Metacridamides A and B, macrocycles from conidia of the entomopathogenic fungus metarhizium acridum

Article abstract of DOI:10.1021/np2007044

Metarhizium acridum, an entomopathogenic fungus, has been commercialized and used successfully for biocontrol of grasshopper pests in Africa and Australia. Its conidia produce two novel 17-membered macrocycles, metacridamides A (1) and B (2), which consist of a Phe unit condensed with a nonaketide. Planar structures were elucidated by a combination of mass spectrometric and NMR techniques. Following hydrolysis of 1, chiral amino acid analysis assigned the l-configuration to the Phe unit. A crystal structure established the absolute configuration of the eight remaining stereogenic centers in 1. Metacridamide A (1) showed cytotoxicity to three cancer lines with IC₅₀'s of 6.2, 11.0, and 10.8 μM against Caco-2 (epithelial colorectal adenocarcinoma), MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell lines, respectively. In addition, metacridamide B (2) had an IC₅₀ of 18.2 μM against HepG2/C3A, although it was inactive at 100 μM against Caco-2 and MCF-7. Neither analogue showed antimicrobial, phytotoxic, or insecticidal activity.

Full text of DOI:10.1021/np2007044

Article
pubs.acs.org/jnp
Metacridamides A and B, Macrocycles from Conidia of the
Entomopathogenic Fungus Metarhizium acridum
Stuart B. Krasnoff,*,† Ulrich Englich,‡ Paula G. Miller,§ Michael L. Shuler,§ Raymond P. Glahn,†
Bruno G. G. Donzelli,⊥ and Donna M. Gibson†
†USDA-ARS-Biological IPM Research Unit, Ithaca, New York 14853, United States
‡The Cornell High Energy Synchrotron Source, Ithaca, New York 14853, United States
§Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853, United States
⊥Department of Plant Pathology, Cornell University, Ithaca, New York 14853, United States
S
* Supporting Information
ABSTRACT: Metarhizium acridum, an entomopathogenic fungus, has been commercialized and
used successfully for biocontrol of grasshopper pests in Africa and Australia. Its conidia produce
two novel 17-membered macrocycles, metacridamides A (1) and B (2), which consist of a Phe unit
condensed with a nonaketide. Planar structures were elucidated by a combination of mass
spectrometric and NMR techniques. Following hydrolysis of 1, chiral amino acid analysis assigned
the L-configuration to the Phe unit. A crystal structure established the absolute configuration of the
eight remaining stereogenic centers in 1. Metacridamide A (1) showed cytotoxicity to three cancer
lines with IC50's of 6.2, 11.0, and 10.8 μM against Caco-2 (epithelial colorectal adenocarcinoma),
MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell lines, respectively. In addition,
metacridamide B (2) had an IC50 of 18.2 μM against HepG2/C3A, although it was inactive at 100
μM against Caco-2 and MCF-7. Neither analogue showed antimicrobial, phytotoxic, or insecticidal
activity.
Metarhizium acridum (Bischoff, Humber, and Rehner) (= M.
anisopliae var. acridum) (Cordicepitaceae) is an entomopatho-
genic fungus with a host-range limited to grasshoppers in the
orthopteran family Acrididae.1 The conidia of M. acridum
constitute the active ingredient of commercially produced
biopesticides, marketed as Green Muscle and Green Guard,
that are used for control of migratory locusts in Africa and
Australia, respectively.2 Application of Green Muscle against
Red Locust over 10 000 ha of Iku-Kitavu National Park,
Tanzania in 2009 was credited with curtailing a potentially
devastating locust outbreak and averting extensive crop losses
that could have affected the food security of ca. 15 million
people in the region.3
We have an ongoing interest in the secondary metabolites of
biocontrol fungi in the genus Metarhizium4,5 with a focus on
the possibility that they contribute to pathogenicity and/or
virulence.6,7 The widespread dissemination of M. acridum for
insect biocontrol compels continued study of its basic biology
to gain insights that can lead to improved efficacy and shelf life
of the formulated product8,9 and to expose potential chemical
risks associated with use of these fungi as biopesticides.5
We recently identified the serinocyclins, cyclic heptapeptides
produced by conidia of M. acridum and M. robertsii, and
identified the gene responsible for their biosynthesis in the
latter.4,6 The discovery of the serinocyclins from conidia as
opposed to mycelium or broth from a liquid culture revealed an
underexploited source of natural product diversity in
Metarhizium fungi. Herein we describe the isolation, character-
ization, and biological activity of metacridamides A (1) and B
(2), two novel cytotoxic macrocycles from the conidia of M.
acridum.
