Secondary metabolites of the sponge-derived fungus acremonium persicinum

Article abstract of DOI:10.1021/np4002114

This study reports the isolation and characterization of six new acremine metabolites, 5-chloroacremine A (4), 5-chloroacremine H (5), and acremines O (6), P (7), Q (8), and R (9), together with the known acremines A (1), F (2), and N (3) from the fungus Acremonium persicinum cultured from the marine sponge Anomoianthella rubra. The relative configuration of acremine F (2) was determined by analyses of proton coupling constant values and NOESY data, and the absolute configuration confirmed as (1S, 4S, 6R) by X-ray crystallographic analysis of the borate ester derivative 15. Acremines O, P, and R were each shown to be of 8R configuration by ¹H NMR analyses of MPA esters. The relative configurations suggested for acremines P and Q were each deduced by molecular modeling together with NOESY and coupling constant data. The ³J_H-C values in acremine P were measured using the pulse sequence EXSIDE, and the observed ³J_H8-C4 of 5.4 Hz and small ³J_H-C values (<1.5 Hz) from H-8 to C-10 and C-11 were fully consistent with stereoisomer 7a. For acremine Q, NOESY data combined with molecular modeling established the preferred diastereomer 8a.

Full text of DOI:10.1021/np4002114

Article
pubs.acs.org/jnp
Secondary Metabolites of the Sponge-Derived Fungus Acremonium
persicinum
Suciati,†,‡ James A. Fraser,† Lynette K. Lambert,§ Gregory K. Pierens,§ Paul V. Bernhardt,†
and Mary J. Garson*,†
†School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4072, Australia
‡Faculty of Pharmacy, Airlangga University, Surabaya, East Java 60286, Indonesia
§Centre for Advanced Imaging, The University of Queensland, Brisbane QLD 4072, Australia
S
* Supporting Information
ABSTRACT: This study reports the isolation and characterization of six new acremine metabolites, 5-chloroacremine A (4), 5-
chloroacremine H (5), and acremines O (6), P (7), Q (8), and R (9), together with the known acremines A (1), F (2), and N
(3) from the fungus Acremonium persicinum cultured from the marine sponge Anomoianthella rubra. The relative configuration of
acremine F (2) was determined by analyses of proton coupling constant values and NOESY data, and the absolute configuration
confirmed as (1S, 4S, 6R) by X-ray crystallographic analysis of the borate ester derivative 15. Acremines O, P, and R were each
shown to be of 8R configuration by 1H NMR analyses of MPA esters. The relative configurations suggested for acremines P and
Q were each deduced by molecular modeling together with NOESY and coupling constant data. The 3JH−C values in acremine P
were measured using the pulse sequence EXSIDE, and the observed 3JH8−C4 of 5.4 Hz and small 3JH−C values (<1.5 Hz) from H-8
to C-10 and C-11 were fully consistent with stereoisomer 7a. For acremine Q, NOESY data combined with molecular modeling
established the preferred diastereomer 8a.
ungi from the genus Acremonium have been reported from
In this report, we describe the isolation, structure elucidation,
and configurations of six new acremines (4−9) along with the
known acremines A (1), F (2), and N (3). Identification of the
ether product 10 as well as the known spiroacremines A (11)
and B (12) during the course of the chromatographic
purification highlights the sensitivity of the oxygenated
cyclohexenone/cyclohexenediol ring systems to the isolation
conditions used.
1−3
and marine sources4−6 and produce
Fboth terrestrial
unique and biologically active secondary metabolites, the most
well known being the antibiotic cephalosporin C.7 Antioxidant
hydroquinone derivatives8 and a chlorinated polyketide6 have
been reported from an algal-derived Acremonium sp., while
isolates derived from marine sponges have yielded alkaloids,4,9
peptides,10,11 or oxygenated metabolites.12
In 2005, Nasini et al. reported a series of 12 meroterpenoids,
including acremines A (1), F (2), and N (3), from an
endophytic strain of Acremonium byssoides isolated from
sporangiospores of Plasmopara viticola in grapevine leaves.1,13,14
Malik et al. described the isolation of a “norterpenoid” from
the plant Periploca aphylla;15 however the structure of this
compound was later revised to that of acremine A (1) based on
a synthetic study.16 Acremine A has also been isolated from the
fungus Myceliopthora lutea by Smetanina et al. along with
isoacremine D and two spiroacremines.17 Although the
RESULTS AND DISCUSSION
■
Structural and Stereochemical Studies. The sponge
Anomoianthella rubra was collected in December 2009 by scuba
from the Gneering reef offshore from Mooloolaba in South
East Queensland. Colonies of Acremonium persicinum were
successfully cultured by streaking small pieces of sterilized
sponge sample onto malt extract and potato dextrose agar
media made up in artificial seawater. Identification of the isolate
was undertaken by performing colony PCR of the rDNA ITS
followed by DNA sequencing. Following preliminary inves-
tigation of a small-scale culture, large-scale fermentation was
1
structure and H NMR data of isoacremine D reported by
the Russian group were identical to those of acremine D
described by the Italian researchers,1 the 13C NMR and melting
point data of the two samples differed. Recently, total syntheses
of acremines A, B, and I have been developed by Mehta et al.18
Received: March 13, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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carried out in malt extract media (4 L) made up in artificial
seawater. After 4 weeks, the culture broth was extracted with
EtOAc followed by n-BuOH to afford extracts from which six
new acremine metabolites (4−9) were isolated together with 9-
O-methylacremine F (10) and spiroacremines A (11) and B
(12) in addition to the known acremines A (1), F (2), and N
(3).
literature. However in this previous work, the relative
configuration at C-1 of acremine F was not defined.1 Treatment
of acremine A (1) with NaBH4 in EtOH gave a mixture of two
compounds in a 2:1 ratio that were separated by RP-HPLC
1
using 15% MeCN/H2O as eluent. The H NMR spectrum of
the major compound, which eluted second from the RP-HPLC
column, was identical to that of acremine F. The minor
compound shared similar 1H NMR features to those of
acremine F, except for the H-2 signal (δH 5.88 (J = 4.8 Hz) vs
δH 5.61 (J = 2.5 Hz) in acremine F), and was assigned as the C-
1 epimer of acremine F (13).
