Aigialomycins and related polyketide metabolites from the mangrove fungus Aigialus parvus BCC 5311

Article abstract of DOI:10.1016/j.tet.2009.03.050

Reinvestigation of the secondary metabolites from the marine mangrove fungus Aigialus parvus BCC 5311 led to the isolation of six new nonaketide metabolites, aigialomycins F (4) and G (5a/5b), 7′,8′-dihydroaigialospirol (7), 4′-deoxy-7′,8′-dihydroaigialos

Full text of DOI:10.1016/j.tet.2009.03.050

Tetrahedron 65 (2009) 4396–4403
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Aigialomycins and related polyketide metabolites from the mangrove fungus
Aigialus parvus BCC 5311
,
a
a
a
a
Masahiko Isaka a , Arunrat Yangchum , Sutichai Intamas , Kanokarn Kocharin , E.B. Gareth Jones ,
*
Palangpon Kongsaeree b c, Samran Prabpai b
,
a National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road,
Klong Luang, Pathumthani 12120, Thailand
b Department of Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
c Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Reinvestigation of the secondary metabolites from the marine mangrove fungus Aigialus parvus BCC 5311
led to the isolation of six new nonaketide metabolites, aigialomycins F (4) and G (5a/5b), 70,80-dihy-
droaigialospirol (7), 40-deoxy-70,80-dihydroaigialospirol (8), and rearranged macrolides 9 and 10, along
with six previously described compounds, hypothemycin (1), aigialomycins A (2) and B (3), aigialospirol
(6), 4-O-demethylhypothemycin (11), and aigialone (12). The structures of the new compounds were
elucidated by analyses of the NMR spectroscopic and mass spectrometry data in combination with
chemical means.
Received 2 December 2008
Received in revised form 6 March 2009
Accepted 19 March 2009
Available online 27 March 2009
Keywords:
Aigialus parvus
Resorcylic acid lactone
Marine fungi
Ó 2009 Elsevier Ltd. All rights reserved.
Aigialomycin
Aigialospirol
1. Introduction
hypothemycin (1), aigialomycins A (2) and B (3), aigialospirol (6), 4-
O-demethylhypothemycin (11),11 and aigialone (12).
Hypothemycin (1)1,2 is one of the highly oxygenated analogues
in the group of 14-membered resorcylic acid lactones.3 Although
only marginal antifungal and cytotoxic activities of 1 were initially
reported,1,2 the recent discovery that hypothemycin (1) and several
closely related compounds are potent protein kinase inhibitors has
stimulated a renewed interest in this class of natural products.4–7
We previously isolated hypothemycin along with five resorcylic
acid lactones aigialomycins A–E from the mangrove fungus Aigialus
parvus BCC 5311.8 In the subsequent studies, extension of the fun-
gus incubation period in a liquid medium from 35 to 80 days
resulted in the production of a hypothemycin-derived spiroacetal
compound, aigialospirol (6), along with a biogenetically different
class of metabolite, aigialone (12).9 Because of the increasing at-
tention of hypothemycin (1) and aigialomycins,3,10 we have rein-
vestigated the fungus BCC 5311. We report herein the isolation and
structure elucidation of six new derivatives, aigialomycins F (4) and
2. Results and discussion
A. parvus BCC 5311 was fermented under static conditions for
two incubation periods of 35 and 80 days in PDB (20 L for each,
80ꢀ250 mL). Although no new hypothemycin/aigialomycin ana-
logue was found in the 35-day culture broth, investigation of the
EtOAc extract of the 80-day culture broth resulted in the isolation of
six new compounds 4, 5a/5b, and 7–10 as minor constituents along
with 1 (most abundant constituent), aigialomycins A (2) and B (3),
and aigialospirol (6). The same extract also provided 4-O-deme-
thylhypothemycin (11) and aigialone (12). Compound 11 was re-
cently isolated from Hypomyces subiculosus DSM 11931 and DSM
11932 as one of the minor co-metabolites with 1.11
Aigialomycin F (4) was the most polar constituent whose mo-
lecular formula was established as C19H26O9 by HRMS (ESI-TOF).
The 1H NMR spectrum in DMSO-d6 showed a chelated phenolic
proton at dH 11.16 (s, 2-OH) and five hydroxy protons (D2O ex-
changeable) of aliphatic secondary alcohols, all appearing as dou-
blets. The structure of the resorcylic acid region was addressed by
HMBC correlations: from H-3 to C-1 and C-2, from H-5 to C-1 and C-
3, from 2-OH to C-1, C-2, and C-3, and from the methoxy protons (dH
G
(5a/5b), 70,80-dihydroaigialospirol (7), 40-deoxy-70,80-dihy-
droaigialospirol (8), and rearranged 13-membered macrolides 9
and 10, together with previously described compounds,
* Corresponding author. Tel.: þ66 25646700x3554; fax: þ66 25646707.
E-mail address: isaka@biotec.or.th (M. Isaka).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.050

M. Isaka et al. / Tetrahedron 65 (2009) 4396–4403
OH CH3
4397
11'
O
OH
O
1'
OH
1
O
1'
CH3
2
8'
5'
2
1
10'
O
3
9'
3
O
2'
OH OH
5'
OH
10'
7'
O
HO
4
2'
6'
H3CO
HO
3'
4
6'
8'
4'
6
R1O
O
6
H3CO
CH3
4'
O
6'
9'
3'
7'
5
5
Y
11'
O
OH
OH
OH
X
OH
4
1 : R1 = CH3
11 : R1 = H
2 : X, Y = O
3 : X = OH, Y = H
OH
O
CH3
10'
OH
O
O
OH O
O
OH
O
OH
6'
6'
H3CO
10'
H3CO
CH3
OH
OH
HO
OH
OH
5a
5b
OH
2
OH
O
O
OH
O
CH3
1
3
O
O
4
1'
10'
8'
O
6
OCH3
OCH3
CH3
5
2'
7'
1'
11'
3'
H3CO
O
OH
OCH3
O
O
O
CH3
O
4'
4'
5'
2'
5'
10'
6'
6'
Z
HO
9'
O
7'
OH
HO
HO
8'
9
7 : Z = OH
8 : Z = H
6
OH
O
1'
CH3
OH
O
8'
H3C
HO
7'
O 10'
2'
OH
CH3
OCH3
4' 5'
H3CO
O
6'
O
H3C
O
O
OH
10
12
Table 1
NMR data for 4 and 5a/5b (500 MHz for 1H and 125 MHz for 13C)
Position
4 (in DMSO-d6)
dC, mult.