RESULTS AND DISCUSSION
■
Low-resolution positive ion ESIMS analyses of crude hexane
extracts of conidia of M. acridum (ARSEF #3341) showed a
predominant signal at m/z 632 consistent with the sodium
adduct [M + Na]+ of a compound with a molecular mass of 609
Da. Negative ion spectra showing peaks at m/z 608 [M − H]−,
644/646 [M + Cl]−, and 654 [M + HCOO]− supported this
conclusion. As there were no other candidates with a unit mass
of 609 Da previously reported from M. acridum, the compound
was earmarked for isolation and characterization.
RP HPLC of the crude hexane extracts produced chromato-
grams with two major peaks at tR = 6.50 and 5.53 min, with the
latter as the minor peak. Preparative RP HPLC led to the
isolation of the major compound 1 at tR = 6.50 min, which was
shown by ESIMS to have a molecular mass of 609 Da.
Compound 2, giving rise to the minor peak, gave a prominent
signal in the positive ion ESIMS at m/z 590, consistent with the
[M + Na]+ of a compound with a molecular mass of 567 Da.
The 42 Da difference between the pseudomolecular ions of 1
and 2 suggested an acetylated compound and its deacetyl
analogue. MS analysis of a sample of 1 dissolved in CH3OD for
Received: September 7, 2011
Published: January 31, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
175
dx.doi.org/10.1021/np2007044 | J. Nat. Prod. 2012, 75, 175−180

Journal of Natural Products
Article
Table 1. NMR Spectroscopic Data for Metacridamides A (1) and B (2)
a
b
metacridamide A (1)
metacridamide B (2)
c
d
e
f
d
e
position
δC, mult.
δH (J in Hz)
HMBC
δC
δH (J in Hz)
HMBC
2
170.9, qC
53.0, CH
173.1
54.4
3
4.94, m
5.91, d (8.6)
2, 5, 25
4.85 br, s
g
4
2, 3, 5, 25
8.21, d (9.1)
3, 5
5
168.7, qC
132.7, qC
172.6
132.5
136.9
36.8
6
g
7
133.7, CH 6.03, dd (9.5, 1.4)
5, 8, 9, 30, 31
6.15, d (9.5)
5, 30
g
h
8
35.5, CH
78.3, CH
137.1, qC
2.84, m
3.95, s
6, 7, 9, 31
2.81,
m
6, 7, 31
gi
,
gi
,
9
7, 8, 10, 11, 31, 32, 33
79.3
3.94, s
7, 8, 10, 11, 31, 32, 33
10
11
12
13
14
15
16
17
18
19
136.3
127.4
40.7
g
g
g
124.3, CH 5.02, m
9, 10, 12, 13, 32, 33
5.26, d (10.5)
9, 32, 33
gi
,
36.6, CH
81.4, CH
130.9, qC
2.83, m
4.92, m
9, 10, 11, 13, 33
2.73, ddd (10.5, 7.0, 3.6) 10, 11, 13, 33
11, 12, 14, 15, 33, 34, Ac CO
81.0
3.84, d (10.2)
11, 12, 14, 15, 33, 34
136.0
130.3
35.5
g
131.0, CH 5.11, dd (10.0)
13, 16, 17, 34, 35
5.12, d (10.2)
13, 16, 17, 34, 35
14, 15, 35
h
33.5, CH
82.7, CH
33.7, CH
40.2, CH2
2.71, m
14, 15, 35
2.81,
m
g
g
4.61, dd (8.8, 2.3)
1.61, m
2, 15, 16, 18, 19, 23, 35
19, 20, 23
83.7
4.68, dd (8.0, 1, 8)
1.70, m
2, 15, 16, 18, 19, 23, 35
g
35.2
19
1.10, ddd (13.3, 9.5, 3.8)
0.84, m
17, 18, 20, 21, 23, 24
17, 18, 20, 21, 23, 24
42.3
1.29, m
18, 20, 21, 23, 24
18, 20, 21, 23, 24
0.91, m
20
21
31.2, CH
27.6, CH2
1.38, m
33.0
29.4
1.43, m
g
g
g
1.28, m
19, 20, 22, 24
1.39, m
19, 20, 22, 24
g
0.84, m
19, 20, 22, 24
20, 21
0.96, m
19, 20, 22, 24
22
23
24
25
10.8, CH3
16.0, CH3
20.5, CH3
36.5, CH2
0.74, t (7.4)
0.75, d (6.7)
0.81, d (6.6)
3.27, dd (14.0, 6.1)
3.20, dd (14.0, 5.8)
11.7
16.3
20.8
37.2
0.85, t (7.4)
0.77, d (6.7)
0.88
20, 21
17, 18, 19
19, 20, 21
2, 3, 26, 27
2, 3, 26, 27
17, 18, 19
19, 20, 21
2, 3, 26, 27
2, 3, 26, 27
3.33, dd (14.1, 5.4)
2.95, dd (14.1, 10.2)
26
137.0, qC
139.5
130.4
129.4
127.6
13.6
27, 27′
28, 28′
29
129.8, CH 7.20, m
128.6, CH 7.27, m
127.0, CH 7.21, m
25, 29
7.233, m
25, 29
26
g
26, 27
7.235, m
g
g
27, 28
7.17, m
27, 28
gj gi gi
, , ,
gi
,
30
13.1, CH3
19.4, CH3
15.0, CH3
16.1, CH3
13.6, CH3
18.2, CH3
169.8, qC
21.4, CH3
1.83, d (1.4)
5, 6, 7, 8, 9, 31
7, 8, 9
1.65, d (1.4)
1.18, d (7.0)