The conformational equilibria for acremine F (2) and 1-epi-
acremine F (13) are considered in Figure 1a. Individual
conformers (2a/b, 13a/b) were modeled using ChemBio3D
Ultra 12.0 (Cambridge) and by applying the MM2 force field
for energy minimization to an RMS gradient of 0.100. In
acremine F (2), the experimental coupling constant values for
H-1/H-2, H-4/H-5a, and H-4/H-5b (2.5, 2.0, and 4.0 Hz) were
in agreement with theoretical values anticipated for conformer
2a (see Table S1 in the Supporting Information) and placed H-
1 and H-4 in pseudoaxial and pseudoequatorial orientations,
respectively. Consistent with this interpretation, 1D-NOESY
irradiation of the proton signal at δH 4.00 (H-1) of 2 led to
enhancement of H-2, H-5b, and Me-12 (Figure 1b). For 1-epi-
acremine F (13), the experimental J values of 4.8, 4.4, and 3.3
Hz for H-1/H-2, H-4/H-5a, and H-4/H-5b matched theoretical
values anticipated for conformer 13a, in which both H-1 and H-
4 were pseudoequatorial. 1D NOESY irradiation of the signal at
δH 3.92 (H-1) of 13 led to enhancement of H-2 and Me-12, but
there was no enhancement of a C-5 proton. A coupling of 1.1
Hz between H-1 and H-5b confirmed the equatorial orientation
of H-5b. In both acremine F and 1-epi-acremine F, conformers
with a cis-1,3-pseudoaxial−axial arrangement between the C-4
and C-6 hydroxy groups were thus favored. In cyclic organic
compounds with a cis-1,3-arrangement of hydroxy groups, the
diequatorial conformer is preferred in polar solvents, but the
diaxial conformer becomes an important contributor to the
overall conformational equilibrium in nonpolar solvents owing
to the intramolecular hydrogen bonding between the two
hydroxy groups.19 In both 2a and 13a there is also the
stabilizing feature of an equatorial Me group at C-6.
The relative and absolute configurational features of 2 were
confirmed by an X-ray crystallographic study conducted on a
derivative prepared from 9-O-methylacremine F (10). For ether
10, a sodiated ion peak at m/z 265.1422 provided the
1
molecular formula C13H22O4. The H NMR data were very
The 1H and 13C NMR data (Tables 1 and 2) for acremine A
similar to those of 2, except for a singlet at δH 3.16 (3H), which
gave HMBC correlations to the methyl groups at C-10 and C-
11, as well as to C-9, and so revealed that a methoxy group had
replaced the hydroxy group at C-9. On exposure to mildly
acidic conditions during isolation, acremine F (2) may form a
stable tertiary allylic carbocation at C-9, which then reacts with
MeOH to form 10. Treatment of 10 with p-bromophenylbor-
onic acid (1.1 equiv) in CH2Cl2 for 24 h gave a mixture of
products, including the desired p-bromophenylboronate ester
14. Recrystallization of the ester mixture from EtOAc gave
single crystals highly suitable for X-ray analysis, except that
these corresponded to the borate ester 15 rather than 14. In its
1H NMR spectrum, 15 lacked signals associated with a phenyl
moiety, while the mass spectrum exhibited an adduct ion at
m/z 291.1350 [M + Na]+ for the molecular formula
C13H21BO5. Boric acid is commonly found as a contaminant
in commercial samples of boronic acid and related substances.20
(1), the major component in the extract, are reported in
1
CDCl3, while the H NMR spectrum was also measured in
acetone-d6 for direct comparison with literature data. Nasini et
al. determined the relative configuration of 1 by X-ray
crystallographic analysis, while the absolute configuration was
deduced as (4S, 6R) following Mosher esterification.1 The
specific rotation of 1 from A. persicinum was measured as +13,
compared to a literature value of +22.3, consistent with a (4S,
6R) configuration.
A full NMR assignment of acremine F (2) was undertaken
using HSQC and HMBC experiments, as the 13C NMR data
were not available from the literature. The signals for H-7 and
H-8 were observed at δH 6.19 and 6.08, respectively; on this
basis the literature assignments for H-7 (δH 6.09) and H-8 (δH
6.17) should be reversed. The specific rotation of 2 from A.
persicinum was +38, compared to a value of +56 reported in the
B
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C
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a,b
Table 2. 13C NMR Assignments for Acremines 1, 2, and 4−9
position
1
2
4
5
6
7
8
9
1
200.1
123.1
157.9
65.6
73.0
130.2
137.7
64.1
198.3
121.5
156.2
72.2
195.5
122.2
155.7
70.5
69.6
75.2
53.0
67.9
67.9
27.5
25.0
23.7
192.6
51.3
83.4
189.1
144.8
147.8
40.1
78.0
86.6
22.8
26.8
14.8
192.3
102.4
162.5
99.0
59.1
57.4
95.0
86.2
78.2
25.8
23.4
14.5
205.3
38.9
43.7
102.7
66.0
60.7
31.7
86.2
71.3
27.7
25.3
14.7
147.8
115.6
116.8
146.3
119.2
123.7
31.3
2
3
4
5
43.9
40.5
70.4
6
73.1
71.1
75.9
7
124.3
147.1
71.4
126.2
138.4
71.1
122.7
149.2
71.3
8
69.8
9
76.3
10
11
12
29.8
30.1
29.6
22.6
29.8
29.7
29.6
24.5
24.1
26.8
21.0
15.7
a
b
Chemical shifts (ppm) taken from 2D spectra referenced to CDCl3 (δC 77.16). At 500 MHz.
Figure 1. (a) Cyclohexene ring conformers for acremine F (2) and 1-epi-acremine F (13) (R = side chain). (b) Energy-minimized model structures
of 2a and 13a showing NOESY correlations observed for 2 and 13.
The absolute configuration of 15 was determined by the
anomalous dispersion method on a highly redundant data set
collected with Cu Kα radiation and confirmed a (1S, 4S, 6R)
configuration (Figure 2).
1.400, 1.39; δC 29.6, 29.6, 21.0], closely resembling those of 11
except that the methylene group at C-5 was replaced by a
methine (δH 4.23, δC 70.4). The chlorine substituent was
placed at C-5 based on HMBC correlations from H-5 to both
C-4 (δC 72.2) and C-6 (δC 75.9). The 8.2 Hz coupling between
H-4 and H-5 established the axial orientation of H-5. In the
preferred conformation, the C-6 hydroxy group is close to
coplanar with the adjacent carbonyl group, resulting in
hydrogen bonding (note that this is also the case for acremine
A). In the side chain of 4, signals at δH 6.49 and 6.70, each a
doublet with a J of 16 Hz and linked to signals at δC 122.7 and
149.2 by HSQC, respectively, confirmed a trans double bond;
HMBC correlations from the alkene proton at δH 6.70 to C-9,
Me-10, and to Me-11 positioned the alkene between C-7 and
C-8.
Figure 2. ORTEP view of borate ester 15 showing absolute and
relative configurations.
