Hamigeromycins C-G, 14-membered macrolides from the fungus Hamigera avellanea BCC 17816

Article abstract of DOI:10.1016/j.tet.2009.11.101

Five new 14-membered macrolides, hamigeromycins C-G, together with the previously described compounds, hamigeromycin A and 89-250904-F1 (radicicol analog A), were isolated from the fungus Hamigera avellanea BCC 17816. Hamigeromycins A, C, D, and E are stereoisomers differing from one another in the absolute configurations of the 4′,5′-diol moiety. Hamigeromycins F and G are unusual 5′-keto-analogs, and they are 6′-epimers to each other. The structures and the stereochemistry of the new compounds were deduced by analyses of the NMR spectroscopic and mass spectrometry data in combination with chemical means.

Full text of DOI:10.1016/j.tet.2009.11.101

Tetrahedron 66 (2010) 955–961
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Tetrahedron
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Hamigeromycins C–G, 14-membered macrolides from the fungus Hamigera
avellanea BCC 17816
*
Masahiko Isaka , Panida Chinthanom, Surisa Kongthong, Sumalee Supothina, Pataranun Ittiworapong
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2009
Received in revised form 2 November 2009
Accepted 20 November 2009
Available online 27 November 2009
Five new 14-membered macrolides, hamigeromycins C–G, together with the previously described
compounds, hamigeromycin A and 89-250904-F1 (radicicol analog A), were isolated from the fungus
Hamigera avellanea BCC 17816. Hamigeromycins A, C, D, and E are stereoisomers differing from one
another in the absolute configurations of the 40,50-diol moiety. Hamigeromycins F and G are unusual 50-
keto-analogs, and they are 60-epimers to each other. The structures and the stereochemistry of the new
compounds were deduced by analyses of the NMR spectroscopic and mass spectrometry data in com-
bination with chemical means.
Keywords:
Hamigera avellanea
Hamigeromycin
Resorcylic acid lactone
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
which was the same as the most abundant macrolide constituent,
hamigeromycin A (1). The UV, IR, and 1H and 13C NMR spectra of 2
Recently, we reported the isolation of two new 14-membered
nonaketide macrolides, hamigeromycins A (1) and B (8), and two
novel cyclopropyl diketones, hamavellones A and B, together with
the known macrolide 89-250904-F1 (radicicol analog A, 7), pseur-
otin A, and four anthraquinone derivatives from the soil fungus
Hamigera avellanea BCC 17816.1 Since this fungal strain proved to be
a potent source of bioactive compounds, we have further chemi-
cally investigated its secondary metabolites by altering a liquid
fermentation medium. When the fungus was fermented in peptone
yeast glucose medium (PYGM, see Experimental Section), the
production of hamigeromycin A (1) and related macrolides was
efficient, whilst metabolites of other chemical classes (hama-
vellones, pseurotin A, and anthraquinones) were absent. A 7 L fer-
mentation broth (PYGM) provided five new macrolides,
hamigeromycins C–G (2–6), along with two major constituents,
hamigeromycin A (1)1 and 89-250904-F1 (7).2,3 This culture extract
lacked hamigeromycin B (8), which was isolated in very low quan-
tity in our previous study. Herein, we report the structure elucida-
tion and assignments of the stereochemistry of the new macrolides.