1.60, d (0.6)
0.93, d (7.0)
1.67, d (1.3)
1.02, d (6.9)
5, 6, 7, 31
gj
,
31
1.19, d (7.0)
1.61, m
19.3
6, 7, 8, 9
9, 10, 11
gj
,
gj
,
32
8, 9, 10, 11, 12
gi
15.2
gi
,
,
33
0.92, d (7.0)
1.63, d (1.3)
0.67, d (6.9)
11, 12, 13, 34
16.1
11, 12, 13, 34
gj
,
gj
,
i
gj
,
gi
,
34
12, 13, 14, 15, 16, 35
15, 16, 17
13.7
12, 13, 14, 15, 35
15, 16, 17
35
19.8
Ac CO
gj
,
Ac CH3
2.04, s
Ac CO, 13
a
b
c
d
e
CDCl3. CD3OD (assignments and correlations for H4 obtained in CD3OH). 150 MHz. 600 MHz. HMBC correlations, optimized for 8 MHz,
are from proton(s) stated to the indicated carbon. 125 MHz. Weak cross-peak. Overlapping signals. 5-Bond correlation (Figures S16, S32). 4-
Bond correlation.
f
g
h
i
j
deuterium exchange showed [M + Na]+ and [M + K]+ at m/z
634 and 650, respectively, indicating the presence of two
carbon signals were identified from the 13C spectrum, three in
the carbonyl region and four in the 131−137 ppm range,
consistent with alkenyl or aromatic carbons.
exchangeable protons.
1H and COSY NMR spectra of 1 (Table 1) indicated the
presence of 53 nonexchangeable protons, including one methyl
triplet, eight methyl doublets (three of which showed coupling
constants of <1 Hz, suggesting a long-range (4J) coupling), and
a methyl singlet. Three well-resolved doublets resonated at δH
6.03, 5.11, and 5.02, suggesting alkenyl protons. Five protons in
the aromatic region suggested a monosubstituted phenyl group.
The 13C spectrum showed signals for 37 carbons. HSQC
data corroborated the presence of 10 methyl carbons (δC 10.8−
21.4) and revealed three sp3 methylenes, six sp3 methine
carbons, three oxymethines, three alkenyl methines, and five
aromatic methine carbons for a total of 30 carbons with
attached protons. By a process of elimination seven quaternary
COSY and HSQC spectra showed three conspicuously
similar spin systems, in each of which a complex multiplet in
the 2.6−2.8 ppm range coupled with (1) a methyl doublet in
the 0.6−1.2 ppm range, (2) a proton in the 3.9−4.9 ppm range,
suggesting an oxygenated methine carbon, and (3) a doublet in
the 5.1−6.1 ppm range, suggesting an olefinic methine proton
vicinal to a quaternary alkenyl carbon. In the HMBC spectra
the alkenyl proton either did not correlate or showed only a
2
weak J correlation to a quaternary olefinic carbon (δC 130.9−
137.1). However, this carbon was correlated with another set of
methyl protons (δH 1.60−1.83) as well as with a methine
proton (δH 2.71−2.84) in all three units. The correlations
176
dx.doi.org/10.1021/np2007044 | J. Nat. Prod. 2012, 75, 175−180

Journal of Natural Products
Article
associated with these three similar units indicated the 6-carbon
substructure depicted in Figure 1.
Chart 1
Figure 1. Six-carbon unit consistent with COSY, HSQC, and HMBC
spectra of 1. Bolded bonds indicate a COSY network. Arrows indicate
HMBC correlations (carbon detected by indicated proton). Double-
headed arrows indicate reciprocal HMBC correlations.
The spin system of a Phe unit was revealed by a proton
doublet signal at δH 5.91 that showed no attached carbon in the
HSQC spectrum. The assignment of this signal to an amide
proton was supported by its COSY correlation with an α-
methine proton at δH 4.94, which in turn coupled with two
geminal β-protons at δH 3.27 and 3.20. HMBC spectra showed
correlations between the β (H25) protons and the γ (C26) and
δ (C27) carbons in the phenyl ring. In addition, in HMBC
spectra, both the α (H3) and amide protons correlated with
both the carbonyl carbon (C2) of the Phe unit and its acylating
carbonyl carbon (C5).