On the basis of comparison of its 1H and 13C NMR data with
those of 4, 5-chloroacremine H (5) was likewise a chlorinated
cyclohexenone; however the prenyl side chain contained an
epoxy group (δH 3.94, 2.99; δC 53.0, 67.9) whose position was
determined by HMBC correlations from both Me-10 and Me-
11 to the epoxy carbon at δC 67.9, as well as from the epoxy
proton (δH 3.94) to the olefinic carbons (C-2 and C-3). HMBC
correlations from the methine proton (δH 4.21) to C-1 (δC
195.5), C-3 (δC 155.7), C-4 (δC 70.5), and C-6 (δC 75.2)
confirmed chlorine substitution at C-5. A 5.7 Hz coupling
between H-4 and H-5 established the equatorial orientation of
H-5. The epoxy protons in 5-chloroacremine H shared a 2.0 Hz
5-Chloroacremine A (4) and 5-chloroacremine H (5) were
obtained from RP-HPLC as a mixture in a 1:1 ratio; ESIMS
suggested a mixture of chlorinated compounds from the ion
clusters at m/z 283/285 and at m/z 299/301, each with an
intensity ratio of 3:1. HRMS measurements provided the
respective molecular formulas as C12H17ClO4 and C12H17ClO5,
respectively. The 1H and 13C NMR data of 5-chloroacremine A
suggested a substituted cyclohexenone ring [δH 6.18 (1H), 4.60
(1H), 4.23 (1H); δC 198.3 (s), 121.5 (d), 156.2 (s), 72.2 (d),
70.4 (d), and 75.9 (s)] with three methyl groups [δH 1.404,
D
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coupling, closely identical to the value in each of acremines H
and L (J = 2.1 Hz),14 which supported a trans configuration for
the epoxy group. The orientation of the epoxy group relative to
the stereogenic centers of the cyclohexenone core could not be
conclusively determined owing to the small amount of material
available; the similarity in the chemical shift of H-7 (δH 3.94) to
the value of H-7 reported for acremine H (δH 3.93)14 may
suggest the same diastereomer.
During NMR analysis of the mixture of 4/5 in CDCl3, it was
noticed that the signal intensity corresponding to 5-
chloroacremine A decreased over time, while signals for a cis
double bond (δH 5.96 and 5.60, d 5.9 Hz), two methines (δH
4.18 and 3.84), and an AB methylene (δH 2.94 and 2.60, d 14.4
Hz) appeared. By comparison with data for spiroacremines A
(11) and B (12), both of which have been identified as
artifacts,17 the new product was identified as the spiro
compound 16. The relative configuration at the spiro center
was not determined due to the small amount of material
available.
to H-7 and by HMBC correlations to C-7 (δC 95.0) and C-8
(δC 86.2); therefore an ether linkage between C-4 and C-8
accounted for the HMBC correlation between H-8 and C-4.
On the basis of its molecular formula, acremine P had six
degrees of unsaturation, of which five had been identified; the
oxygen substituents at C-4 and C-9 were therefore connected
to form a peroxy ring, as shown in 7.
Reduction of 7 under mild conditions (H2, Pd/C, EtOAc, 24
h) gave acremine A (1) as the sole product. The specific
rotation of the synthetic sample was +25, comparable to the
value of +13 for the natural isolate from A. persicinum. The
absolute configuration at C-7 was pursued by preparation of the
(R)- and (S)-O-methylmandelate (MPA) esters 18a/18b,
respectively. The ΔδRS values of the MPA esters (where ΔδRS
= δR − δS) were positive for H-2 and Me-12 and negative for
H-8, Me-10, and Me-11 and established a 7R configuration.
With this information in hand, and recognizing the stereo-
chemical constraint imposed by the 2,3,8-trioxabicyclo[3.2.1]-
octane ring system, two candidate diastereomers, 7a and 7b
(Figure 3a), were next considered. In the 1H NMR spectrum of
The final chlorinated compound isolated from the A.
persicinum extract was named acremine O (6). The mass
spectrum of this compound exhibited a typical ion cluster for a
chlorinated molecule at m/z 281/283 [M + Na]+ in a ratio of
3:1; a molecular formula of C12H15ClO4 was determined by
1
HRESIMS. The H NMR spectrum showed signals for one
oxymethine (δH 4.15), two methylene groups (δH 3.26, 3.02
and δH 3.01, 1.84), and three methyl singlets (δH 2.16, 1.27,
1.16). In the 13C NMR spectrum, there were two carbonyl
groups (δC 189.1, 192.6), while the downfield chemical shift of
C-3 (δC 83.4) suggested a spiro ring.17 The carbonyl at δC
192.6 was placed at C-1 based on an HMBC correlation to Me-
12 (δH 2.16), while the carbonyl group at δC 189.1 was assigned
to C-4 based on HMBC correlations to the methylene protons
of C-7. In the side chain, a hydroxy group was placed at C-8
based on HMBC correlations from the methyl groups (δH 1.27,
1.16) to C-8 (δC 78.0). As in 4 and 5, the chlorine substituent
was therefore placed at C-5. NOESY correlations between Me-
11/H-8 and Me-11/H-7a indicated that these protons were all
on the same face of the tetrahydrofuran ring. The NOESY
correlation between H-2a and H-7b then established the
relative configuration at the spiro centre. When 6 was esterified
at C-8 to its (R)- and (S)-O-methylmandelate (MPA) esters
17a and 17b, the ΔδRS values (where ΔδRS = δR − δS) were
positive for H2-2 and H2-7, while negative for Me-10 and Me-
11, which was consistent with an 8R configuration. The
absolute configuration of acremine O is thus (3R, 8R). The
natural product status of acremine O is unclear.
Figure 3. (a) Structures of candidate diastereomers 7a and 7b for
acremine P. (b) Energy-minimized model structures of diastereomers
of acremine P showing NOESY correlations for preferred conformer
7a.
A molecular formula of C12H14O6 was determined for
acremine P (7) from the adduct ion at m/z 277.0673 [M +
Na]+. The 1H and 13C NMR spectra supported the presence of
a substituted cyclohexenone ring [δH 5.20 (1H); δC 192.3 (s),
162.5 (s), 102.4] containing epoxy [δH 3.57 (1H); 59.1 (d),
57.4 (s)] and acetal (δC 99.0) groups. Three methyl groups
observed at δH 1.51, 1.47, and 1.43 were linked to signals at δC
14.5, 25.8, and 23.4 by HSQC, respectively, and there were two
oxymethines (δH 5.83, 4.15; δC 95.0, 86.2) and one other
oxygenated carbon (δC 78.2). HMBC correlations from the
epoxy proton to C-3 (δC 162.5), C-4 (δC 99.0), C-6 (δC 57.4),
and Me-12 (δC 14.5) secured the position of the epoxy ring
between C-5 and C-6. Three other signals (H-2, H-7, H-8), as
well as Me-12, all showed HMBC correlations to the acetal
signal at δC 99.0, which was therefore assigned to C-4. A
hydroxy group (δH 3.17) was located at C-7 from its coupling
7, the signals for H-2 and H-8 were noticeably sharp. The zero
coupling between H-7 and H-8 indicated a dihedral angle of
approximately 90° and so revealed that these two protons were
on opposite faces of the ether ring, as in 7a rather than 7b.