5a/5b (in acetone-d6)
dH, mult (J in Hz)
HMBC
1,2
dC, mult.
dH, mult (J in Hz)
dC
dH, mult (J in Hz)
1-COO–
1
2
2-OH
3
4
168.5, C
100.6, C
163.6, C
168.5, C
100.0, C
164.1, C
168.6
100.1
164.1
11.16, s
6.48, br s
11.22, s
11.26, s
6.43
100.5, CH
166.2, C
56.3, CH3
105.3, CH
145.6, C
99.9, CH
166.5, C
55.3, CH3
104.3, CH
145.3, C
67.4, CH
6.43, d (2.4)
99.8
166.5
55.3
104.4
145.3
67.6
4-OCH3
5
3.83, s
6.59, br s
4
1,3
3.88, s
6.69, dd (2.4, 0.7)
3.88, s
6.69
6
10
67.2, CH
4.51, dd (6.6, 6.1)
6.07, d (6.1)
4.59, m
1.84, m
1.70, m
1,6,30
20
4.68, br dd (8.3, 6.1)
5.28, br d (6.1)
4.68
10-OH
20
5.29, m
4.68
2.24, m
1.99, m
4.17, m
4.80, br d (4.7)
3.20, t (7.9)
3.72, m
80.8, CH
36.0, CH2
80.0, CH
35.0, CH2
4.64, ddd (10.0, 8.3, 2.4)
2.13, ddd (14.3, 10.8, 2.4)
1.76, ddd (14.3, 10.0, 1.9)
4.16, m
4.37, br d (5.7)
4.11, m
80.0
36.9
30
40
66.4, CH
77.6, CH
71.5, CH
133.9, CH
127.9, CH
42.7, CH2
66.4, CH
23.5, CH3
3.72, m
68.3, CH
80.2, CH
211.6, C
67.2
77.7
98.0
29.7
18.4
32.9
65.9
21.4
40-OH
50
4.82, d (6.3)
3.07, dt (3.1, 6.8)
4.40, d (6.8)
4.08, m
4.48, d (6.0)
5.52, dd (15.5, 5.4)
40,50
50-OH
60
40,50,60
50,60
4.48, br d (5.1)
60-OH
70
5.28, m
1.73, m
1.59, m
1.87, m
1.59, m
1.57, m
1.15, m
4.08, m
39.3, CH2
19.4, CH2
38.6, CH2
66.5, CH
23.1, CH3
2.72, ddd (17.9, 8.3, 6.5)
2.63, ddd (17.9, 8.2, 6.3)
1.67, m
1.58, m
1.40, m
1.38, m
3.71, m
3.49, br d (3.8)
1.10, d (6.2)
80
90
5.56, dt (15.5, 6.3)
2.10, m
2.00, m
3.61, m
4.43, d (4.4)
1.02, d (6.1)
70,80,100,110
70,80,100,110
100
100-OH
110
100,110
90,100
1.07, d (6.3)

4398
M. Isaka et al. / Tetrahedron 65 (2009) 4396–4403
OH
CH3
OH
O
1'
O
2'
3'
4' 5'
6'
H3CO
O
O
2''
2'''
3'''
O
O
CH3
H3C
1''
CH3
CH3
1'''
3''
13
OH
O
O
2'
3'
4'
1'
H3CO
O
O
5'
6'
3''
7'
2''
CH3
CH3
8'
O
O
11'
CH3
10'
1''
9'
14
Figure 1. Selected NOESY correlations for 13.
OH
O
O
OH
OH
CH3
3.83, 3H, s) to C-4. The side-chain structure from C-10 to C-110,
through a trans-olefin (C-70/C-80, J¼15.5 Hz), was elucidated pri-
marily by COSY correlations, which also revealed that five hydroxy
groups were attached to C-10, C-40, C-50, C-60, and C-100. HMBC
correlations from H-10 to C-1 (dC 100.6) and C-6 (dC 145.6) indicated
the connection of C-10 to C-6. The downfield H-20 (dH 4.59, m) and
X
Y
6'
8'
H3CO
7'
OH
OH
15 : X = OH, Y = H
16 : X = H, Y = OH
C-20 (dC 80.8) strongly suggested the
d-lactone form, which was also
required from the molecular formula (HRMS). The 1H–1H J1 ,2
0
0
values of 6.6 Hz in DMSO-d6 and 7.7 Hz in acetone-d6–D2O sug-
gested a trans relation of these protons. The remaining stereo-
chemistry was addressed by derivatization and correlation to
aigialomycin G (5a/5b) as described below.
The molecular formula of aigialomycin G was determined by
HRMS as C19H26O9. The 1H and 13C NMR spectra indicated that it
exists as a linear form (5a) and a hemiacetal form (5b), which in-
terconvert each other. The 5a/5b ratio in acetone-d6 was probably
sensitive to concentrations of the compound and trace H2O, as it
varied in each sample from 3:1 to 1.3:1 (1H NMR spectroscopy). The
1H and 13C resonances of 5a and 5b were superimposed for the
resorcylic acid moiety, whereas the resonances were clearly re-
solved for the C-10–C-110 portion. The connectivity of the ketone 5a
from C-10 to C-50, and from C-70 to C-110 was addressed by analysis
of COSY spectrum. The ketone (dC 211.6) was placed at the C-60
position on the basis of the HMBC correlations from four methylene
protons, H-70 (dH 2.72 and 2.63) and H-80 (dH 1.67 and 1.58) to this
Figure 3. Crystallographic structure of 2-O-(4-bromobenzyl)aigialospirol (17). The
ORTEP diagram is drawn at 50% probability level.
0
0
carbon. The J1 ,2 value of 8.3 Hz indicated the pseudoaxial orien-
tation of these protons. The hemiacetal form 5b was apparent from
the presence of a quaternary carbon, which resonated at dC 98.0 (C-
60) and showed HMBC correlations from H-50 and dH 5.28 hydroxy
proton (60-OH). This hemiacetal hydroxy proton also showed HMBC
correlation to C-70. Although the key HMBC correlation from H-100
Figure 2. Selected NOESY correlations for 14.
Figure 4. Selected NOESY correlations for 8.

M. Isaka et al. / Tetrahedron 65 (2009) 4396–4403
4399
moiety as depicted. Proton H-50 showed a NOESY correlation to one
of the five-membered acetonide methyl (dH 1.46, H-1%), whereas
H-60 showed a NOESY cross-peak with the other acetonide methyl
at dH 1.43 (H-3%). Under the acetonide-forming reaction conditions
(p-TsOH, 2,2-dimethoxypropane, rt, 4 h), aigialomycin G (5a/5b)
was converted into a spiroacetal–acetonide derivative 14. The five-
membered acetonide due to the 40,50-diol had a cis-form, since both
H-40 and H-50 showed NOESY correlations to the same acetonide
methyl at dH 1.32 (H-300), but not to the other (dH 1.40, H-100). Intense
NOESY cross-peaks between H-50 and H-100 revealed the configu-
ration of the spiroacetal carbon (C-60) (Fig. 2). Finally, 4 and 5a/5b
were chemically correlated. Hydrogenation of 4 (H2, Pd/C, THF, rt,
3 h) gave the 70,80-dihydro derivative 15. NaBH4 reduction of 5a/5b
(THF, rt, 30 min) gave a 1:5 mixture of two epimeric C-60 alcohols
(15 and 16), wherein 1H NMR (CD3OD) spectroscopic data for the
minor isomer were identical to 15. These results revealed that
aigialomycins F (4) and G (5a/5b) possess the same relative ste-
reochemistry. On the basis of these experimental data and by bio-
genetic correlation to aigialomycin B (3), as discussed below, the
Figure 5. Probable stereo structure and key NOESY correlations of 9. The absolute
configuration of the tertiary alcohol (C-50) is uncertain, therefore, the methoxycarbonyl
group and the hydroxyl group attached to this carbon could be interchanged.