were similar to those of 1 (Tables 1 and 2). The planar structure of 2
was elucidated on the basis of the 2D NMR (COSY, NOESY, HMQC,
and HMBC) spectroscopic data (Fig. 1). Thus, the structure of the
pentasubstituted benzene ring was addressed from the HMBC
correlations: from a chelated OH proton (dH 11.41, 2-OH) to C-1, C-2,
and C-3, from H-3 (dH 6.44, s) to C-1, C-2, C-4, and C-5, and from
methoxy protons resonating at dH 3.88 (3H, s) and 3.60 (3H, s),
respectively, to C-4 and C-5. The aromatic methine proton (H-3)
showed a weak four-bond correlation to the dC 170.6 carbonyl,
which confirmed the attachment of the ester carbonyl to C-1. The
benzene ring was attached to a trans-olefin (C-10/C-20) at C-6 po-
sition as indicated by the HMBC correlations from the olefinic
proton H-10 (dH 6.72, d, J¼16.3 Hz) to C-1, C-5, and C-6, and from H-
20 (dH 6.03, ddd, J¼16.3, 7.8, 6.2 Hz) to C-6. The connection from C-10
to C-50 as well as the local structure from C-70 to C-110 were
addressed on the basis of the COSY correlations. The dC 210.9 ketone
was placed in the C-60 position, since the HMBC spectrum exhibited
correlations from H-50, H2-70, and H2-80 to this carbon. The mac-
rolactone ring was required to account for a downfield shift of H-100
(dH 5.31) and from the molecular formula (HRMS). Thus, the planar
structure of hamigeromycin C (2) was established to be the same as
that of hamigeromycin A (1). Hamigeromycins D (3) and E (4) also
possessed the same molecular formula (C20H26O8, HRESIMS), and
detailed analyses of the NMR spectroscopic data in the similar
manner as described above led to the establishment of the same
planar structure as 1 and 2 (Tables 1 and 2). These results implied
that compounds 1–4 are diastereomers differing from each one
other by the absolute configurations of the 40,50-diol chiral centers.
2. Results and discussion
Hamigeromycin C (2) was isolated as a colorless solid, and its
molecular formula was established to be C20H26O8 by HRESIMS,
* 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.2009.11.101

956
M. Isaka et al. / Tetrahedron 66 (2010) 955–961
11'
Table 2
1H NMR data for hamigeromycins A (1), C (2), D (3), and E (4) (CDCl3, 500 MHz)
OH
O
CH3
OH
O
CH3
2
1
3
9'
8'
10'
Position
1
2
3
4
O
O
2-OH
3
4-OCH3 3.89, s
5-OCH3 3.60, s
10
20
30
11.94, s
6.44, s
11.41, s
6.44, s
3.88, s
3.60, s
6.72, d (16.3)
10.96, s
6.46, s
3.89, s
3.64, s
6.73, d (16.3)
10.68, s
6.43, s
3.89, s
3.62, s
6.43, d (16.0)
1'
4
6
7'
6'
H3CO
HO
H3CO
H3CO
HO
5
H3CO
2'
5'
4'
O
O
3'
6.64, dd
(15.9, 1.9)
6.03, ddd
(15.9, 10.7, 3.1) (16.3, 7.8, 6.2)
2.70, m
OH
OH
1
2
6.04, ddd
5.98, ddd
(16.3, 8.4, 6.3)
2.71, dt
(13.9, 8.8)
2.61, m
6.39, ddd
(16.0, 8.5, 3.8)
2.68, m
2.74, m
OH
O
CH3
OH
O
CH3
2.18, ddd
(15.8, 10.7, 5.0)
4.11, m
2.57, m
2.26, ddd
(15.4, 8.5, 5.0)
4.21, m
4.45, d (2.4)
2.68, m; 2.64, m
O
O
40
50
70
3.99, m
4.33, m
4.04, m
4.45, d (2.7)
H3CO
HO
H3CO
H3CO
HO
4.42, d (1.7)
H3CO
2.81, m; 2.42, m 3.01, m; 2.41, m 2.88, ddd
(15.6, 6.8, 4.7)
O
O
OH
2.43, ddd
(15.6, 10.7, 6.7)
OH
3
4
80
1.88, m; 1.76, m 1.89, m; 1.78, m 1.90, m; 1.77, m 1.87, m; 1.82, m
1.75, m; 1.73, m 1.77, m; 1.76, m 1.74, m; 1.69, m 1.84, m; 1.70, m
90
100
110
5.16, m
1.40, d (6.2)
5.31, m
1.40, d (6.3)
5.25, m
1.33, d (6.2)
5.13, m
1.38, d (6.2)
OH
O
CH3
OH
O
CH3
O
O
H
O
H3CO
H3CO
H3CO
H3CO
O
COSY
HMBC
2
OH
OH
1
O
HO 8'
O
O
6
5
6
7'
1'
H3CO
H3CO
5'
OH
O
CH3
O
2'
6'
O
OH
O
CH3
O
OH
O
HO
H3CO
H3CO
O
Figure 1. Proposed planar structure of hamigeromycin C (2).
