scraped out, mixed
with sterile water, and filtered through a 100 μm filter to separate
spores from the mycelia. Absorbance of the spore solution was
measured (at 600 nm) and adjusted to between 0.3 and 0.5. This spore
solution was used to inoculate 2 × 2.0 L Erlenmeyer flasks, each
holding 1 L of the medium (PBD) and incubated at 160 rpm and 28
°C. The glucose level in the medium was monitored using glucose
strips (URISCAN), and after 7 months, the strip gave a green color for
the glucose test, indicating the absence of glucose in the medium.
1-O-Acetylgeopyxin A (12): white, amorphous solid; IR (KBr) νmax
3467, 2955, 2878, 1726, 1701, 1643, 1445, 1416, 1247, 1240, 1215,
1
1151, 1149, 1088, 1024, 984 cm−1; H NMR data, see Supporting
Information S8; 13C NMR data, see Supporting Information S9;
APCIMS (−) mode m/z 389 [M − H]−.
367
dx.doi.org/10.1021/np200769q | J. Nat. Prod. 2012, 75, 361−369

Journal of Natural Products
Article
1
1H NMR data of 2b (400 MHz, pyridine-d5): δ 6.083 (1H, s, H-17a),
5.322 (1H, s, H-17b), 3.814 (1H, dd, J = 3.6, 2.0 Hz, H-7), 2.956 (1H,
m, H-13), 2.167 (2H, dd, J = 12.8, 1.8 Hz, H-3a), 1.858 (1H, d, J =
12.0 Hz, H-14a), 1.790 (3H, m, H-2a, H-6a, and H-9), 1.650 (1H, m,
H-12a), 1.623 (1H, m, H-1a), 1.500 (1H, m, H-12b), 1.483 (1H, m,
H-11a), 1.451 (1H, m, H-2b), 1.369 (1H, m, H-6b), 1.316 (1H, m, H-
14b), 1.315 (1H, m, H-11b), 1.218 (3H, s, H3-18), 1.058 (1H, m, H-
3b), 0.745 (1H, td, 13.2, 4.0 Hz, 1b), 0.679 (3H, s, H3-20).
1,7-O-Diacetylgeopyxin A (13): white, amorphous solid; H NMR
data, see Supporting Information S8; 13C NMR data, see Supporting
Information S9; APCIMS (−) mode m/z 431 [M − H]−.
Oxidation of Geopyxin A. To a solution of 1 (10.0 mg) in dry
CH2Cl2 (0.5 mL) at 0 °C was added pyridinium chlorochromate (30.0
mg), and the mixture was stirred at 0 °C. After 3 h the reaction
mixture was chromatographed over a column of silica gel (200 mg)
made up in CH2Cl2 and eluted with 2% MeOH in CH2Cl2 to give 3
(6.2 mg).
Acetylation of Methylgeopyxin A. To a solution of 7 (10.0 mg)
in pyridine (0.5 mL) was added Ac2O (0.2 mL), and the mixture was
stirred at 25 °C. After 48 h pyridine and excess Ac2O were evaporated
under reduced pressure and by adding EtOH, and the residue was
separated by preparative TLC (silica gel) using 8% MeOH in CH2Cl2
as eluant to give 14 (6.1 mg) and 15 (3.8 mg).
ASSOCIATED CONTENT
■
S
* Supporting Information
Selected HMBC correlations of compounds 4 and 6. Selected
NOEs observed in the NOEDIFF experiments of compounds 4
and 5. Cytotoxic activity of compounds 1−15 against a panel of
five cancer cell lines. Majority-rule consensus tree indicating
placement of strains AZ0484 and AZ0066 within Pyronema-
taceae (Pezizomycetes). Tables giving 1H and 13C NMR
1-O-Acetylmethylgeopyxin A (14): white, amorphous solid; 1H
NMR data, see Supporting Information S8; 13C NMR data, see
Supporting Information S9; APCIMS (+) mode m/z 405 [M + H]+.