(
dH 4.08, m) to C-60 was not observed at long range JCH of 15.4 and
10 Hz, this proton (H-100) was downfield shifted when compared to
the linear form 5a (dH 3.71).
absolute configurations of
4 and 5a were deduced to be
10R,20S,40S,50S,60S,100S and 10R,20S,40S,50S,100S, respectively.
The stereochemistry of aigialomycins F (4) and G (5a/5b) was
addressed by conversion to acetonide derivatives. We first exam-
ined preparation of two five-membered acetonide derivatives of
aigialomycin F (4), due to the involvement of 40,50-diol and 50,60-
diol. Unexpectedly, treatment of 4 with p-TsOH in 2,2-dimethoxy-
propane (rt, 4 h) gave a bis-acetonide derivative 13 as the sole
product. The structure of the seven-membered acetonide moiety
was confirmed by the HMBC correlations from H-10 and H-40 to the
acetal carbon (dC 102.2, C-200). The large J value of 10.6 Hz for H-10
Compound 7 was isolated as a colorless solid, which has the
molecular formula C19H24O8 (HRESIMS). The 1H and 13C NMR spectra
were very similar to those of aigialospirol (6). The only differences
were the absence of the olefin (C-70/C-80) in 7, instead of the presence
of two additional methylenes. A dihydroisobenzofuranone structure
was confirmed by the downfield shift of H-10 (dH 5.33) and HMBC
correlations from this proton to the lactone carbonyl (dC 171.2), C-1, C-
5, and C-6. The linkage from C-10 to C-110, through a quaternary acetal
carbon (C-60, dC 101.39), was established by analysis of the 2D NMR
spectroscopic data, including the HMBC correlations from H-20 (dH
3.94, ddd, J¼12.0, 6.5, 2.2 Hz) and Hax-70 (dH 2.08, dt, J¼4.9,13.6 Hz) to
C-60. Analysis of the 1H–1H J values and NOESY correlations revealed
the relative configuration of the spirocyclic region and its rigid con-
formation, demonstrating that both tetrahydropyran rings adopt
a chair conformation. Thus, an intense NOESY cross-peak between
one of the C-30 methylene protons at dH 1.88 (Hax-30) and H-50 in-
dicated the 1,3-diaxial relationship of these protons. The axial
and H-20 indicated the rigid
d-lactone conformation, placing both
these protons as pseudoaxial (Fig. 1). One of the H-30 methylene
protons that resonated at dH 1.90 was coupled to H-20 and H-40, both
with J¼11.2 Hz and lacked NOESY correlation to these protons,
hence there was an antiperiplanar relation. This proton (Hb-30)
showed an intense NOESY correlation to H-10, which was consistent
with their synfacial relation. On the other hand, an intense NOESY
cross-peak was observed between H-20 and H-40. These data in-
dicated the relative configuration of the seven-membered ring
Table 2
NMR data for 7 and 8 in CDCl3 (500 MHz for 1H, and 125 MHz for 13C)
Position
7
8
dC, mult.
dH, mult. (J in Hz)
HMBC
dC, mult.
dH, mult. (J in Hz)
HMBC
1-COO-
1
2
171.2, C
104.5, C
157.8, C
171.5, C
104.5, C
157.7, C
3
4
101.43, CH
167.3, C
56.1, CH3
101.9, CH
149.2, C
82.6, CH
65.5, CH
6.48, d (1.5)
COO,1,2,4,5
101.4, CH
167.2, C
56.0, CH3
101.8, CH
150.0, C
83.1, CH
70.2, CH
6.48, s
COO,1,2,4,5
4-OCH3
5
6
10
20
3.88, s
6.67, br s
4
3.88, s
6.71, s
4
1,3,4,10
1,3,4,10
5.33, d (6.5)
3.94, ddd
COO,1,5,6,20,30
6,10,60
5.20, d (7.3)
3.62, m
COO,5,6,20
6,10
(12.0, 6.5, 2.2)
2.16, ddd
(14.0, 5.2, 2.2)
1.88, m
30
34.4, CH2
68.5, CH
40,50
27.2, CH2
27.6, CH2
1.96, m
10,20
20
1.64, m
1.93, m
40
4.07, m
50
50
1.73, m
3.32, dd (11.2, 4.8)
50
60
70
71.1, CH
101.39, C
29.2, CH2
3.29, d (3.4)
70
71.6, CH
98.0, C
29.7, CH2
2.08, m
1.51, m
1.85, m
1.68, m
1.59, m
1.27, m
50,60,80,90
60,90
1.99, dt (4.7, 13.4)
1.54, m
1.88, tq (3.8, 13.3)
1.69, ddd (12.0, 3.2, 2.6)
1.57, m
1.19, dq (3.8, 12.2)
3.57, m
1.07, d (6.2)
60,80
60,90
70
80
90
18.2, CH2
31.8, CH2
18.8, CH2
32.4, CH2
80
110
100
100
110
67.5, CH
21.9, CH3
3.68, m
1.14, d (6.3)
80,110
90
66.2, CH
21.8, CH3
90,100

4400
M. Isaka et al. / Tetrahedron 65 (2009) 4396–4403
OH
O
CH3
8'
OH
7'
O
O
OH
OH
OH
1'
path a
path c
2'
H3CO
H3CO
O
O
OH
1'
H
c
OCH3
2'
H O
OH
O
6, 7
HO
HO
HO
6'
OH
O
OH
O
CH3
10'
CH3
8'
10'
OH
2'
7'
6'
CH3
O
HO
2'
1'
O
6'
H3CO
O
1'
HO
O
O
a
OH
HO
b
O
H
H
OH
path b
OH
O
CH3
5a/5b
O
OH
OH
OH
2'
H3CO
O
1'
OH
Scheme 1. Possible biosynthetic pathways from hypothemycin/dihydrohypothemycin to dihydroisobenzofuranone–spiroacetal and dihydroisicoumarin derivatives.