H3CO
H3CO
O
OH
OH
gave respective acetonide derivatives 9–12 (Fig. 2). Interestingly,
the major reaction product from hamigeromycins C (2) and D (3)
were the C-60 dimethyl acetal derivatives, whereas the ketone
functionality was retained in the reactions of hamigeromycins A (1)
and E (4). The acetonide derivatives 9–12 were purified by
8
7
The 40S,50S-configuration of hamigeromycin A (1) was pre-
viously established by chemical correlation with 7.1 To assign the
absolute configurations of the other three isomers, compounds 1–4
were treated with p-TsOH$H2O in 2,2-dimethoxypropane, which
OH
O
CH3
OH
O
CH3
O
O
Table 1
H3CO
H3CO
H3CO
H3CO
13C NMR data for hamigeromycins A (1), C (2), D (3), and E (4) (CDCl3, 125 MHz)
4'
S
H
O
H
O
5'S
OCH3
O
OCH3
Position
Mult.
1
2
3
4
2"
H
H
1"
O
O
CH3
1-COO–
1
2
3
C
C
C
CH
C
CH3
C
CH3
C
CH
CH
CH3
CH
CH
C
CH3
CH2
CH2
CH
CH3
171.2
103.6
161.5
99.8
158.9
55.9
140.0
60.4
133.5
126.9
130.4
38.0
73.3
79.8
209.7
39.5
20.9
170.6
103.8
160.7
99.9
158.7
55.9
140.5
60.3
133.4
128.7
129.3
36.0
71.4
75.5
210.9
40.2
19.0
170.7
104.7
159.8
99.9
158.2
55.9
140.6
60.2
133.2
128.7
129.9
38.2
71.1
75.7
211.2
38.7
20.8
170.6
105.3
159.4
99.7
158.4
55.9
140.7
59.9
131.7
127.2
130.4
38.0
72.8
80.4
210.1
39.6
20.2
3"
CH3
H3C
H3C
10
9
4
4-OCH3
5
5-OCH3
OH
O
CH3
OH
O
CH3
6
O
10
O
20
H3CO
H3CO
30
H CO
3
H
O
4'R
5'R
OCH3
OCH3
H
O
40
H3CO
O
H
50
H
O
CH3
60
O
CH3
H3C
70
H3C
80
90
34.9
73.0
20.1
35.0
72.7
20.2
34.9
73.0
20.7
35.1
73.3
19.6
11
12
100
110
Figure 2. Structures and key NOESY correlations of acetonide derivatives 9–12.

M. Isaka et al. / Tetrahedron 66 (2010) 955–961
957
had to be the 40R,50S-isomer and the other the 40S,50R-isomer.
Conformational analysis of their acetonide derivatives, 10 and 11,
based on the available NMR spectroscopic data (1H–1H J-values and
NOESY correlations) did not lead to a conclusive proposal of their
stereochemistry. Therefore, we employed the exciton chirality
method.4 Compounds 2 and 3 were converted to their tris-p-
dimethylaminobenzoate derivatives 13 and 14, respectively. The
circular dichroism (CD) spectrum of 13 in MeOH showed a negative
first Cotton effect (330 nm, D3¼ꢀ16.0) and a positive second Cotton
effect (307 nm, D3¼þ22.4) centered at the p-dimethylamino-
benzoate chromophore lmax (318 nm). In contrast, the CD spectrum
of 14 exhibited a positive first Cotton effect (327 nm, D3¼þ26.1)
OR
O
CH3
OR
O
CH3
O
H
O
H
H3CO
H3CO
H3CO
H3CO
4'R
5'S
4'S
5'R
O
O
RO
RO
H
H
RO
RO
13
14
R = p-dimethylaminobenzoyl
O
and a negative second Cotton effect (304 nm, D3¼ꢀ8.5). The 1H
Ar
O
3
H4'
C6'
C3'
0
0
NMR spectra of 13 and 14 in CD3OD showed JH4 –H5 values of
4.5 and 4.8 Hz, respectively. Figure 3 shows Newman projections
of four possible conformations for the 40R,50S-isomer; A1, A2, B,
and C. Examinations of the macrolide ring conformations with
a molecular model strongly suggested that conformations cor-
responding to the projections B and C are disfavored due to
steric hindrance of the two p-dimethylaminobenzoyloxy groups,
which would be forced into close contact with the macrocyclic
ring. On the basis of the exciton chirality rule, compound
13, exhibiting negative chirality, was proposed to possess the
40R,50S configuration, while in turn 14 was assigned as the
40S,50R-isomer.