A COSY correlation between the methyl triplet at δH 0.74
(H22) and a pair of methylene protons at δH 1.28 and 0.84
(H21a,b) provided an entry point for deciphering a seven-
carbon aliphatic group from NMR data as a 4-methylhexan-2-yl
group.
Direct chiral amino acid analysis by Cu2+ ligand-exchange
chromatography indicated the presence of L-Phe in 1. The
absolute configuration of the Phe unit of 1 was identified by co-
chromatography of the hydrolysis product of 1 with a standard
mixture consisting of D- and L-Phe (Figure S35).
The crystal structure of 1 was determined using X-ray
diffraction and standard techniques (Figure 2).10 This structure,
HSQC and HMBC correlations readily confirmed the acetate
group predicted from MS data as the R-group in one of the
units depicted in Figure 1. In HMBC spectra the acetate methyl
protons (δH 2.04) correlated with the acetate carbonyl carbon
(δC 169.8) as did the methine proton at δH 4.92, which HSQC
data indicated is attached to the oxygenated carbon at δC 81.4
(C13). The methine proton at δH 3.95 (H9) correlated with
the quaternary alkenyl carbon at δC 137.1 (C10) and its
adjacent methyl carbon at δC 15.0 (C32). These correlations
thus linked the oxymethine carbon of a second unit with the
quaternary alkenyl carbon of the unit carrying the acetyl group.
An analogous set of correlations linked the acetylated carbon
(C13) with the alkenyl quaternary carbon (C14) of the third
unit. In HMBC spectra the oxymethine proton of this third unit
at δ 4.61 (H17) correlated with the carbonyl carbon of the Phe
group (C2) at δC 170.9 as well as with the methine carbon of
the 4-methylhexan-2-yl unit (C18) at δC 33.7. The COSY
correlation of the methine proton at δH 1.61 (H18) with H17,
the HMBC correlation H23→C17, and its complement H17→
C23 confirmed the linkage of C17 to C18 of the 4-
methylhexan-2-yl group.
The HMBC correlation between the H30 methyl protons
(δH 1.83) and the C5 carbonyl carbon, which acylates the
amide nitrogen of the Phe unit, as well as the correlation
between the H7 olefinic proton (δH 6.03) and this carbonyl,
established the bond between the quaternary alkenyl carbon
(C6) and C5 and, thus, completed the 17-member macrocycle
of 1 (Chart 1; Table 1).
HRESIMS data for 1 indicating the molecular formula
C37H55NO6 and an index of unsaturation of 11 were consistent
with the planar structure deduced from NMR data and also
allowed for two deuterium-exchanged protons, the amide
proton and the hydroxyl proton on the C9 oxygen.
Figure 2. Crystal structure of 1, with 50% probability thermal
ellipsoids for non-H atoms.
combined with the results of chiral amino acid analysis,
established the absolute configuration of all nine stereocenters
as well as the geometry of the three double bonds in 1 as
3S,6E,8S,9R,10E,12S,13S,14E,16S,17R.
HRESIMS data for 2 were consistent with the molecular
formula C35H53NO5, confirming that 2 differed from 1 by the
replacement of the acetyl group in 1 with a hydroxyl group.
NMR data for 2 lacked all signals and correlations associated
with the acetyl group of 1. Because 2 was isolated in much
smaller quantities than 1, attempts were made to conserve
material for bioassays by finding an aprotic NMR solvent other
than d6-DMSO. However, 2 was insoluble in all common low-
boiling aprotic solvents tried (CDCl3, CD2Cl2, CD3CN, d6-
177
dx.doi.org/10.1021/np2007044 | J. Nat. Prod. 2012, 75, 175−180

Journal of Natural Products
Article
acetone) and was, therefore, dissolved in CD3OD for NMR
studies. As expected, the amide proton of the Phe unit of 2 was
not detected in NMR experiments in CD3OD because of
deuterium exchange. An abbreviated series of experiments using
CD3OH revealed the amide proton (δH 8.21) and its
correlations (Table 1, Figures S29, S33). Most of the key
correlations observed in 2-D spectra of 1 were also observed for
2 (Table 1). The absolute configuration of 2 was deduced by
analogy to 1.
The transannular ROESY correlations between the C7
olefinic methine proton and the C35 methyls were observed
in the spectra of both 1 (Figure S17, S36) and 2 (Figure S34).
In this respect the solution structures were consistent with the
crystal structure of 1, in which the H35 methyl group is the
only one of the 10 in the molecule that projects into the
interior of the macrocycle, and its protons can approach within
3 Å of the H7 proton.