orientation of H-20 was indicated by its large 1H–1H coupling
(J¼12.0 Hz) to Hax-30. The small J value (3.4 Hz) for H-40 and H-50, and
NOESY cross-peaks from H-40 to Hax-30, Heq-30, and H-50 with close
intensity placed H-40 in an equatorial position. A chair conformation
of the outer tetrahydropyran ring in CDCl3 was suggested by NOESY
correlations between Hax-70 and Hax-90, and between Hax-80 and
H-100, therefore, the C-110 methyl group occupied an equatorial po-
sition. To confirm the identical stereochemistry of 7 with aigialospirol
(6), these compounds were chemically correlated. Hydrogenation of
6 (H2, Pd/C, EtOAc, rt,15 h) gave a single product whose ESIMS and 1H
NMR spectroscopic data were identical to those of 7, thus, compound
7 is designated as 70,80-dihydroaigialospirol. In the previous report,9
the C-10 configuration of aigialospirol (6) was proposed solely based
on the observation of a weak NOESY cross-peak of H-5 and H-100, and
the absolute configuration of this molecule was proposed by analogy
to hypothemycin. Recently, Hsung and co-workers reported total
synthesis of 6.12 Since the synthetic route involves a 10-OH secondary
alcohol intermediate, which quickly epimerized under the reaction
conditions, the chirality of the starting material cannot be correlated
to the C-10 absolute configuration of the natural product. Fortunately,
the 2-O-(4-bromobenzyl) derivative 17, prepared from 6, upon crys-
tallization in MeOH, provided colorless needles suitable for X-ray
diffraction analysis (Fig. 3). This experiment also unambiguously
confirmed the absolute configuration of aigialospirol (6) as depicted
(10S,20R,40S,50S,60R,100S).
oxygen atom from 7. The 1H and 13C NMR spectra were closely re-
lated to those of 7, except for the replacement of an oxymethine (C-
40) to a methylene at dC 27.6 (dH 1.93 and 1.73). The gross structure of
8 was deduced by detailed analysis of COSY, HMQC, and HMBC data
in similar fashion as described for 7. The relative configuration of the
spirocyclic region was determined on the basis of the NOESY cor-
relations: H-20 to Hax-40, Hax-30 to H-50, H-50 to Heq-70, and Hax-80 to
H-100 (Fig. 4). Therefore, both tetrahydropyran rings adopt chair
conformation wherein H-20, H-50, and H-100 are placed in axial po-
sition. Aweak NOESY cross-peak of H-5 and H-100, as also found for 6
and 7, indicated the 10S-configuration. Compound 8 is, hence, des-
ignated as 40-deoxy-70,80-dihydroaigialospirol. The absolute config-
uration of 8 should be identical to that of the co-metabolites 6 and 7.
The molecular formula of compound 9 was determined to be
C20H24O9 by HRMS. The 1H and 13C NMR data and HMBC correlations
forthe resorcylicacidmoietywere consistent with thoseof 1and 2. A
trans-epoxide (C-10/C-20), attached to C-6, was connected at the
other side with a methylene (C-30), which was further bonded to an
oxymethine (C-40). The substructure from C-70 to C-110, including
a trans-olefin (C-70/C-80, J¼15.5 Hz), was addressed from COSY cor-
relations. The remaining fragments were a tertiary alcohol (dC 81.0)
and a methoxycarbonyl group (dC 174.2; dC 53.6, dH 3.83). Therefore,
the quaternarycarbon (dC 81.0, C-50)shouldlinkC-30 andC-70, and also
substituted with the methoxycarbonyl group. The absolute configu-
ration of the epoxide carbons (C-10 and C-20) and the lactone-linking
oxymethine (C-100) should be identical to all other co-metabolites
(10R,20R,100S-configuration). The 40S-isomer with a conformation
showninFigure 5 isconsistentwiththeobservedNOESY correlations;
The HRESIMS and 13C NMR spectroscopic data of compound 8
indicated its molecular formula to be C19H24O7, which is lacking one
OH
O
CH3
2
Table 3
O
H
Antimalarial and cytotoxic activities of aigialomycin-related compounds
7'
6'
CH3OH
Compound
Antimalarial (IC50
P. falciparum K1
,
m
g/mL)a
Cytotoxicity (IC50, m
g/mL)b
H3CO
OH
O
H
4'
5'
O
KB
BC
NCI-H187
Vero
2.1
OH
O
1'
CH3
OH
H O OCH3
1
4
2.8
>10
>10
>10
>10
>10
3.0
>20
>20
>20
>20
>20
>20
>20
dc
2.0
>20
>20
>20
>20
>20
3.6
19
O
H
>20
>20
>20
>20
>20
>50
>50
>50
>50
>50
5a/5b
6
7
8
7'
6'
H3CO
9
CH3OH
O
H
5'
OH
O
CH3
O
4'
O
O
11
2.6
0.77
18
7'
H
a
H3CO
OH
Inhibition of the proliferation of P. falciparum K1. Standard antimalarial drug,
g/mL.
The IC50 values of a standard compound, doxorubicin, against KB, BC, and NCI-
H187 were 0.18, 0.13, and 0.40 g/mL, respectively. Ellipticine was used as a standard
H
O
H
4'
5'
dihidroartemisinin, showed an IC50 value of 0.0012
m
O
6'
b
OCH3
O
m
20
compound for the cytotoxicity assay against Vero cells (IC50 0.40
m
g/mL).
c
Not tested.
Scheme 2. Proposed mechanisms for the production of 9 from aigialomycin A (2).

M. Isaka et al. / Tetrahedron 65 (2009) 4396–4403
4401
however, the configuration of the tertiary alcohol (C-50) could not be
proposed from available NMR spectroscopic data.
activities. For comparison, hypothemycin (1) and aigialospirol (6)
were also subjected to the same assays (Table 3). Non-macrolide
derivatives 4, 5a/5b, 6, 7, and 8 were inactive in these assays. Bio-
logical activities of 4-O-demethylhypothemycin (11) were similar to
those of hypothemycin (1).
Compound 10 possessed the same molecular formula as 9,
C20H24O9 (HRMS). The UV, IR, and IR data were similar to those of 9.
The only difference was that compound 10 possessed cis-olefinic
geometry (C-70/C-80, J¼12.4 Hz in acetone-d6, and J¼11.3 Hz in CDCl3).
Significant peak broadening was observed for the macrolactone
moiety in the 1H and 13C NMR spectra acquired in acetone-d6 and
CDCl3, in particular, the resonances of the olefinic carbons (C-70 and
C-80) were not detected and the HMQC spectra lacked corresponding
H–C correlations. Due to sample limitation, further NMR studies at
variable temperature or chemical correlation to 9 (by hydrogenation)
were notexamined, therefore, theconfigurationsatC-40 and C-50 of 10
remain unassigned.
3. Conclusion
In conclusion, reinvestigation of the mangrove fungus A. parvus
BCC 5311 resulted in the isolation of six new aigialomycin-related
compounds, including two dihydroisocoumarin-type derivatives,
aigialomycins F (4) and G (5), and their post-PKS biogenetic re-
lations were proposed. The compositions of the metabolites are
significantly influenced by the duration of incubation and also
differ in each fermentation batch. Taking together with our pre-
vious studies,8,9 the rarely investigated fungal species has been
proved to be a potent source of novel bioactive compounds.