H4'
O
Ar
O
O
C6'
C3'
O
O
H5'
O
H5'
Ar
Ar
A2
A1
H4'
H5'
Ar
H4'
H5'
C6'
O
O
O
O
C3'
O
C3'
O
O
C6'
The molecular formula of hamigeromycin F (5) was determined
by HRESIMS to be C20H26O7, which was lacking one oxygen atom
compared to compounds 1–4. The 1H and 13C NMR, DEPT135, and
HMQC spectroscopic data demonstrated that an oxymethine (C-40)
of 1 was replaced by a methylene in 5. The HMBC correlations
(Table 3) revealed that the pentasubstituted benzene moiety was
identical to 1. The connection from C-10 to C-40 and the linkage from
C-60 to C-110 was addressed by COSY and HMBC correlations. HMBC
correlations from H2-30 (dH 2.81 and 2.55), H-70 (dH 2.06), and 60-OH
to the ketone carbon at dC 211.5 indicated that this ketone should be
placed in the C-50 position. Consequently, hamigeromycin F (5) was
identified as a 40-deoxy derivative of 1–4, and the positions of the
ketone and the remaining secondary alcohol functions were ex-
changed. The molecular formula of hamigeromycin G (6) was de-
termined to be C20H26O7 (HRESIMS), which was the same as 5.
Detailed analyses of the NMR spectroscopic data (Table 3) resulted
Ar
Ar
Ar
O
B
C
Figure 3. Newman projections showing possible local conformations (A1, A2, B, and C)
in the 40R,50S-isomer (13).
preparative HPLC (ODS column) and the gross structures were
confirmed by HRMS and interpretation of the 2D NMR spectro-
scopic data. The NOESY correlations indicated that 9 and 12 were
cis-acetonide derivatives, whereas 10 and 11 exhibited trans-ace-
tonide configurations (Fig. 2). Since hamigeromycin A (1) possesses
40S,50S configuration, the other cis-acetonide from hamigeromycin
E (4) should be the 40R,50R-isomer. What remained then was the
assignment of the absolute configurations of 2 and 3, one of which
Table 3
NMR data for hamigeromycins F (5) and G (6) in CDCl3 (500 MHz for 1H, and 125 MHz for 13C)
Position
5
6
dC, mult.
dH, mult. (J in Hz)
HMBC
dC, mult.