Weak but clearly resolved ROESY correlations to the H27
(δ-Phe) and H28 (ε-Phe) protons from H35, H31, and H7
were observed for 1 (but not for 2 possibly because the sample
was much more dilute). In the crystal structure all interatomic
distances involved in these correlations are greater than 6 Å
and, thus, would be unlikely to produce a ROESY cross-peak
(Figure S37). Using a computer-generated 3-D model (Chem-
3-D Pro) of the crystal structure of 1 all the relevant
interatomic distances can be brought into the 2−4 Å range
by rotations around the C3−C25 (Phe α−β) and C-25−C26
(Phe β−γ) bonds (Figure S38). Thus, the ROESY data suggest
that these bonds rotate freely in solution.
Metacridamide A (1) showed no antimicrobial activity at 100
μg/disk, no insecticidal activity against Drosophila melanogaster
at a 100 μg dosage, no larvicidal activity against Aedes aegypti at
100 μg/mL, and no phytotoxicity activity at 50 μg/mL against
lettuce seedlings. However, it did show moderate cytotoxicity
against three cancer lines, with IC50 values of 6.2, 11.0, and 10.8
μM against Caco-2 (epithelial colorectal adenocarcinoma),
MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell
lines, respectively. In addition, metacridamide B (2) had an
IC50 of 18.2 μM against HepG2/C3A, although it was inactive
at 100 μM against Caco-2 and MCF-7.
biosynthetic pathways that are similar, if not homologous, to
those of the metacridamides. A compound from the patent
literature derived from an unidentified soil fungus combines a
Phe unit with a heptaketide.17 The torrubiellutins combine an
N-methylated Phe unit with a presumptive hexaketide.18
Intriguingly, the torrubiellutins, like the metacridamides, derive
from an entomopathogenic fungus. Other PKS−NRPS hybrid
macrocycles incorporating a Phe unit include hapalosin19 and
the cytochalasins.20 The chondropsins,21 although lacking a Phe
unit, contain three iterations of the substructure depicted in
Figure 1, two of which are contiguous.
As with many microbial natural products, the biological
activity of the metacridamides was revealed in this study in
assays that can have little if any relevance to the context in
which these compounds evolved. There is no obvious adaptive
rationale for the cytotoxicity of a compound produced by the
spores of fungal pathogens of insects. There are now many
compounds known from entomopathogenic Metarhizium fungi
with a wide range of biological activities ascribed to them,22 but
virtually nothing is known about how they function for the
organisms that produce them. The looming challenge is to
advance our understanding of the biology of these compounds
to a point in line with our ability to define them chemically.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured at 25 °C in MeOH on a Rudolph Autopol IV polarimeter
1
at 589 nm with a 100 mm cell. H, 13C, gCOSY, DQCOSY, ROESY,
HSQCAD, and gHMBCAD NMR experiments were carried out on
Varian Inova 400, 500, or 600 spectrometers using either HSW, DBG
broad-band decoupler, 5 mm switchable, or HCN inverse detection
probes. Pulse sequences were provided in Varian VnmrJ 2.2D/
Chempack 4.1. HSQC and HMBC spectra were acquired at 600 MHz
1
n
(1H-dimension) and were optimized for JCH = 150, 190 Hz and JCH
= 8.0, 5.0, respectively. ROESY spectra were acquired with a mixing
time of 0.2 s.
NMR data were recorded for samples dissolved in CDCl3, CD3OD,
1
or CD3OH. H chemical shifts were referenced to the singlet at δH
7.26 or center of the residual CHD2OD quintuplet at δH 3.31. 13C
chemical shifts were referenced to the center of the 13CD3OD
septuplet at δC 49.15 or the CDCl3 triplet at δC 77.16. Low-resolution
ESI mass spectra were acquired by infusion of methanolic solutions at
5 μL/min by a syringe pump (Harvard Apparatus) into a Micromass
ZMD 4000 spectrometer. High-resolution ESI mass spectra were
similarly acquired in positive ion mode on a Waters XEVO-G2
spectrometer using lock-mass correction with leu-enkephalin as the
external lock-mass standard.
The uninterrupted 18-carbon chain starting at C5 and ending
at C22 in both 1 and 2 bears the hallmarks of a polyketide.11
The carbon skeleton, as well as all of the modifications of this
putative nonaketide moiety, can be accounted for as products of
the well-characterized functionalizing enzymes associated with
type I fungal polyketide synthases (ketoreductase, dehydratase,
enol-reductase, and a C-methyl transferase employing S-
adenosyl methionine as a source of methyl groups). The acetyl
group at C13 of 1 could also be the product of a closely linked
or heterologous acetyl transferase.