We previously proposed that aigialospirol (6) should be bio-
genetically derived from hypothemycin (1).9 Similarly, likely pre-
cursors for dihydroisobenzofuranone–spiroacetal type compounds 7
and 8 are dihydrohypothemycin (known as a co-metabolite of 1 from
Hypomyces trichothecoides)1 and 40-deoxy-dihydrohypothemycin, re-
spectively, although we have not isolated these compounds in extracts
of A. parvus BCC 5311 (Scheme 1). One of the possible mechanisms
accounting for the production of the dihydroisobenzofuranone–spi-
roacetal derivatives is the hydrative epoxide cleavage by attack of
a hydroxyl oxygen to the benzylic carbon (C-10) with stereo inversion
(SN2) (path a), giving a 10S,20R-diol, which should subsequently un-
dergo intramolecular trans-lactonization and spiroacetal formation. An
alternate pathway involving hydrolysis of the ester linkage (path c) and
subsequent attack at the epoxide (C-10), giving the same final precursor
as path a, may also be possible. In the present study dihy-
droisocoumarin derivatives 4 and 5a/5b, possessing a 10R,20S-config-
uration, have been additionally isolated. The stereochemistry of 4
and 5a/5b can be correlated to aigialomycin B (3) and dihy-
drohypothemycin, respectively, with only difference was the inversion
of C-20 configuration. The dihydroisocoumarin formation can be
explained by two routes: (1) hydrative epoxide cleavage at C-20 (path b)
and subsequent intramolecular trans-lactonization, and (2) initial
macrolactone hydrolysis (path c) and attack of the resulting carboxyl
oxygen to the epoxide at C-20. Path c accounts for the appropriate
stereochemistry of both dihydroisobenzofuranone–spiroacetal (C-10
inversion) and dihydroisocoumarin (C-20 inversion) derivatives; how-
ever, this route is not consistent with the composition of isolated
compounds. Thus, 70Z-olefinic derivatives are absent in the dihy-
droisocoumarin series, whereas no 70E-olefinic derivative was isolated
in the dihydroisobenzofuranone series. The selectivity of dihy-
droisobenzofuranone and dihydroisocoumarin production could be
better explained by considering the competition of path a and path b,
which should be significantly influenced by the macrolide conforma-
tion. Further experimental evidences would be necessary for conclu-
sive proposal of the mechanisms of the transformations.
HPLC/UV analysis of an EtOAc extract from culture broth of BCC
5311 revealed the presence of aigialospirol (6) and aigialomycin F (4)
along with 1, 2, and 3, which was confirmed by co-injections of the
standard compounds. These results strongly suggested that both
dihydroisobenzofuranone–spiroacetal and dihydroisocoumarin de-
rivatives are not isolation artifacts. On the other hand, 13-membered
macrolides 9 and 10 were possibly produced, respectively, from
aigialomycin A (2) and hypothemycin (1), during extensive silica gel
(eluent, MeOH/CH2Cl2) and Sephadex LH-20 (eluent, MeOH) column
chromatography. Possible mechanisms for the production of 9 from
aigialomycin A (2) are proposed in Scheme 2. Air oxidation of 2 will
give a diketone 18, which should form hemiacetal 19 and/or 20 in the
presence of MeOH. Rearrangement from a 14-membered to 13-
membered macrolide could be explained by carbon migration of
hemiacetal 19 or 20.
4. Experimental
4.1. General procedures
Melting points were measured with an Electrothermal IA9100
digital melting point apparatus. Optical rotations were measured
with a JASCO D-1030 digital polarimeter. UV spectra were recorded
on an analytikjena SPEKOL 1200 UV–visible spectrophotometer.
FTIR spectra were taken on a Perkin–Elmer 2000 spectrometer.
NMR spectra were recorded on Bruker DRX400 and AV500D
spectrometers. ESI-TOF mass spectra were measured with Micro-
mass LCT and Bruker micrOTOF mass spectrometers.
4.2. Fungal material
A. parvus was collected, identified, and isolated from mangrove
wood by one of the authors (E. B. G. Jones). This fungus was de-
posited at the Thailand BIOTEC Culture Collection as BCC 5311 in
June 1999.9
4.3. Fermentation and isolation
A. parvus BCC 5311 was fermented in 80ꢀ1 L Erlenmeyer flasks
each containing 250 mL of potato dextrose broth (PDB) under static
conditions at 25 ꢁC for 80 days. The cultures were filtered, and the
filtrate (20 L) was extracted with an equal volume of EtOAc and
concentrated to a brown solid (5.41 g). This crude extract was
passed through a Sephadex LH-20 column (4.0ꢀ60 cm) using
MeOH as eluent to obtain a major fraction containing a complex
mixture of compounds (5.01 g) and a late-eluting fraction (314 mg)
mostly composed of hypothemycin. The former was subjected to
column chromatography (CC) on Si gel (5.0ꢀ20 cm, step gradient
elution with 0–40% MeOH/CH2Cl2) to furnish 12 fractions. Fraction
3 (192 mg, major components were hypothemycin and aigialo-
mycin A) was purified by combination of preparative HPLC using
a reverse phase column (NovaPak HR C18, 10
m
m, 4.0ꢀ10.0 cm;
MeCN/H2O¼40:60; flow rate, 20 mL/min) and CC on Si gel (5–30%
EtOAc/CH2Cl2) to furnish compound 8 (4.2 mg). Fraction 4 (1.16 g)
was triturated in MeOH and filtered. The filtrate was subjected to
preparative HPLC (MeCN/H2O¼25:75, then MeOH/H2O¼35:65) to
afford 7 (35.4 mg). The residual solid (877 mg) was mainly com-
posed of hypothemycin (1) and aigialomycin A (2) (ca. 2:1, 1H
NMR). Further fractionation of this material by repeated CC on Si
gel (CH2Cl2/MeOH and CH2Cl2/EtOAc) and preparative HPLC
(MeOH/H2O) provided 1 (522 mg), 2 (22 mg), 9 (2.5 mg), and 10
(1.2 mg). It was observed that aigialomycin A (2) was unstable
under these Si gel column chromatographic conditions, although
we did not experience such phenomenon in the previous studies.
Four new compounds 4, 5a/5b, 7, and 8, and 4-O-demethylhy-
pothemycin (11) were tested for antimalarial and cytotoxic

4402
M. Isaka et al. / Tetrahedron 65 (2009) 4396–4403
Fraction 5 (321 mg) was subjected to preparative HPLC (MeCN/
H2O¼50:50) and CC on Si gel (5–30% acetone in CH2Cl2) to obtain 6
(98 mg). Fraction 6 (272 mg) was fractionated by CC on Si gel
(3.0ꢀ15 cm, 1–25% MeOH in CH2Cl2) to furnish 12 (11.6 mg) and 5a/
5b (20.5 mg). Fraction 7 (253 mg) was repeatedly fractionated by
CC on Si gel (2–10% MeOH in CH2Cl2) and preparative HPLC (MeCN/
H2O and MeOH/H2O) to furnish 7 (21.9 mg) and 12 (44.8 mg).
Fraction 8 (1.18 g) was repeatedly fractionated by CC on Si gel
(MeOH/CH2Cl2) to afford 11 (98.9 mg), 3 (203 mg), and 4 (23.6 mg).
Fraction 10 (427 mg) also provided 4 (57.4 mg).