Other pieces of spectroscopic information that pointed to 7a
included (i) NOESY correlations from H-8 to H-7, Me-10, and
Me-11 and (ii) the absence of coupling between H-2 and H-7.
Also there was no evidence of NOESY correlations between H-
2 and H-7, although such information must be interpreted with
caution. Structure 7a was modeled using ChemBio3D Ultra
with MM2 software for energy minimization to an RMS
gradient of 0.100 (Figure 3b). On the basis of the measured
3
dihedral angles, diagnostic JH−C values were predicted by
application of the Karplus equation and revealed that
3
stereoisomer 7a should give a medium JH8−C4 value (4−5
Hz) and small couplings (0−3 Hz) from H-8 to Me-10 and to
E
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Figure 4. (a) Candidate diastereomers of acremine Q. (b) Energy-minimized model structures of the preferred diastereomer of acremine Q (8a)
showing observed NOESY correlations and hydrogen bonding between the C-4 and C-9 hydroxy groups (dashed line).
Me-11.21 The 3JH−C values in acremine P were measured using
of the global energy minimum for 8a and 8d, respectively. The
Boltzmann-weighted chemical shifts were calculated, but the
values for 8a and 8d were similar and neither set could be
conclusively assigned to acremine Q. However the Boltzmann-
weighted proton NMR coupling constants calculated under
vacuum using the method of Jain et al.25 gave values that
supported 8a as the preferred diastereomer (see Table S3 in the
Supporting Information). The noticeable difference in chemical
shift values for the Me-10 and Me-11 signals (δH 1.32 and 1.14
in CDCl3) may be a consequence of the hydrogen bonding
between the C-4 and C-9 hydroxy groups. The absolute
configuration of acremine Q was assigned (3R, 4R, 5S, 6R, 8R)
as shown in 8. Attempted reduction of acremine Q using Pd/C
in EtOAc led to a mixture of products in insufficient quantity
for individual identification.
the pulse sequence EXSIDE;22 the observed JH8−C4 of 5.4 Hz
3
3
and small JH−C values (<1.5 Hz) from H-8 to C-10 and C-11
were fully consistent with stereoisomer 7a.
Also isolated from the large-scale extract of A. persicinum was
acremine Q (8), with a molecular formula of C12H18O5
indicated by HRESIMS. The 1H and 13C NMR spectra
revealed a saturated ketone (δC 205.3 (s)), an acetal (δC
102.7 (s)), an epoxy group [δH 3.51 (1H); 66.0 (d), 60.7
(s)], an oxymethine (δH 3.98; δC 86.2), and one other
oxygenated carbon (δC 71.3). Three methyl groups at δH 1.44,
1.14, and 1.32 were linked to signals at δC 14.7, 25.3, and 27.7
by HSQC, respectively. Because the molecular formula implied
two rings, a comparison of HMBC data with those of 7 was
undertaken, leading to the planar structure suggested. Key
HMBC correlations included those from the epoxy proton (H-
5) to C-3, C-4, C-6, and Me-12, from H-8 to C-3 and C-4, and
from H-7 to C-4 and C-9. The signal for H-7b, which was
obscured by the H2O peak in the 1H NMR spectra recorded in
CDCl3, was detected when the spectrum was rerun in acetone-
d6 or in benzene-d6. In acetone-d6, signals for the hydroxy
groups at C-4 and C-9 were observed at δH 5.73 and 3.87,
respectively.
Acremine R (9) was isolated as a colorless oil from RP-
HPLC and had the same molecular formula (C12H16O3) as that
1
for acremine N (3) from HRESIMS measurements. The H
and 13C NMR spectra of this compound also revealed a close
relationship to acremine N, notably the signals for a
tetrasubstituted benzene ring [δH 6.61 (1H), 6.49 (1H); δC
147.8 (s), 146.3 (s), 123.7 (s), 119.2 (d), 116.8 (s), 115.6 (d)],
a quaternary carbon (δC 76.3), one oxymethine (δH 3.73, δC
69.8), one methylene group (δH 2.99, 2.69; δC 31.3), and three
methyl singlets (δH 2.17, 1.34, 1.27; δC 15.7, 22.6, 24.5). Two
methyl groups (δH 1.34, 1.27) and the oxymethine showed
HMBC correlations to the quaternary carbon at δC 76.3,
assigned as C-9. The downfield chemical shift of C-9 suggested
a chromane ring.26 The signal at δC 69.8 was assigned as C-8
based on HMBC correlations to Me-10 (δH 1.34), Me-11 (δH
1.27), and the methylene group (δH 2.99, 2.67). A DQFCOSY
experiment located the hydroxy group (δH 1.75 d, 8.0) at C-8.
When 9 was converted to its (R)- and (S)-O-methylmandelate
(MPA) esters 19a and 19b, the ΔδRS values (where ΔδRS = δR
− δS) were positive for H-2 and H2-7 and negative for H-5,
Me-10, Me-11, and Me-12. These data supported an 8R
configuration. Also isolated from the fungal extract was
The relative configuration was suggested from a combination
of NOESY data, molecular modeling, and coupling constant
values. Only diastereomers with a (5S, 6R) configuration were
considered on the reasonable expectation that 8 would share
the same configuration at C-6 as other acremines. In CDCl3, H-
3 showed couplings of 7.5, 6.0, 7.4, and 4.4 Hz to H-2a, H-2b,
H-7a, and H-7b, respectively, which suggested an equatorial
rather than an axial orientation for H-3. On stereoelectronic
grounds, the C-4 OH was anticipated to be axial. As a
consequence of the flexible nature of the cis-fused ring junction,
the coupling constant values for H-3 represented conforma-
tionally averaged values. A key piece of information was a
pronounced NOESY correlation between H-2b and H-8. Four
possible diastereomers, 8a−8d, with a cis ring junction were
considered (Figure 4a), and minimum energy conformations
calculated using ChemBio3D Ultra with MM2 software for
energy minimization to an RMS gradient of 0.100. Stereo-
isomers 8a and 8d had interproton distances of 2.4 and 2.7 Å
between H-2b and H-8, respectively; for each of the two
diastereomers 8b and 8c, the interproton distance was >4 Å.
NOESY correlations between H-2b/H-7b, H-7a/Me-11, and
H-7b/Me-11 were in complete agreement with either 8a or 8d.