1,7-O-Diacetylmethylgeopyxin A (15): white, amorphous solid; 1H
NMR data, see Supporting Information S8; 13C NMR data, see
Supporting Information S9; APCIMS (+) mode m/z 387 [M + H −
CH3COOH]+.
spectroscopic data for compounds 7−15. H and 13C NMR,
1
1H−1H COSY, HSQC, and HMBC spectra of compounds 1, 4,
1
1
and 5; H and 13C NMR, H −1H COSY, and HMBC spectra
of compound 2; 1H and 13C NMR spectra of compounds 3 and
7−15. 1H and 13C NMR, HSQC, and HMBC spectra of
compound 6. This material is available free of charge via the
Internet at http://pubs.acs.org.
Preparation of the (S)- and (R)-MTPA Ester Derivatives of
Geopyxin A (1) by a Convenient Mosher Ester Procedure.
Geopyxin A (1, 1.0 mg) was transferred into a clean NMR tube and
was dried completely under the vacuum of an oil pump. Deuterated
pyridine (0.5 mL) and (R)-(+)-α-methoxy-α-trifluoromethylphenyla-
cetyl chloride (MTPA-Cl) (4.0 μL) were added into the NMR tube
immediately under a stream of N2, and then the NMR tube was shaken
carefully to mix the sample and MTPA chloride evenly. The reaction
in the NMR tube was monitored immediately by 1H NMR and left at
25 °C for 14 h to give the bis-(S)-MTPA ester derivative (1a) of 1. 1H
NMR data of 1a (400 MHz, pyridine-d5; data were assigned on the
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 520-621-9932. Fax: 520-621-8378. E-mail: leslieg@cals.
arizona.edu.
1
basis of the correlations of the H−1H COSY, HSQC, and HMBC
ACKNOWLEDGMENTS
spectra): δ 6.035 (1H, s, H-17a), 5.771 (1H, dd, J = 9.6, 5.2 Hz, H-7),
5.341 (1H, d, J = 0.8 Hz, H-17b), 4.407 (1H, t, J = 2.8 Hz, H-1), 2.852
(1H, dd, J = 8.8, 4.4 HZ, H-13), 2.442 (1H, dd, J = 9.6, 5.2 Hz, H-6a),
2.293 (1 h, dd, J = 12.8, 6.8 Hz, H-5), 2.126 (1H, m, H-6b), 2.080
(2H, J = 12.0, H-12a, H-14a), 1.928 (1H, dd, J = 13.6, 4.0 Hz, H-9),
1.866 (1H, dd, J = 12.0, 4.8 Hz, H-14b), 1.815 (2H, m, H2-2), 1.584
(1H, m, H-11a), 1.528 (1H, m, H-3a), 1.479 (1H, m, H-3b), 1.358
(1H, m, H-12b), 1.321 (1H, m, H-11b), 1.030 (3H, s, H3-18), 1.013
(3H, s, H3-20). In the same manner described for 1a another portion
of 1 (1.0 mg) was reacted in a second NMR tube with (S)-(−) MTPA-
Cl (4.0 μL) at 25 °C for 14 h using d5-pyridine (0.5 mL) as solvent, to
■
Financial support for this work from the National Cancer
Institute (grant R01 CA90265) and National Institute of
General Medical Sciences (grant P41 GM094060) are gratefully
acknowledged. We also thank the National Science Foundation
for supporting collection and identification of fungal strains
(grant DEB-064099 to A.E.A.), the NSF IGERT Program in
Genomics at The University of Arizona for a graduate
fellowship (J.M.U.), and the College of Agriculture and Life
Sciences at the University of Arizona for institutional support of
E.M.K.W. and M.K.G., and T. Bolton and L. West for their
assistance in preparing large-scale cultures.
1
afford the bis-(R)-MTPA ester derivative (1b). H NMR data of 1b
(400 MHz, pyridine-d5): δ 5.970 (1H, s, H-17a), 5.273 (1H, s, H-17b),
5.669 (1H, dd, J = 10.0, 5.6 Hz, H-7), 4.421 (1H, t, J = 2.8 Hz, H-1),
2.778 (1H, dd, J = 9.2, 4.8 Hz, H-13), 2.520 (1H, dd, J = 12.8, 9.6 Hz,
H-6a), 2.338 (1H, dd, J = 12.8, 6.8 Hz, H-5), 2.249 (1H, dd, J = 12.8,
5.6 Hz, H-6b), 2.060 (1H, m, H-12a), 2.000 (1H, d, J = 12.0 Hz, H-
14a), 1.919 (1H, dd, J = 13.6, 4.0 Hz, H-9), 1.828 (2H, m, H2-2),
1.660 (1H, dd, J = 12.0, 4.8 Hz, H-14b), 1.551 (1H, m, H-11a), 1.526
(1H, m, H-3a), 1.455 (1H, m, H-3b), 1.341 (1H, m, H-12b), 1.286
(1H, m, H-11b), 1.074 (3H, s, H3-18), 1.010 (3H, s, H3-20).