J¼2.7 Hz, H-3), 5.72 (1H, br, H-80), 5.57 (1H, m, H-100), 5.52 (1H, br d,
J¼12.4 Hz, H-70), 4.44 (1H, br s, 50-OH), 4.39 (1H, m, H-40), 4.29 (1H,
br d, J¼7.3 Hz, 40-OH), 4.17 (1H, br s, H-10), 3.83 (3H, s, 4-OCH3), 3.74
(3H, s, –COOCH3), 2.84 (1H, m, H-20), 2.52–2.48 (2H, m, H-90), 2.09–
2.05 (2H, m, H-30), 1.49 (3H, d, J¼6.4 Hz, H-110); 13C NMR (125 MHz,
acetone-d6) d
173.7 (C, C-60),171.3 (C, –COO–),166.1 (C, C-2),165.1 (C,
C-4), 143.1 (C, C-6), 103.8 (CH, C-5), 103.6 (C, C-1), 100.2 (CH, C-3),
81.6 (C, C-50), 73.6 (CH, C-40), 72.5 (CH, C-100), 62.1 (CH, C-20), 58.1
(CH, C-10), 55.0 (CH3, 4-OCH3), 51.9 (CH3, –COOCH3), 36.4 (CH2, C-30),
34.1 (CH2, C-90), 17.8 (CH3, C-110), resonances for C-70 and C-80 were
not clearly detected because of the peak broadening; HRMS (ESI-
TOF) m/z 431.1322 [MþNa]þ (calcd for C20H24O9Na 431.1318).
4.3.1. Aigialomycin F (4)
27
Colorless solid; mp 186–187 ꢁC; [
a]
ꢂ39 (c 0.10, MeOH); UV
D
(MeOH) lmax (log
3
) 214 (4.42), 266 (4.15), 301 (3.85) nm; IR (KBr)
4.4. Synthesis of the acetonide derivatives 13 and 14
nmax 3363, 1637, 1379, 1202, 1060, 1034, 712 cmꢂ1
;
1H NMR
(500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see
Table 1; HRMS (ESI-TOF) m/z 421.1464 [MþNa]þ (calcd for
C19H26O9Na 421.1469).
To a suspension of aigialomycin F (4, 2.0 mg) in 2,2-dimethoxy-
propane (0.5 mL) was added p-TsOH$H2O (ca. 0.5 mg), and the
mixture was stirred at room temperature for 4 h. The mixture was
diluted with EtOAc and washed with satd NaHCO3. The organic
layer was concentrated under reduced pressure to leave a colorless
solid, which was purified by preparative HPLC using a reverse phase
column (MeCN/H2O¼45:55) to furnish compound 13 (1.2 mg) as
a colorless solid. Similarly, compound 14 (0.3 mg) was synthesized
from aigialomycin G (5a/5b, 1.0 mg).
4.3.2. Aigialomycin G (5a/5b)
27
Colorless solid; mp 94–95 ꢁC; [
a
]
ꢂ35 (c 0.10, MeOH); UV
D
(MeOH) lmax (log
nmax 3424, 1713, 1677, 1650, 1629, 1584, 1372, 1254, 1206, 1162,
1032 cmꢂ1 1H NMR (500 MHz, acetone-d6) and 13C NMR
3) 213 (4.46), 267 (4.18), 303 (3.89) nm; IR (KBr)
;
(125 MHz, acetone-d6) data, see Table 1; HRMS (ESI-TOF) m/z
421.1461 [MþNa]þ (calcd for C19H26O9Na, 421.1469).
4.4.1. Compound 13
Colorless solid; 1H NMR (500 MHz, CDCl3)
d 11.19 (1H, s, 2-OH),
4.3.3. 70,80-Dihydroaigialospirol (7)
6.52 (1H, dd, J¼2.4, 1.2 Hz, H-5), 6.44 (1H, d, J¼2.4 Hz, H-3), 5.88 (1H,
dt, J¼15.4, 7.3 Hz, H-80), 5.63 (1H, dd, J¼15.4, 7.5 Hz, H-70), 4.89 (1H, d,
J¼10.6 Hz, H-10), 4.35 (1H, dt, J¼5.6, 10.6 Hz, H-20), 4.42 (1H, t,
J¼7.7 Hz, H-60), 4.03 (1H, dd, J¼11.2, 5.2 Hz, H-40), 3.89 (1H, m, H-100),
3.88 (3H, s, 4-OCH3), 3.75 (1H, dd, J¼7.9, 5.2 Hz, H-50), 2.38 (1H, dd,
J¼13.5, 5.6 Hz, Ha-30), 2.28–2.26 (2H, m, H-90), 1.90 (1H, dt, J¼13.5,
11.2 Hz, Hb-30),1.49 (3H, s, H-100),1.48 (3H, s, H-300),1.46 (3H, s, H-1%),
1.43 (3H, s, H-3%), 1.24 (3H, d, J¼6.3 Hz, H-110); 13C NMR (125 MHz,
28
Colorless solid; mp 80–83 ꢁC; [
a
]
þ14 (c 0.10, CHCl3); UV
D
(MeOH) lmax (log 3) 217 (4.47), 257 (4.20), 290 (3.81) nm; IR (KBr)
nmax 3459 (br),1749,1614,1216,1158,1073 cmꢂ1; 1H NMR (500 MHz,
CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 2; HRMS (ESI-
TOF) m/z 403.1372 [MþNa]þ (calcd for C19H24O8Na 403.1369).
4.3.4. 40-Deoxy-70,80-dihydroaigialospirol (8)
28
Colorless solid; mp 70–71 ꢁC; [
a]
D
þ12 (c 0.10, CHCl3); UV
CDCl3) d 168.4 (C, –COO–), 165.1 (C, C-4), 164.7 (C, C-2), 143.1 (C, C-6),
(MeOH) lmax (log
3
) 217 (4.44), 257 (4.05), 292 (3.64) nm; IR (KBr)
131.5 (CH, C-80),131.3 (CH, C-70),109.3 (C, C-2%),104.4 (CH, C-5),102.2
(C, C-200), 99.8 (C, C-1), 99.8 (CH, C-3), 82.5 (CH, C-50), 79.1 (CH, C-60),
79.0 (CH, C-20), 67.5 (CH, C-10), 67.5 (CH, C-40), 67.2 (CH, C-100), 55.6
(CH3, 4-OCH3), 42.1 (CH2, C-90), 37.0 (CH2, C-30), 27.1 (CH3, C-1%), 26.8
(CH3, C-3%), 24.9(CH3, C-100), 24.9(CH3, C-300), 23.0 (CH3, C-110);HRMS
(ESI-TOF) m/z 501.2095 [MþNa]þ (calcd for C25H34O9Na 501.2101).
nmax 3488, 3385,1733,1614,1221,1168,1085,1075,976 cmꢂ1;1HNMR
(500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 2;
HRMS (ESI-TOF) m/z 387.1412 [MþNa]þ (calcd for C19H24O7Na
387.1420).