Candidate diastereomers 8a and 8d were modeled using
1
acremine N (3), with H and 13C NMR data identical to
those reported by Nasini and co-workers. The [α]D was
measured as +15, in reasonable agreement with the literature
value ([α]D +35). On the basis of a comparison of its ECD data
with those of other 2,3-dihydrobenzofurans, Nasini et al.
proposed that (+)-acremine N has an 8S configuration.14
CONCLUSIONS
■
This study reported six new acremine metabolites, 5-
chloroacremine A (4), 5-chloroacremine H (5), and acremines
O (6), P (7), Q (8), and R (9), together with the known
acremines A (1), F (2), and N (3). The relative configuration
Macromodel version 9.9 (Schrodinger LLC),23 and the
̈
structures further optimized with Gaussian version 09,24
resulting in twelve and eight conformations within 3 kcal/mol
F
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Journal of Natural Products
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Fractions 8 and 9 (45 mg) were combined and subjected to NP-
HPLC, employing 40% EtOAc/hexanes, flow rate 2 mL/min over 40
min, to afford acremine N (3) (2.4 mg), together with a mixture of
acremine R (9) and spiroacremine A (11) (7.5 mg), then a fraction
containing acremine P (7) (3.0 mg), and finally acremine Q (8) (1.7
mg) in order of elution. The mixture of 9 and 11 was further purified
by RP-HPLC using gradient elution from 60% to 80% MeOH/H2O
(20 min), followed by isocratic 80% MeOH/H2O (10 min), flow rate
1.5 mL/min, yielding spiroacremine A (11) (1.1 mg) and acremine R
(9) (1.1 mg) in order of elution. The fraction containing acremine P
(3.0 mg) was purified with NP-HPLC using isocratic EtOAc/Hex
(30:70), flow rate 2 mL/min, yielded 7 (1.1 mg). Fractions 11−13 of
the NP flash column chromatography were combined (0.7 g) and
subjected to NP flash column chromatography, eluting with CH2Cl2/
MeOH in order of increasing polarity, to yield six fractions, coded
F13-1 to F13-7. Fraction F13-2 (259 mg) was further chromato-
graphed on an NP flash column employing hexanes/EtOAc in order of
increasing polarity to obtain nine fractions. Acremine A (1) (137 mg)
was identified in fraction 5, while the third fraction (3.8 mg) was
purified on RP-HPLC using gradient elution from 10% to 40%
MeOH/H2O (20 min), followed by isocratic 40% MeOH/H2O (20
min), flow rate 1.5 mL/min, UV 254 nm, to obtain a mixture of 5-
chloroacremine H (5), 5-chloroacremine A (4) (0.5 mg), and 9-O-
methylacremine F (10) (1.5 mg) in order of elution. 5-
Chloroacremine A (4) decomposed to 5-chlorospiroacremine (16)
during storage in CDCl3 for NMR analysis. Fraction F13-3 (154 mg)
was also subjected to RP-HPLC employing isocratic 15% MeCN/H2O
(20 min), followed by a gradient of 15−30% MeCN/H2O (10 min),
then isocratic 30% MeCN/H2O (10 min), flow rate 1.5 mL/min, to
obtain acremine A (1) (22.3 mg), acremine F (2) (13.9 mg), and 9-O-
methylacremine F (10) (17.7 mg) in order of elution.
of acremine F (2) was determined by analyses of proton
coupling constant values and NOESY data, and the absolute
configuration was confirmed as (1S, 4S, 6R) by X-ray
crystallographic analysis of borate ester 15. Acremines O, P,
and R were all shown to have an 8R configuration by MPA
ester determination. The relative configurations of acremines P
and Q were each investigated by molecular modeling together
with NOESY and coupling constant data.
EXPERIMENTAL SECTION
■
General Experimental Procedures. These have been reported
previously.27 NMR spectra were recorded at ambient probe temper-
ature on a Bruker Avance 500 spectrometer unless otherwise stated.
EXSIDE22 data were acquired on a 750 MHz Bruker Avance NMR
spectrometer. In this two-dimensional experiment, a selective pulse
was centered on the single proton of interest (H-8 of 7) such that
proton-coupled peaks were not excited. Cross-peaks appeared as
doublets at the frequency of the coupled carbon resonances in F1. A J-
scaling factor of 15 was applied to increase the magnitude of the
splitting in F1, and this enabled couplings to be obtained with a
resolution of 0.6 Hz.
Biological Material. Sponge specimens were collected at the
Gneering reef offshore from Mooloolaba, QLD, in December 2009.
The sponges were cut into small pieces, rinsed three times with sterile
artificial seawater (ASW, Ocean Nature), and then cut into smaller
cubes. Individual cubes were smeared across malt extract and potato
dextrose agar (PDA) media or were shaken with sterile glass beads in a
Falcon tube containing sterile ASW (5 mL). The cell suspension was
then spread onto plates containing malt extract and PDA using a
sterile cotton bud. The plates were then incubated at 27 °C for 1−2
weeks. Individual fungal strains were isolated and streak purified onto
new media several times. Fungal identification was undertaken using
the colony polymerase chain reaction performed with an Eppendorf
MasterCycler Epgradient 28 (Eppendorf). Standard reaction mixes
contained 0.25 μL of each primer designed to amplify the conserved
rDNA ITS, 5 μL of template DNA in distilled H2O, 2.5 μL of 10×
buffer, 2 μL of dNTPs, and 0.125 μL of Taq polymerase, with distilled
H2O added to make 25 μL of total volume. PCR products were
electrophoresed on 1× TAE 1% agarose gels, stained with ethidium
bromide, and visualized on a UV transilluminator. The PCR products
were purified using a QIAquick gel extraction kit (QIAGEN GmbH,
Hilden DE), then sent to the Australian Genome Research Facility
(AGRF) for sequencing (GenBank accession number KF 017582).
Small-Scale Fermentation. Cultures (50 mL) of A. persicinum
were cultivated in malt extract media made in artificial seawater and
were kept in a shaker incubator (180 rpm) at 28 °C for 2 weeks. The
culture broths were separated from the mycelia by filtration and then
extracted with EtOAc (3 × 50 mL). The organic layers were collected
and dried over anhydrous MgSO4, then concentrated under reduced
pressure to afford brown extracts. The EtOAc extract (30 mg) was
then subjected to NP flash column chromatography, eluting with
hexanes/EtOAc in order of increasing polarity, to give acremine A (1)
(4.3 mg) and spiroacremine B (12) (1.9 mg).