Fungal Material and Culture Conditions. Conidia of M.
acridum were produced using a published protocol.8 Briefly,
cryogenically preserved mycelial material (ARSEF #3341 and #7486,
both derived from IMI-330189, which was isolated from an
unidentified grasshopper in Niger) was used to inoculate 100 mL
liquid cultures (20 g glucose, 20 g yeast extract/L H2O) in 250 mL
It seems likely that the Phe group, which forms the 17-
membered macrocycle by an ester linkage to O1 at its C-
terminal and an amide linkage to C5 at its N-terminal, is
incorporated by a nonribosomal peptide synthetase (NRPS).12
Hybrid PKS−NRPS genes are widespread in fungi.7,13−15 We
expect that recently published genome sequence data16 will
facilitate identification of hybrid synthetases in M. acridum and
that a functional analysis using a gene knockout approach will
identify the gene responsible for the metacridamide bio-
synthetic pathway.
Erlenmeyer flasks, which were shaken at 150 rpm for 3 days at 20
3
°C. The liquid starter culture was then used to inoculate solid cultures
on barley (100 g rolled barley flakes moistened with 60 mL H2O) in
autoclavable perforated polyethylene “spawn bags” (Myco Supply).
Extraction and Isolation. A simple extraction protocol was
developed to optimize the yield of 1 and 2. Conidia were suspended in
hexane (200 mL/13 g conidia) in 250 mL polypropylene centrifuge
bottles. The suspensions were vortexed for 30 s, sonicated for 30 min,
and then centrifuged to pellet conidia. The supernatants were
decanted and concentrated in vacuo. About 160 g of conidia afforded
ca. 156 mg of a whitish solid residue. This was then vortexed
vigorously in MeOH, and the supernatant was filtered and
chromatographed by semipreparative reversed-phase HPLC (Phenom-
enex Prodigy ODS3; 10 × 250 mm; 5 μm; 100 Å) eluting at 4 mL/
A search of the literature revealed two examples of recently
discovered fungal natural products whose structures and
patterns of oxygenated carbons and alkenyl groups suggest
178
dx.doi.org/10.1021/np2007044 | J. Nat. Prod. 2012, 75, 175−180

Journal of Natural Products
Article
min using an isocratic mixture of MeOH (95)/H2O (5) and UV
detection (λ = 215 nm). Fractions containing 1 and 2 were polished
using the same HPLC column with a mobile phase consisting of
MeCN (9)/H2O (1). The mixture furnished 1 (18 mg) and 2 (6 mg)
of >99% purity (tR = 6.50 and 5.53 min, respectively).
noma), MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell
lines using the XTT assay30 with the XTT cell proliferation kit II
(Roche Applied Science).
ASSOCIATED CONTENT
* Supporting Information
■
Metacridamide A (1): colorless plates (MeOH); [α]26 −10.0 (c
D
S
1
1.20, MeOH); UV (MeOH) λmax (log ε) 233 (3.34); H, 13C NMR,
1H, 13C, COSY, HSQC, HMBC, and ROESY spectra of 1 and
2, chiral HPLC amino acid analysis data for 1, 3-D models of 1
with interatomic distances for selected protons, tabulated X-ray
data for 1, and tabulated interatomic distance data for selected
proton pairs of 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
HSQC, HMBC, ROESY data, see Table 1 and the Supporting
Information; HRESIMS obsd m/z 632.3925 [M + Na]+; calcd for
C37H55NO6 + Na, 632.3927; HPLC tR = 6.50 min.
Metacridamide B (2): colorless oil; [α]24 +1.3 (c 0.34, MeOH);
D
UV (MeOH) λmax (log ε) 236 (3.36); 1H, 13C NMR, HSQC, HMBC,
ROESY data, see Table 1 and the Supporting Information; HRESIMS
obsd m/z 590.3820 [M + Na]+; calcd for C35H53NO5 + Na, 590.3821;
HPLC tR = 5.53 min.
AUTHOR INFORMATION
Corresponding Author
■
Chiral Amino Acid Analysis. A 0.7 mg sample of 1 was
hydrolyzed in a 1:1 mixture of 12 M HCl and propionic acid at 110 °C
for 15 h and then dried in vacuo to yield a white solid and a brown
gum. The latter was removed by trituration with 0.4 mL of
dichloromethane. The solid white residue was taken up in 0.25 mL
of H2O. Aliquots of 10 μL were co-chromatographed with standard D-
and L-Phe (Sigma-Aldrich) using a Cu2+ ligand-exchange HPLC
column (Phenomenex Chirex (D)-penicillamine; 4.6 × 250 mm; 5
μm) eluted at 1 mL/min with 2 mM CuSO4 in MeOH/H2O (3:7);
detection by UV absorption at 254 nm.
*Tel: 607-255-2026. E-mail: stuart.krasnoff@ars.usda.gov;
sbk1@cornell.edu.
ACKNOWLEDGMENTS
■
We gratefully acknowledge R. Vaughan and Mary Bodis
(USDA-ARS), J. Y. Maeng, J. Zuk (Cornell), and I. Keresztes
(Cornell Chemistry and Biology) for technical assistance and I.