4.3.5. Compound 9
25
Colorless solid; mp 128–129 ꢁC; [
a
]
þ8 (c 0.06, MeOH); UV
4.4.2. Compound 14
D
(MeOH) lmax (log
nmax 3444, 1734, 1645, 1615, 1257, 1159 cmꢂ1
500 MHz)
3
) 214 (4.17), 265 (3.83), 305 (3.52) nm; IR (KBr)
Colorless solid; 1H NMR (500 MHz, CDCl3)
d 11.14 (1H, s, 2-OH),
;
1H NMR (CDCl3,
6.68 (1H, dd, J¼2.4, 1.3 Hz, H-5), 6.40 (1H, d, J¼2.2 Hz, H-3), 5.10
(1H, d, J¼10.9 Hz, H-10), 4.52 (1H, dt, J¼2.5, 10.9 Hz, H-20), 4.51 (1H,
m, H-40), 4.24 (1H, d, J¼7.9 Hz, H-50), 3.94 (1H, m, H-100), 3.85 (3H, s,
4-OCH3), 2.54 (1H, ddd, J¼14.2, 11.3, 2.3 Hz, Ha-30), 2.46 (1H, ddd,
J¼14.2, 4.5, 2.5 Hz, Hb-30), 2.03 (1H, m, Ha-70), 1.96 (1H, m, Ha-80),
1.93 (1H, m, Hb-70),1.72 (1H, m, Ha-90),1.70 (1H, m, Hb-80),1.40 (3H,
s, H-100), 1.39 (1H, m, Hb-90), 1.32 (3H, s, H-300), 1.20 (3H, d, J¼6.2 Hz,
d
11.75 (1H, s, 2-OH), 6.51 (1H, d, J¼2.7 Hz, H-5), 6.42 (1H,
d, J¼2.7 Hz, H-3), 6.21 (1H, ddd, J¼15.5, 8.2, 5.6 Hz, H-80), 5.52 (1H,
m, H-100), 5.51 (1H, br d, J¼15.5 Hz, H-70), 4.55 (1H, d, J¼1.7 Hz, H-
10), 4.05 (1H, m, H-40), 3.83 (3H, s, –COOCH3), 3.82 (3H, s, 4-OCH3),
2.72 (1H, dt, J¼8.9, 2.1 Hz, H-20), 2.57 (1H, m, Ha-90), 2.44 (1H, ddt,
J¼15.7,1.6, 5.6 Hz, Hb-90), 2.37 (1H, br d, J¼10.7 Hz, 40-OH), 2.07 (1H,
dt, J¼15.5, 2.7 Hz, Ha-30), 2.03 (1H, ddd, J¼15.5, 8.9, 5.6 Hz, Hb-30),
H-110); 13C NMR (125 MHz, CDCl3)
d 169.4 (C, –COO–),166.5 (C, C-4),
1.45 (3H, d, J¼6.5 Hz, H-110); 13C NMR (CDCl3, 125 MHz)
d
174.2 (C,
164.4 (C, C-2), 144.3 (C, C-6), 107.7 (C, C-200), 103.4 (CH, C-5), 100.5
(C, C-1), 100.0 (C, C-60), 99.5 (CH, C-3), 78.8 (CH, C-50), 77.2 (CH, C-
20), 69.8 (CH, C-40), 69.8 (CH, C-100), 67.5 (CH, C-10), 55.5 (CH3, 4-
OCH3), 35.2 (CH2, C-30), 30.2 (CH2, C-70), 30.0 (CH2, C-90), 26.2 (CH3,
C-100), 23.9 (CH3, C-300), 21.8 (CH3, C-110), 16.6 (CH2, C-80); HRMS
(ESI-TOF) m/z 443.1686 [MþNa]þ (calcd for C22H28O8Na 443.1682).
C-60), 170.7 (C, –COO–), 165.4 (C, C-2), 165.1 (C, C-4), 142.3 (C, C-6),
129.8 (CH, C-70), 127.3 (CH, C-80), 104.4 (C, C-1), 104.0 (CH, C-5),
100.8 (CH, C-3), 81.0 (C, C-50), 72.8 (CH, C-40), 70.7 (CH, C-100), 63.4
(CH, C-20), 58.2 (CH, C-10), 55.5 (CH3, 4-OCH3), 53.6 (CH3, –COOCH3),
36.6ꢀ2 (CH2, C-30; and CH2, C-90), 19.0 (CH3, C-110); HRMS (ESI-TOF)
m/z 431.1315 [MþNa]þ (calcd for C20H24O9Na 431.1318).
4.5. Hydrogenation of aigialomycin F (4)
4.3.6. Compound 10
25
Colorless solid; mp 156–159 ꢁC; [
a
]
þ21 (c 0.065, MeOH); UV
To a solution of aigialomycin F (7, 3.0 mg) in THF (0.5 mL) was
added 10% Pd/C (5 mg), and the mixture was vigorously stirred
under hydrogen for 3 h. The suspension was filtered, and the filtrate
was concentrated in vacuo to leave a colorless solid, which was
D
(MeOH) lmax (log
nmax 3460,1747,1644,1618,1260 cmꢂ1; 1H NMR (500 MHz, acetone-
d6)
12.15 (1H, s, 2-OH), 6.52 (1H, d, J¼2.7 Hz, H-5), 6.41 (1H, d,
3) 217 (4.17), 265 (3.87), 305 (3.56) nm; IR (KBr)
d

M. Isaka et al. / Tetrahedron 65 (2009) 4396–4403
4403
purified by preparative HPLC using a reverse phase column (MeCN/
H2O¼25:75) to furnish 15 (1.4 mg) as a colorless gum.
OH or 40-OH), 2.03–1.89 (4H, m, H-30 and H-90), 1.23 (3H, d,
J¼6.3 Hz, H-110); HRMS (ESI-TOF) m/z 569.0785 and 571.0771
[MþNa]þ (calcd for C26H27O879BrNa 569.0782; calcd for
C26H27O881BrNa 571.0765).
4.5.1. 70,80-Dihydroaigialomycin F (15)
Colorless gum; 1H NMR (500 MHz, CD3OD)
d 6.66 (1H, d,
J¼2.3 Hz, H-5), 6.46 (1H, d, J¼2.3 Hz, H-3), 4.68 (1H, ddd, J¼10.6,
7.4, 2.4 Hz, H-20), 4.58 (1H, d, J¼7.4 Hz, H-10), 3.98 (1H, ddd, J¼10.9,
7.5, 1.8 Hz, H-40), 3.86 (3H, s, 4-OCH3), 3.80 (1H, m, H-60), 3.73 (1H,
m, H-100), 3.21 (1H, dd, J¼7.5, 2.2 Hz, H-50), 2.06 (1H, d, J¼14.6, 10.6,
1.8 Hz, Ha-30), 1.90 (1H, ddd, J¼14.6, 10.9, 2.4 Hz, Hb-30), 1.58–1.55
(2H, m, H-70), 1.54 (1H, m, Ha-80), 1.48–1.46 (2H, m, H-90), 1.39 (1H,
m, Hb-80), 1.15 (3H, d, J¼6.2 Hz, H-110); 13C NMR (125 MHz, CD3OD)
4.9. X-ray crystallographic analysis of 17
Crystal data forcompound 17 at 298(2) K: C26H27O8Br, Mr¼547.40,
monoclinic, space group P21 (No. 4) with a¼6.2147(1) Å,
b¼14.5504(7) Å, c¼13.5813(6) Å,
b
¼90.669(3)ꢁ, V¼1228.02(8) Å3,
Z¼2, Dcalcd¼1.480 Mg/m3. F000¼564,
l(Mo
Ka
)¼0.71073 Å,
m
¼1.72 mmꢂ1
.