DEDICATION
■
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, Frederick, Mary-
land, for his pioneering work on the development of natural
product anticancer agents.
Preparation of the (S)- and (R)-MTPA Ester Derivatives of
Geopyxin B (2) by a Convenient Mosher Ester Procedure. In
the same manner described for 1a and 1b, portions (1.0 mg each) of
geopyxin B were reacted in two NMR tubes with (R)-(+)-MTPA-Cl
(4 μL) and (S)-(−)-MTPA-Cl (4.0 μL) at 25 °C for 14 h using d5-
pyridine (0.5 mL) as solvent, to afford (S)-MTPA and (R)-MTPA
REFERENCES
■
(1) Studies on Arid Lands Plants and Microorganisms, Part 23. For
Part 22, see ref 3.
(2) Paranagama, P. A.; Wijeratne, E. M. K.; Burns, A. M.; Marron, M.
T.; Gunatilaka, M. K.; Arnold, A. E; Gunatilaka, A. A. L. J. Nat. Prod.
2007, 70, 1700−1705. Ding, G.; Li, Y.; Fu, S.; Liu, S.; Wei, J.; Che, Y. J.
Nat. Prod. 2009, 72, 182−186. Zhang, F.; Liu, S.; Lu, X.; Guo, L.;
Zhang, H.; Che, Y. J. Nat. Prod. 2009, 72, 1782−1785. Wang, Y.;
Zheng, Z.; Liu, S.; Zhang, H.; Li, E.; Guo, L.; Che, Y. J. Nat. Prod.
2010, 73, 920−924. Wang, Y.; Niu, S.; Liu, S.; Guo, L.; Che, Y. Org.
Lett. 2010, 12, 5081−5083.
1
ester derivatives, 2a and 2b, respectively. H NMR data of 2a (400
MHz, pyridine-d5): δ 6.085 (1H, s, H-17a), 5.317 (1H, s, H-17b),
3.706 (1H, dd, J = 3.6, 2.0 Hz, H-7), 2.959 (1H, m, H-13), 2.156 (1H,
d, J = 12.8 Hz, H-3a), 2.154 (1H, dd, J = 12.8 Hz, H-5), 2.036 (1H, d,
J = 12.0 Hz, H-14a), 1.809 (1H, d, J = 8.0 Hz, H-9), 1.741 (1H, m, H-
12a), 1.714 (1H, m, H-2a), 1.670 (2H, m, H-1a), 1.526 (3H, m, H-6a,
H-11a, and H-12b), 1.334 (3H, m, H-2b, H-11b, and H-14b), 1.258
(1H, dd, J = 11.6, 2.0 Hz, H-6b), 1.156 (3H, s, H3-18), 1.044 (1H, m,
H-3b), 0.976 (3H, s, H3-20), 0.765 (1H, td, J = 13.6, 4.0 Hz, H-1b).
(3) Wang, X.-N.; Bashyal, B. P.; Wijeratne, E. M. K.; U’Ren, J. M.;
Liu, M. X.; Gunatilaka, M. K.; Arnold, A. E.; Gunatilaka, A. A. L. J. Nat.
Prod. 2011, 74, 2052−2061.
368
dx.doi.org/10.1021/np200769q | J. Nat. Prod. 2012, 75, 361−369

Journal of Natural Products
Article
(4) Turbyville, T. J.; Wijeratne, E. M. K.; Liu, M. X.; Burns, A. M.;
Seliga, C. J.; Luevano, L. A.; David, C. L.; Faeth, S. H.; Whitesell, L.;
Gunatilaka, A. A. L. J. Nat. Prod. 2006, 69, 178−184.
(33) Niu, X. M.; Li, S. H.; Mei, S. X.; Na, Z.; Zhao, Q. S; Lin, Z. W.;
Sun, H. D. J. Nat. Prod. 2002, 65, 1892−1896.
(34) Wang, J.; Zhao, Q. S.; Lin, Z. W.; Sun, H. D. Acta. Bot. Yunnan.