Large-Scale Fermentation. The isolated strain of A. persicinum
was cultured in malt extract media (4 L) made in artificial seawater in a
shaker incubator (180 rpm) at 28 °C for 4 weeks. Culture medium and
mycelia were then separated by filtration, and then the broth was
extracted with EtOAc (3 × 2 L). The aqueous layer was further
extracted with n-BuOH (3 × 1 L). The collected organic layers were
then dried over anhydrous MgSO4 and concentrated in vacuo to obtain
EtOAc (0.3 g) and BuOH (0.5 g) extracts. The EtOAc and BuOH
extracts had a very similar profile by NMR and TLC and were
combined for chemical investigation. The combined extract (0.8 g)
was subjected to NP flash column chromatography, using a stepwise
elution with hexanes/EtOAc, to afford 14 fractions. Fraction 6 was
subjected to NP-HPLC using RI detection, eluting with 25% EtOAc/
hexanes, flow rate 2 mL/min over 40 min, to obtain acremine O (6)
(0.8 mg). Fraction 7 was purified through NP-HPLC using the same
procedure as for fraction 6 and yielded acremine N (3) (2.1 mg).
Acremine A (1) (ref 1): colorless needles (137 mg); [α]24D +13 (c
2.52, MeOH), lit.1 +22.3 (c 0.04, MeOH); 1H and 13C NMR see Table
1 and Table 2; HRESIMS m/z 249.1102 [M + Na]+ (calcd for
C12H18NaO4, 249.1097).
Acremine F (2) (ref 1): yellow oil (19.2 mg); [α]24 +38 (c 0.47,
D
1
1
CHCl3), lit. +56 (c 0.2, CHCl3); H and 13C NMR see Table 1 and
Table 2; LRESIMS m/z 251.2 [M + Na]+.
Acremine N (3) (ref 14): brown oil (2.1 mg); [α]24D +15 (c 0.08,
CHCl3), lit. +35 (c 0.2, MeOH); 1H and 13C NMR see ref 14;
LRESIMS m/z 231.1 [M + Na]+.
5-Chloroacremine A (4): colorless oil (0.5 mg) mixture with 5;
1H and 13C NMR see Table 1 and Table 2; HRESIMS m/z 283.0716
[M + Na]+ (calcd for C12H17ClNaO4, 183.0708).
5-Chloroacremine H (5): colorless oil (0.5 mg) mixture with 4;
1H and 13C NMR see Table 1 and Table 2; HRESIMS m/z 299.0645
[M + Na]+ (calcd for C12H17ClNaO5, 299.0657).
Acremine O (6): colorless oil (0.8 mg); [α]24D −8 (c 0.02, CHCl3);
1H and 13C NMR see Table 1 and Table 2; HRESIMS m/z 281.0565
[M + Na]+ (calcd for C12H15ClNaO4, 281.0551).
Acremine P (7): colorless oil (1.1 mg); [α]24 −43 (c 0.05,
D
1
CHCl3); H and 13C NMR see Table 1 and Table 2; HRESIMS m/z
277.0673 [M + Na]+ (calcd for C12H14NaO6, 277.0683).
Acremine Q (8): colorless oil (1.7 mg); [α]24 −12 (c 0.08,
D
CHCl3); 1H and 13C NMR (CDCl3) see Table 1 and Table 2;
HRESIMS m/z 265.1042 [M + Na]+ (calcd for C12H18NaO5,
265.1046).
Acremine R (9): colorless oil (1.5 mg); [α]24D −8 (c 0.1, CHCl3);
1H and 13C NMR see Table 1 and Table 2; HRESIMS m/z 231.0991
[M + Na]+ (calcd for C12H16NaO3, 231.09992).
9-O-Methylacremine F (10): yellow oil (17.7 mg); [α]24D +38 (c
1
1.27, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.09 (1H, d, J = 16.0,
H-7), 5.90 (1H, d, J = 16.0, H-8), 5.61 (1H, d, J = 2.5, H-2), 4.43 (1H,
m, H-4), 3.99 (1H, dd, J = 9.0, 2.5, H-1), 3.59 (1H, s, 6-OH), 3.48
(1H, d, J = 7.5, 4-OH), 3.16 (3H, s, OMe), 2.85 (1H, d, J = 9.0, 1-
OH), 2.22 (1H, dd, J = 15.0, 2.0, H-5a), 1.80 (1H, dd, J = 15.0, 4.0, H-
5b), 1.31 (3H, s, Me-12), 1.30 (6H, s, Me-11 and Me-10; 13C NMR
(CDCl3, 125 MHz) δ 137.7 (C-3), 136.0 (C-8), 130.5 (C-2), 128.7
(C-7), 75.2 (C-9), 73.0 (C-1), 71.0 (C-6), 64.1 (C-4), 50.6 (OMe),
G
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(26.8 (Me-12), 26.0 (Me-10 and Me-11); HRESIMS m/z 265.1422
(OMe), 40.4 (C-5), 26.7 (C-12), 26.0 (C-10), 26.0 (C-11); LRESIMS
m/z 291.1 [M + Na]+; HRESIMS m/z 291.1350 [M + Na]+ (calcd for
C13H21BNaO5, 291.1374).
[M + Na]+ (calcd for C13H22NaO4, 265.1410).
Spiroacremine A (11) (ref 17): white solid (1.4 mg); [α]24 +15
D
(c 0.09, CHCl3), lit.19 +18 (c 0.3, EtOH); 1H and 13C NMR see ref 17;
HRESIMS m/z 249.1125 [M + Na]+ (calcd for C12H18NaO4,
249.1097).
Crystallographic data of the borate ester 15: C13H21BO5, M
268.11, T 293(2) K, monoclinic, space group C2, a 22.3089(7) Å, b
7.0719(2) Å, c 9.8434(3) Å, V 1486.28(8) Å3, Dc (Z = 4) 1.198 g
cm−3, F(000) 576, μ(Cu Kα) 0.738 mm−1, 10 093 data (2θmax = 62°),
Rint 0.0204, 2282 with I > 2σ(I); R 0.0248 (obsd data), wR2 0.0659 (all
data), goodness of fit 1.068. CCDC number 928199. Data were
collected on an Oxford Diffraction Gemini CCD diffractometer with
Cu Kα radiation (1.5418 Å). The structure was solved by direct
methods and refined with SHELX.28 A complete sphere of data was
collected, and the absolute structure was determined by analysis of
1001 Bijvoet pairs using the method of Hooft et al.29 implemented
within PLATON.30 The probability of the correct enantiomer (P2)
was 1.000 using Student’s t-statistics with a ν value of 10 and a Hooft
parameter of −0.01(6). All calculations were carried out within the
WinGX31 program, and the thermal ellipsoid plot (Figure 2) was
produced with ORTEP3.32
Preparation of MPA Esters of Acremine O (17a/17b),
Acremine P (18a/18b), and Acremine R (19a/19b). Acremine
O (6) (0.6 mg) was divided into two portions, and each sample
(approximately 0.3 mg) was treated with either (R)- or (S)-MPA (0.4
mg, 2 equiv), followed by DCC (0.5 mg, 2 equiv) and DMAP (0.3 mg,
2 equiv) in dry CH2Cl2 (0.5 mL). The reaction was stirred overnight
at rt, which was then filtered through a small plug of silica eluting with
CHCl3. The solvent was then dried in vacuo, and each product was
then purified by RP-HPLC, eluting with 60−100% MeCN/H2O for 35
min, flow rate 1.5 mL/min, to yield the (R)-MPA ester (17a) (0.2 mg)
and (S)-MPA ester (17b) (0.2 mg). A portion of acremine P (7) was
likewise divided into two, and each sample (approximately 0.4 mg)
was treated with either (R)- or (S)-MPA, using the same procedures
for 6 to obtain the (R)-MPA ester (18a) (0.4 mg) and (S)-MPA ester
(18b) (0.4 mg). A portion of acremine R (9) (0.5 mg each) was
reacted with either (R)- or (S)-MPA using the same procedure as for 6
and 7 to obtain the (R)-MPA ester (19a) (0.3 mg) and (S)-MPA ester
(19b) (0.3 mg).