Data collection and reduction: crystal size
range 1.00–26.02ꢁ, 9204 reflection collected,
(I):
d
168.5 (C, –COO–), 166.7 (C, C-4), 164.0 (C, C-2), 144.7 (C, C-6), 104.5
0.10ꢀ0.15ꢀ0.20 mm,
q
(CH, C-5), 100.3 (C, C-1), 99.9 (CH, C-3), 80.3 (CH, C-20), 76.1 (CH, C-
50), 70.0 (CH, C-60), 67.5 (CH, C-10), 67.1 (CH, C-100), 66.8 (CH, C-40),
54.9 (CH3, 4-OCH3), 38.8 (CH2, C-90), 35.8 (CH2, C-30), 33.3 (CH2, C-
70), 22.1 (CH3, C-110), 21.8 (CH2, C-80); HRMS (ESI-TOF) m/z 401.1813
[MþHþ] (calcd for C19H29O9 401.1811).
4250 independent reflections (Rint¼0.048), final R indices I>2
s
0.0434, wR2¼0.1234 for 316 parameters, GOF¼1.021. Flack parame-
ter¼0.019(11). Intensity data were measured on a Bruker–Nonius
kappaCCDdiffractometer. Crystallographicdata forthe structure 17 in
this paper have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication number CCDC-711607.
Copies of the data can be obtained, free of charge on application to
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: þ44 (0)1223
336033 or e-mail: deposit@ccdc.cam.ac.uk].
4.6. NaBH4 reduction of aigialomycin G (5a/5b)
To a mixture of NaBH4 (2 mg) in THF (0.2 mL) was added
aigialomycin G (1.0 mg) in THF (0.2 mL), and the mixture was stir-
red for 30 min. The reaction was terminated by addition of H2O, and
the resulting mixture was partially concentrated by evaporation.
The residue was extracted twice with EtOAc, and the combined
organic layer was concentrated in vacuo to obtain a colorless gum
(0.9 mg). The 1H NMR (500 MHz, CD3OD) spectrum of the crude
product suggested the presence of a 1:5 mixture of the di-
astereomeric C-60 alcohols. The resonances of the minor isomer
were identical to those of 15. 1H NMR spectral data (500 MHz,
CD3OD) for the major isomer 16 were assigned by 1H–1H COSY;
4.10. Biological assays
Assay for activity against Plasmodium falciparum (K1, multi-drug
resistant strain) was performed using the microculture radioiso-
tope technique described by Desjardins et al.13 Cytotoxicity against
KB cells (oral human epidermoid carcinoma), BC cells (human
breast cancer), NCI-H187 cells (human small-cell lung cancer), and
Vero cells (African green monkey kidney fibroblasts) were evalu-
ated using the resazurin microplate assay.14
d
6.66 (1H, d, J¼2.3 Hz, H-5), 6.46 (1H, d, J¼2.3 Hz, H-3), 4.68 (1H,
m, H-20), 4.58 (1H, d, J¼7.4 Hz, H-10), 4.03 (1H, m, H-40), 3.86 (3H, s,
4-OCH3), 3.74 (1H, m, H-100), 3.58 (1H, m, H-60), 3.39 (1H, t,
J¼6.3 Hz, H-50), 1.99–1.98 (2H, m, H-30), 1.70–1.42 (6H, m, H-70, H-80,
and H-90), 1.16 (3H, d, J¼6.2 Hz, H-110).
Acknowledgements
P.K. would like to acknowledge the Center of Excellence for In-
novation in Chemistry (PERCH-CIC), Commission on Higher Edu-
cation, Ministry of Education for financial support.
4.7. Hydrogenation of 6
References and notes
To a solution of aigialospirol (6, 3.0 mg) in EtOAc (0.5 mL) was
added 10% Pd/C (5 mg), and the mixture was vigorously stirred
under hydrogen for 15 h. The suspension was filtered, and the fil-
trate was concentrated in vacuo to leave a colorless solid (2.8 mg).
The 1H NMR (400 MHz, CDCl3) spectra and ESIMS data were
identical to those of 7.
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R.; Carey, S. T.; James, J. C. Tetrahedron 1981, 37, 2445–2449.
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4.8. Synthesis of compound 17
To a solution of aigialospirol (6, 5.0 mg) and 4-bromobenzyl
bromide (8.0 mg) in 2-butanone (0.3 mL) was added K2CO3
(10 mg), and the mixture was stirred at room temperature for 3
days. The mixture was diluted with EtOAc and washed with H2O.
The organic layer was concentrated under reduced pressure to
leave a pale brown solid (11.7 mg), which was purified by CC on Si
gel (MeOH/CH2Cl2) to furnish compound 17 (5.9 mg) as a colorless
solid. Recrystallization in MeOH gave colorless needles.
5. Ninomiya-Tsuji, Y.; Kajino, T.; Ono, K.; Ohtomo, T.; Matsumoto, M.; Shiina, M.;
Mihara, M.; Tsuchiya, M.; Matsumoto, K. J. Biol. Chem. 2003, 278, 18485–18490.
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aranonth, Y. J. Org. Chem. 2002, 67, 1561–1566.
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10. Total synthesis of aigialomycin D: (a) Geng, X.; Danishefsky, S. J. Org. Lett. 2004,
6, 413–416; (b) Barluenga, S.; Dakas, P.-Y.; Ferandin, Y.; Meijer, L.; Winssinger, N.
Angew. Chem., Int. Ed. 2006, 45, 3951–3954; (c) Lu, J.; Ma, J.; Xie, X.; Chen, B.;
She, X.; Pan, X. Tetrahedron: Asymmetry 2006, 17, 1066–1073; (d) Vu, N. Q.; Chai,
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4.8.1. 2-O-(4-Bromobenzyl)aigialospirol (17)
Colorless needles (MeOH); mp 173–175 ꢁC; 1H NMR (500 MHz,
CD3OD)
d
7.51 (2H, d, J¼8.4 Hz, 4-bromobenzyl), 7.37 (2H, d, J¼
8.4 Hz, 4-bromobenzyl), 6.62 (1H, br s, H-5), 6.41 (1H, d, J¼1.6 Hz,
H-3), 6.15 (1H, m, H-80), 5.66 (1H, m, H-70), 5.24 (1H, d, J¼6.2 Hz,
H-10), 5.20 (2H, s, 4-bromobenzyl), 4.09–4.03 (2H, m, H-20 and
H-40), 3.97 (1H, m, H-100), 3.80 (3H, s, 4-OCH3), 3.51 (1H, m, H-50),
3.41 (1H, d, J¼10.6 Hz, 40-OH or 50-OH), 2.56 (1H, d, J¼10.4 Hz, 50-