1997, 19, 191−195.
(5) Dai, C.; Whitesell, L.; Rogers, A. B.; Lindquist, S. Cell 2007, 130,
1005−1018.
(35) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem.
2000, 267, 5421−5426.
(6) Santagata, S.; Xu, Y.; Wijeratne, E. M. K.; Kontnik, R.; Rooney,
C.; Perley, C. C.; Kwon, H.; Clardy, J.; Kesari, S.; Whitesell, L.;
Lindquist, S.; Gunatilaka, A. A. L. ACS Chem. Biol. 2011, in press
(dx.doi.org/10.1021/cb200353m).
(7) Xu, Y.; Marron, M. T.; Seddon, E.; McLaughlin, S. P.; Ray, D. T.;
Whitesell, L.; Gunatilaka, A. A. L. Bioorg. Med. Chem. 2009, 17, 2210−
2214.
(8) Kawaide, H. Biosci., Biotechnol., Biochem. 2006, 70, 583−590.
(9) (a) Garcia, P. A.; Oliveira, A. B.; de; Batista, R. Molecules 2007,
12, 455−483. (b) Schmelz, E. A.; Kaplan, F.; Huffaker, A.; Dafoe, N. J.;
Vaughan, M. M.; Ni, X.; Rocca, J. R.; Alborn, H. T.; Teal, P. E. Proc.
Natl. Acad. Sci. U. S. A. 2011, 108, 5455−5460.
(10) Hou, A. J.; Li, M. L.; Jiang, B.; Lin, Z. W.; Ji, S. Y.; Zhou, Y. P.;
Sun, H. D. J. Nat. Prod. 2000, 63, 599−601.
(11) Rosselli, S.; Bruno, M.; Maggio, A.; Bellone, G.; Chen, T. H.;
Bastow, K. F.; Lee, K. H. J. Nat. Prod. 2007, 70, 347−352.
(12) Bai, N.; He, K.; Zgou, Z.; Tsai, M. L.; Zhang, L.; Quan, Z.; Shao,
X.; Pan, M. H.; Ho, C.-T. Planta Med. 2010, 76, 140−145.
(13) Li, L. M.; Weng, Z. Y.; Huang, S. X.; Pu, J. X.; Li, S. H.; Huang,
H.; Yang, B. B.; Han, Y.; Xiao, W. L.; Li, M. L.; Han, Q. B.; Sun, H. D.
J. Nat. Prod. 2007, 70, 1295−1301.
(14) Weng, Z. Y.; Huang, S. X.; Li, M. L.; Zeng, Y. Q.; Han, Q. B.;
Ríos, J. L.; Sun, H. D. J. Agric. Food Chem. 2007, 55, 6039−6043.
(15) Yej, S. H.; Chang, F. R.; Wu, Y. C.; Yang, Y. L.; Zhuo, S. K.;
Hwang, T. L. Planta Med. 2005, 71, 904−909.
(36) Bagatell, R.; Paine-Murrieta, G. D.; Taylor, C. W.; Pulcini, E. J.;
Akinaga, S.; Benjamin, I. J.; Whitesell, L. Clin. Cancer Res. 2000, 6,
3312−3318.
(37) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.;
Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B.
F. Nature 2010, 468, 790−795.
(38) Hooper, P. L.; Tytell, M.; Vigh, L. Cell Stress Chaperones 2010,
15, 761−770.
(39) Howitz, K. T.; Sinclair, D. A. Cell 2008, 133, 387−391.
(40) Chouchani, E. T.; James, A. M.; Fearnley, I. M.; Lilley, K. S.;
Murphy, M. P. Curr. Opin. Chem. Biol. 2011, 15, 120−128.
(41) Dennehy, M. K.; Richards, K. A. M.; Wernke, G. R.; Shyr, Y.;
Liebler, D. C. Chem. Res. Toxicol. 2006, 19, 20−29.
(42) U’Ren, J. M.; Lutzoni, F.; Miadlikowska, J.; Arnold, A. E. Microb.
Ecol. 2010, 60, 340−353.
(43) Maddison, W. P.; Maddison, D. R. Mesquite, 2011, http://www.
mesquiteproject.org (accessed Sept 22, 2011)
(44) Hansen, K.; LoBuglio, K. F.; Pfister, D. H. Mol. Phylogenet. Evol.