Spiroacremine B (12) (ref 17): yellow oil (4.7 mg); [α]24D −17 (c
0.18, CHCl3), lit.19 +4.2 (c 0.24, EtOH); 1H and 13C NMR see ref 17;
HRESIMS m/z 249.1100 [M + Na]+ (calcd for C12H18NaO4,
249.1097).
5-Chlorospiroacremine (16): colorless oil (0.5 mg) mixture with
1
4 and 5; H NMR (CDCl3, 500 MHz) δ 6.70 (1H, d, J = 16.0, H-8),
6.49 (1H, d, J = 16.0, H-7), 6.18 (1H, d, J = 1.8, H-2), 4.60 (1H, m, H-
4), 4.23 (1H, d, J = 8.2, H-5), 2.90 (1H, d, J = 4.8, 4-OH), 1.404 (3H,
s, Me-10), 1.400 (3H, s, Me-11), 1.39 (3H, s, Me-12); 13C NMR
(CDCl3, 500 MHz) δ 198.3 (C-1), 156.2 (C-3), 149.2 (C-8), 122.7
(C-7), 121.5 (C-2), 75.9 (C-6), 72.2 (C-4), 71.3 (C-9), 70.4 (C-5),
29.6 (2C, Me-10 and Me-11), 21.0 (C-12).
Reduction of Acremine A with NaBH4. Acremine A (1) (7.0
mg) dissolved in EtOH (1 mL) was treated with NaBH4 (1.3 mg, 1.1
equiv) at room temperature (rt). After 1 h, 1 mL of acetone was
added, followed by H2O (2 mL) and extraction with EtOAc (3 × 2
mL). The organic layer was then collected, dried over MgSO4, and
concentrated in vacuo to obtain a mixture of acremine F (2) and 1-epi-
acremine F (13) in a 2:1 ratio. The mixture (5 mg) was then subjected
to RP-HPLC, UV 254 nm, using isocratic 15% MeCN/H2O (25 min),
flow rate 1.5 mL/min, to yield acremine F (2.0 mg) and 1-epi-
acremine F (0.7 mg).
1-epi-Acremine F (13): colorless oil (0.7 mg); [α]24D −18 (c 0.05,
1
CHCl3); H NMR (CDCl3, 500 MHz) δ 6.24 (1H, d, J = 16.1, H-7),
6.16 (1H, d, J = 16.1, H-8), 5.88 (1H, d, J = 4.8, H-2), 4.49 (1H, ddd, J
= 7.0, 4.4, 3.0, H-4), 3.92 (1H, br t, J = 5.4, H-1), 2.75 (1H, d, J = 7.0,
4-OH), 2.59 (1H, s, 6-OH), 2.01 (1H, J = dd, 14.4, 4.4, H-5a), 1.96
(1H, ddd, J = 14.4, 3.3, 1.1, H-5b), 1.53 (1H, d, J = 6.3, 1-OH), 1.38
(3H, s, Me-10), 1.37 (3H, s, Me-11), 1.36 (3H, s, Me-12); LRESIMS
m/z 251.2 [M + Na]+.
Preparation of p-Bromophenylboronate and Borate Esters
of 9-O-Methylacremine F. 9-O-Methylacremine F (10) (2.6 mg)
was treated with p-bromophenylboronic acid (2.4 mg, 1.1 equiv) in
CH2Cl2 (1 mL) at rt. After 24 h, the sample was evaporated under N2
to obtain a mixed ester product (4.4 mg) containing the p-
bromophenylboronate ester of 9-O-methylacremine F (14). The
mixture was then subjected to NP pipet column chromatography,
employing gradient elution with hexanes/EtOAc to obtain nine
fractions. Fractions 7 to 9 were combined (2.1 mg) to give a mixture of
the boronate ester 14 and the borate ester 15 in a 3:2 ratio. This
sample was subjected to recrystallization without further purification in
EtOAc using the vapor diffusion method, yielding single crystals of the
borate ester 15.
(R)-MPA ester (17a): colorless oil; 1H NMR (CDCl3, 500 MHz) δ
7.46−7.35 (5H, m, MPA phenyl protons), 5.16 (1H, dd, J = 5.8, 2.0,
H-8), 4.79 (1H, CH of MPA), 3.43 (3H, s, OMe of MPA), 3.22 (1H,
dd, J = 14.6, 5.8, H-7a), 3.12 (1H, d. J = 16.1, H-2a), 2.89 (1H, d, J =
16.1, H-2b), 2.15 (3H, s, Me-12), 1.80 (1H, dd, J = 14.6, 2.0, H-7b),
1.08 (3H, s, Me-10), 0.80 (3H, s, Me-11); LRESIMS m/z 429.2 [M +
Na]+.
(S)-MPA ester (17b): colorless oil; 1H NMR (CDCl3, 500 MHz) δ
7.46−7.35 (5H, m, MPA phenyl protons), 5.15 (1H, dd, J = 5.8, 1.5,
H-8), 4.80 (1H, CH of MPA), 3.42 (3H, s, OMe of MPA), 3.19 (1H,
dd, J = 15.0, 5.8, H-7a), 2.73 (1H, d. J = 16.3, H-2a), 2.51 (1H, d, J =
16.3, H-2b), 2.13 (3H, s, Me-12), 1.48 (1H, dd, J = 15.0, 1.5, H-7b),
1.18 (3H, s, Me-10), 1.13 (3H, s, Me-11); LRESIMS m/z 429.2 [M +
Na]+.
p-Bromophenylboronate ester of 9-O-Methylacremine F
1
(14): white, amorphous solid; H NMR (CDCl3, 500 MHz) δ 7.62
(R)-MPA ester (18a): colorless oil; 1H NMR (CDCl3, 500 MHz) δ
7.48−7.37 (5H, phenyl protons), 6.603 (1H, s, H-7), 5.22 (1H, s, H-
2), 4.83 (1H, s, CH of MPA), 4.17 (1H, s, H-8), 3.44 (3H, s, OMe of
MPA), 1.51 (3H, s, Me-12), 1.45 (3H, s, Me-10), 1.42 (3H, s, Me-11);
LRESIMS m/z 425.0 [M + Na]+.