X-ray Crystallography of 1. X-ray crystallographic data for
compound 1 were collected at the F1 beamline at the Cornell High
Energy Synchrotron Source using an ADSC Quantum 270 CCD
detector that was offset to allow collection of data up to a resolution of
0.87 Å.23
A single crystal with dimensions of 100 × 10 × 5 μm was obtained
by dissolving the sample in MeOH, adding 2% HCOOH dropwise to
induce precipitation, clearing the solution with another drop of
HCOOH, and then slowly evaporating the sample at 20 °C. The
crystal was then mounted on a Mitegen micro mesh mount24 using a
small amount of highly viscous NVH oil (Hampton Research). Data
were collected at 100 K at a wavelength of λ = 0.9177 Å. Using a
microcrystal setup with focusing glass capillary (focal spot diameter 18
μm) 75 φ-wedges, 5 deg each with 4 s exposure time, were recorded.
Data were integrated with XDS and scaled with XSCALE;25,26
15 274 observations were merged into 4991 unique reflections (space
group P212121, Rint = 4.32%, 96.4% completeness).
́
Molnar (University of Arizona) for helpful discussion. The L.
Harrington and B. Lazzaro laboratories (Cornell Entomology)
kindly provided insects for bioassays. X-ray diffraction data
were collected at the Cornell High Energy Synchrotron Source,
which is supported by the National Science Foundation under
award DMR-0225180, using the MacCHESS facility, supported
by award RR-01646 from the National Institutes of Health.
Mention of a trademark, proprietary product, or vendor does
not constitute a guarantee or warranty of the product by the
U.S. Department of Agriculture and does not imply its approval
to the exclusion of other products or vendors that may also be
suitable.
REFERENCES
■
(1) Bischoff, J. F.; Rehner, S. A.; Humber, R. A. Mycologia 2009, 101,
512−530.
The crystal structure was solved by direct methods (SHELXS-97)
and refined using full-matrix least-squares methods on F2 (SHELXL-
97).27 Hydrogen atoms were generated geometrically and included in
the refinement in the riding model approximation. All non-hydrogen
atoms were refined anisotropically. Final results for 398 parameters: R1
= 5.31%, wR2 = 13.15%, GooF = 1.056.
(2) Lomer, C. J.; Bateman, R. P.; Johnson, D. L.; Langewald, J.;
Thomas, M. B. Annu. Rev. Entomol. 2001, 46, 667−702.
(3) Anonymous. New Agriculturalist, September 2009, http://www.
new-ag.info/developments/devItem.php?a=943.
(4) Krasnoff, S. B.; Keresztes, I.; Gillilan, R. E.; Szebenyi, D. M. E.;
Donzelli, B. G. G.; Churchill, A. C. L.; Gibson, D. M. J. Nat. Prod.
2007, 70, 1919−1924.
(5) Krasnoff, S. B.; Sommers, C. H.; Moon, Y.-S.; Donzelli, B. G. G.;
Vandenberg, J. D.; Churchill, A. C. L.; Gibson, D. M. J. Agric. Food
Chem. 2006, 54, 7083−7088.
(6) Moon, Y. S.; Donzelli, B. G. G.; Krasnoff, S. B.; McLane, H.;
Griggs, M. H.; Cooke, P.; Vandenberg, J. D.; Gibson, D. M.; Churchill,
A. C. L. Appl. Environ. Microbiol. 2008, 74, 4366−4380.
(7) Donzelli, B. G. G.; Krasnoff, S. B.; Churchill, A. C. L.;
Vandenberg, J. D.; Gibson, D. M. Curr. Genet. 2010, 56, 156−162.
(8) Faria, M.; Hajek, A. E.; Wraight, S. P. Biol. Control 2009, 51,
346−354.
(9) Roberts, D. W.; St. Leger, R. J. In Adv. Appl. Microbiol. Laskin, A.
I., Bennet, J. W., Gadd, G., Eds.; Elsevier Inc.: London, 2004; Vol. 54,
p. 1−70.
(10) CCDC 840273 contains the crystallographic data for
metacridamide A (1). These data can be obtained, free of charge,
from The Cambridge Crystallographic Data Centre via http://www.
ccdc.cam.ac.uk.
(11) Schuemann, J.; Hertweck, C. In The Mycota XV: Physiology and
Genetics; Anke, T., Weber, D., Eds.; Springer-Verlag: Berlin, 2009; Vol.
XV, pp 331−351.