2005, 36, 1−23.
(45) Perry, B. A.; Hansen, K.; Pfister, D. Mycol. Res. 2007, 111, 549−
571.
(46) Zwickl, D. J. Genetic algorithm approaches for the phylogenetic
analysis of large biological sequence datasets under the maximum
likelihood criterion. Ph.D. Dissertation, The University of Texas at
Austin, Austin, TX, 2008; pp 1−114.
(16) Hwang, B. Y.; Lee, J. H.; Koo, T. H.; Kim, H. S.; Hong, Y. S.;
Ro, J. S.; Lee, K. S.; Lee, J. J. Planta Med. 2001, 67, 406−410.
(17) Meade-Tollin, L. C.; Wijeratne, E. M. K.; Cooper, D.; Guild, M.;
Jon, E.; Fritz, A.; Zhou, G. X.; Whitesell, L.; Liang, J. Y.; Gunatilaka, A.
A. L. J. Nat. Prod. 2004, 67, 2−4.
(47) Posada, D.; Crandall, K. A. Bioinformatics 1998, 14, 817−818.
(18) Saepou, S.; Pohmakotr, M.; Reutrakul, V.; Yoosook, C.; Kasisit,
J.; Napaswad, C; Tuchinda, P. Planta Med. 2010, 76, 721−725.
̈
(19) Ertas, A.; Ozturk, M.; Bog
72, 500−502.
a, M.; Topcu
̧
, G. J. Nat. Prod. 2009,
(20) Dao, T. T.; Le, T. V. T.; Nguyen, P. H.; Thuong, P. T.; Minh, P.
T. H.; Woo, E. R.; Lee, K. Y.; Oh, W. K. Planta Med. 2010, 76, 1011−
1014.
(21) Takeda, Y.; Otsuka, H. Recent Advances in the Chemistry of
Diterpenoids from Rabdosia Species. In Studies in Natural Products
Chemistry; Atta-ur-Rahman, Ed.; Elsevier: Amsterdam., 1995; Vol. 15.
(22) Aquila, S.; Weng, Z. Y.; Zeng, Y. Q.; Sun, H. D.; Ríos, J. L. J.
Nat. Prod. 2009, 72, 1269−1272.
(23) Zhang, C. L.; Wu, L. J.; Tashiro, S.; Onodera, S.; Ikejima, T.
Acta Pharmacol. Sin. 2004, 25, 691−698.
(24) Schubert, W. M.; Sweeney, W. A. J. Am. Chem. Soc. 1955, 77,
2297−2300.
(25) Herz, W.; Sharma, R. P. J. Org. Chem. 1976, 41, 1021−1026.
(26) Zhao, Q. S.; Lin, Z. W.; Sun, H. D. Yunnan Zhiwu Yanjiu 1996,
18, 234−238.
(27) Sun, H. D.; Lin, Z. W.; Fu, J.; Zheng, X.; Gao, Z. Huaxue Xuebao
1985, 43, 353−359.
(28) Qu, J. B.; Zhu, R. L.; Zhang, Y. L.; Guo, H. F.; Wang, X. N.; Xie,
C. F.; Yu, W. T.; Ji, M.; Lou, H. X. J. Nat. Prod. 2008, 71, 1418−1422.
(29) Hutchison, M.; Lewer, P.; MacMillan, J. J. Chem. Soc., Perkin
Trans. 1 1984, 10, 2363−2366.
(30) Santos, H. S.; Barros, F. W. A.; Albuquerque, M. R. J. R.;
Banderia, P. N.; Pessoa, C.; Braz-Filho, R.; Monte, F. J. Q.; Leal-
Cardoso, J. H.; Lemos, T. L. G. J. Nat. Prod. 2009, 72, 1884−1887.
(31) (a) Kusumi, T.; Ohtani, I.; Inouye, Y.; Kakisawa, H. Tetrahedron
Lett. 1988, 37, 4731−4734. (b) Ohtani, I.; Kusumi, T.; Kashman, Y.;
Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096.
(32) Wagoner, R. M. V.; Satake, M.; Bourdelais, A. J.; Baden, D. G.;
Wright, J. L. C. J. Nat. Prod. 2010, 73, 1177−1179.
369
dx.doi.org/10.1021/np200769q | J. Nat. Prod. 2012, 75, 361−369