(2H, m, Ph), 7.47 (2H, m, Ph), 6.06 (2H, s, H-7 and H-8), 5.61 (1H,
d, J = 2.5, H-2), 4.81 (1H, t, J = 2.7, H-4), 4.14 (1H, dd, J = 11.7, 2.5,
H-1), 3.18 (3H, s, OMe), 2.18 (1H, dd, J = 14.0, 3.3, H-5a), 2.16 (1H,
d, J = 11.7, 1-OH), 2.04 (dd, J = 14.0, 2.4, H-5b), 1.54 (3H, s, Me-12),
1.33 (3H, s, Me-10), 1.33 (3H, s, Me-11); 13C NMR (CDCl3, 500
MHz) δ 138.5 (C-3), 136.8 (C-8), 135.5 (Ph), 131.0 (Ph), 130.8 (Ph),
130.1 (C-2), 127.9 (C-7), 125.9 (Ph), 75.0 (C-9), 74.7 (C-1), 72.6 (C-
6), 63.4 (C-4), 50.5 (OMe), 38.5 (C-5), 26.4 (C-11), 25.2 (C-10),
25.1 (C-12); LRESIMS m/z 429.2/431.1 [M + Na]+, 445.1/457.1 [M
+ K]+; HRESIMS m/z 429.0858/431.0848 [M + Na]+ (calcd for
C19H24BBrNaO4, 429.0849/431.0828).
Borate ester of 9-O-methylacremine F (15): colorless crystals;
1H NMR (CDCl3, 500 MHz) δ 6.10 (1H, d, J = 16.4, H-7), 5.92 (1H,
d, J = 16.4, H-8), 5.63 (1H, d, J = 2.5, H-2), 4.47 (1H, m, H-4), 4.01
(1H, dd, J = 8.8, 2.5, H-1), 3.17 (3H, s, OMe), 2.56 (1H, dd, J = 14.9,
2.2, H-5a), 2.41 (1H, d, J = 8.8, 1-OH), 1.82 (dd, J = 14.9, 4.3, H-5b),
1.33 (3H, s, Me-12), 1.314 (3H, s, Me-10), 1.309 (3H, s, Me-11); 13C
NMR (CDCl3, 500 MHz) δ 137.6 (C-3), 136.1 (C-8), 130.4 (C-2),
128.5 (C-7), 75.0 (C-9), 72.9 (C-1), 70.7 (C-6), 64.0 (C-4), 50.5
(S)-MPA ester (18b): colorless oil; 1H NMR (CDCl3, 500 MHz) δ
7.48−7.38 (5H, phenyl protons), 6.597 (1H, s, H-7), 5.19 (1H, s, H-
2), 4.82 (1H, s, CH of MPA), 4.34 (1H, s, H-8), 3.43 (3H, s, OMe of
MPA), 1.45 (3H, s, Me-12), 1.48 (3H, s, Me-10), 1.47 (3H, s, Me-11);
LRESIMS m/z 425.0 [M + Na]+.
(R)-MPA ester (19a): colorless oil; 1H NMR (CDCl3, 500 MHz) δ
7.43−7.30 (5H, m, phenyl protons), 6.58 (2H, br s, H-2 and H-5),
4.92 (1H, t, J = 5.5, H-8), 4.75 (1H, CH of MPA), 3.50 (3H, s, OMe
of MPA), 3.03 (1H, dd, J = 17.3, 5.5, H-7a), 2.71 (1H, dd, J = 17.3,
5.5, H-7b), 1.75 (3H, s, Me-12), 1.02 (3H, s, Me-10), 0.92 (3H, s, Me-
11); LRESIMS m/z 379.1 [M + Na]+.
(S)-MPA ester (19b): colorless oil; 1H NMR (CDCl3, 500 MHz) δ
7.43−7.30 (5H, m, phenyl protons), 6.59 (1H, s, H-5), 6.37 (1H, s, H-
2), 4.98 (1H, t, J = 5.1, H-8), 4.75 (1H, CH of MPA), 3.50 (3H, s,
H
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Journal of Natural Products
Article
OMe of MPA), 3.87 (1H, dd, J = 17.2, 5.1, H-7a), 2.32 (1H, dd, J =
17.2, 5.1, H-7b), 1.78 (3H, s, Me-12), 1.28 (3H, s, Me-10), 1.23 (3H,
s, Me-11); LRESIMS m/z 379.1 [M + Na]+.
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Catalytic Hydrogenation of Acremine P (7) and Acremine Q
(8). Acremine P (7) (0.7 mg) was first dried under N2 followed by
high-vacuum treatment overnight. The round-bottom flask (5 mL)
containing the sample was flushed with N2 for about 2 min. Dry
EtOAc (2 mL) was then added, and reaction flushed with H2 gas for 2
min. Catalyst Pd/C (1.3 mg) was then added, and the reaction flushed
with H2 gas for about 5 min to remove air. After 22 h, the product was
filtered through a short plug of Celite eluting with EtOAc (15 mL).
The collected product was then dried under N2. When 1H NMR
showed that no reaction had occurred, hydrogenation was carried out
for a further 24 h under the same conditions. The reaction was filtered
through Celite eluting with EtOAc to give acremine A (1), which was
purified by RP-HPLC, using a gradient elution of MeCN/H2O, to
obtain acremine A (1) (0.5 mg). Acremine Q (0.5 mg) was treated
similarly, but the reaction product (0.5 mg) obtained contained a
mixture of products.
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ASSOCIATED CONTENT
(23) MacroModel, version 9.9; Schrodinger LLC: New York, NY,
̈
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2012.
S
* Supporting Information
(24) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennuci, B.;
Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, P.;
Ixmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jamarillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;
Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,
Revision A.02; Gaussian, Inc.: Wallingford, CT, 2009.
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Mukhtar, M. R.; Awang, K. Bot. Mar. 2010, 53, 507−513.
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Figures S1−S25. 1H and selected 2D NMR data for compounds
1−19, molecular modeling details for 2 and 8, and crystallo-
graphic data for the borate ester 15. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*(M.J.G.) Tel: +61-7-3365 3605. Fax: +61-7-3365 4273. E-
mail: m.garson@uq.edu.au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Australian Research Council and The University
of Queensland for financial support, and Prof. M. G. Banwell
(Australian National University) for valuable discussions. M.
Mayhew and S. Graham assisted with sample collection.
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