Bioassays. Metacridamide A (1) was tested against a panel of
bacterial and fungal targets in disk-diffusion assays. Escherichia coli and
Bacillus cereus were grown on nutrient agar, Colletotrichum acutatum,
Botrytis cinerea, and Beauveria bassiana (ARSEF #8354) on potato
dextrose agar, and Saccharomyces cerevisiae on 1% yeast extract, 2%
peptone, 1.5% agar, supplemented with either 2% dextrose or 2%
glycerol. Plates were spread with 0.6 mL aliquots of a liquid bacterial
culture grown for 24 h in nutrient broth, 0.6 mL of a fungal conidial
suspension (ca. 1 × 106 conidia/mL H2O), or with 0.5 mL of a yeast
suspension in H2O adjusted to OD600 = 0.5. Inoculated plates were
allowed to dry for 1 h. Test samples were applied to 7 mm filter paper
disks in 10 μL of MeOH. Disks were air-dried for 1 h and then placed
on assay plates. Cultures were monitored for zones of inhibition every
24 h for 4 days. Tetracycline and filipin were used at 10 μg/disk as
positive controls for the bacterial and fungal targets, respectively.
MeOH was used at 10 μL/disk as a negative control.
Using previously published methods 1 was also tested for
phytotoxicity in a lettuce seedling elongation assay28 and for
insecticidal activity against Drosophila melanogaster adults29 and
Aedes aegypti larvae.4
Cytotoxicity Assays. Metacridamides A and B (1 and 2) were
assayed for toxicity against Caco-2 (epithelial colorectal adenocarci-
179
dx.doi.org/10.1021/np2007044 | J. Nat. Prod. 2012, 75, 175−180

Journal of Natural Products
Article
(12) Strieker, M.; Tanovic,
2010, 20, 234−240.
́
A.; Marahiel, M. A. Curr. Opin. Struct. Biol.
(13) Song, Z.; Cox, R. J.; Lazarus, C. M.; Simpson, T. J.
ChemBioChem 2004, 5, 1196−1203.
(14) Eley, K. L.; Halo, L. M.; Song, Z.; Powles, H.; Cox, R. J.; Bailey,
A. M.; Lazarus, C. M.; Simpson, T. J. ChemBioChem 2007, 8, 289−
297.
(15) Isaka, M.; Chinthanom, P.; Supothina, S.; Tobwor, P.; Hywel-
Jones, N. L. J. Nat. Prod. 2010, 73, 2057−2060.
(16) Gao, Q.; Jin, K.; Ying, S.-H.; Zhang, Y.; Xiao, G.; Shang, Y.;
Duan, Z.; Hu, X.; Xie, X.-Q.; Zhou, G.; Peng, G.; Luo, Z.; Huang, W.;
Wang, B.; Fan, W.; Wang, S.; Zhong, Y.; Ma, L.-J.; St. Leger, R. J.;
Zhao, G.-P.; Pei, Y.; Feng, M.-G.; Xia, Y.; Wang, C. PLoS Genet. 2011,
7, e1001264.
(17) Jacquot, D.; Poeschke, O.; Burger, C. International Patent WO/
2009/146,772, 2009.
(18) Pittayakhajonwut, P.; Usuwan, A.; Intaraudom, C.; Khoyaiklang,
P.; Supothina, S. Tetrahedron 2009, 65, 6069−6073.
(19) Stratmann, K.; Burgoyne, D. L.; Moore, R. E.; Patterson, G. M.
L.; Smith, C. D. J. Org. Chem. 1994, 59, 7219−7226.
(20) Fex, T. Tetrahedron Lett. 1981, 22, 2703−2706.
(21) Cantrell, C. L.; Gustafson, K. R.; Cecere, M. R.; Pannell, L. K.;
Boyd, M. R. J. Am. Chem. Soc. 2000, 122, 8825−8829.
́
(22) Molnar, I.; Gibson, D. M.; Krasnoff, S. B. Nat. Prod. Rep. 2010,
27, 1241−1275.
(23) Szebenyi, D. M. E.; Arvai, A.; Ealick, S.; LaIuppa, J. M.; Nielsen,
C. J. Synchrotron Radiat. 1997, 4, 128−135.
(24) Thorne, R. E.; Stum, Z.; Kmetko, J.; O’Neill, K.; Gillilan, R. J.
Appl. Crystallogr. 2003, 36, 1455−1460.
(25) Kabsch, W. J. Appl. Crystallogr. 1993, 26, 795−800.
(26) Kabsch, W. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2010, D66,
125−132.
(27) Sheldrick, G. M. Acta Crystallogr. Sect. A: Found. Crystallogr.
2008, A64, 112−122.
(28) Krasnoff, S. B.; Lobkovsky, E. B.; Wach, M. J.; Loria, R.; Gibson,
D. M. J. Agric. Food Chem. 2005, 53, 9446−9451.
(29) Krasnoff, S. B.; Gibson, D. M.; Belofsky, G. N.; Gloer, K. B.;
Gloer, J. B. J. Nat. Prod. 1996, 59, 485−489.
(30) Roehm, N. W.; Rodgers, G. H.; Hatfield, S. M.; Glasebrook, A.
L. J. Immunol. Methods 1991, 142, 257−265.
180
dx.doi.org/10.1021/np2007044 | J. Nat. Prod. 2012, 75, 175−180