dH, mult. (J in Hz)
HMBC
1-COO-
1
2
171.1, C
104.0, C
160.9, C
168.8, C
105.8, C
158.3, C
2-OH
3
4
4-OCH3
5
5-OCH3
11.70, s
6.41, s
1,2,3
1,2,4,5,COO
10.06, s
6.40, s
1,2,3
1,2,4,5,COO
99.6, H
99.6, CH
157.7, C
55.8, CH3
140.6, C
60.3, CH3
133.8, C
125.9, CH
132.9, CH
27.2, CH2
37.2, CH2
211.8, C
76.0, CH
158.6, C
55.8, CH3
140.3, C
60.5, CH3
133.6, C
125.7, CH
132.1, CH
28.4, CH2
37.7, CH2
211.5, C
76.6, CH
3.87, s
3.59, s
4
3.87, s
3.62, s
4
5
5
6
10
6.62, d (16.1)
1,5,6,20,30
6.54, br d (15.8)
5.75, ddd (15.8, 6.8, 6.4)
2.78, m; 2.50, m
1,5,30
6,30
10,20,40
30
20
5.94, ddd (16.1, 8.2, 4.1)
2.81, m; 2.55, m
2.84, m; 2.58, m
30
10,50
20,30
40
2.80, m; 2.52, m
50
60
4.36, m
4.40, m
60-OH
70
3.51, d (4.6)
2.06, m; 1.84, m
1.50, m; 121, m
1.69, m; 1.65, m
4.99, m
50,60
50,60,80
70
3.57, d (4.9)
2.11, m; 1.81, m
1.63, m; 1.04, m
1.77, m; 1.57, m
5.30, m
50
50,60
33.0, CH2
20.1, CH2
36.1, CH2
73.5, CH
20.7, CH3
33.0, CH2
18.0, CH2
35.5, CH2
71.4, CH
20.0, CH3
80
90
100
110
80
70,80,100
80
90,100
1.36, d (6.2)
90,100
1.36, d (6.3)

958
M. Isaka et al. / Tetrahedron 66 (2010) 955–961
OH
O
CH3
O
CH3
-0.01
CH3
MTPA
O
O
10'
O
O
15a: (
S
)-MTPA ester
-0.20
15b: (R
)-MTPA ester
9'
8'
-0.12
O
H3CO
HO
HO
6'
-0.03
-0.04
+0.02
H3CO
H3CO
OH
OH
+0.03
+0.14
-0.01
6'
-0.02
O
O
H3CO
+0.10
O MTPA
+0.01
+0.10
+0.18
17
18
O
(relative configuration)
OH
O
CH3
+0.03
CH3
MTPA
O
O
16a: (
S
)-MTPA ester
O
-0.13
16b: (
R
)-MTPA ester
9'
8'
-0.13
O
H3CO
H3CO
HO
+0.04
+0.08
-0.03
H3CO
O
-0.07
-0.02
-0.01
6' +0.04
OH
H3CO
-0.02
O MTPA
-0.01
-0.06
0.00
19
O
(relative configuration)
Figure 4. Dd-Values (dS–dR) of the bis-(S)- and bis-(R)-MTPA esters 15a and 15b of
hamigeromycin F (5), and 16a and 16b of hamigeromycin G (6). The assignments of
proton resonances are based on the COSY data. Accurate chemical shifts of H2-80 and
H2-90 of these compounds could not be estimated due to the signal overlap with H2O
or lipid.
Macrolides 1–5 were subjected to a cytotoxicity assay against
three human cancer cell lines (KB, MCF-7, and NCI-H187) and
noncancerous Vero cells (African green monkey kidney fibro-
blasts). All compounds were inactive against cancer cell lines at
a concentration of 50
weak growth inhibition against Vero cells with respective IC50
values of 42 and 13 g/mL. All compounds were inactive in an
antimalarial activity assay against Plasmodium falciparum K1 at
10 g/mL.
mg/mL, whilst compounds 1 and 2 showed
in establishing the same planar structure as 5, which implied that 5
and 6 are C-60 epimers. The absolute configuration of these com-
pounds was addressed by application of modified Mosher
method.5,6 Hamigeromycin F (5) was reacted with (R)- and (S)-
MTPACl to give, respectively, bis-(S)- and bis-(R)-MTPA ester
m
m
3. Experimental
derivatives (15a and 15b). Based on the Dd values (dS–dR) of the bis-
MTPA ester derivatives (Fig. 4), the 60S configuration of 5 was
established. Similarly hamigeromycin G (6) was converted to its
bis-(S)- and bis-(R)-MTPA ester derivatives 16a and 16b, the 1H
NMR spectroscopic data of which unambiguously indicated the 60R
configuration of 6.
3.1. General procedures
Melting points were measured with an Electrothermal IA9100
digital melting point apparatus. Optical rotations were measured
with a JASCO P-1030 digital polarimeter. UV spectra were recorded
on a GBC Cintra 404 spectrophotometer. CD spectra were recorded
on a JASCO J-180 spectropolarimeter. FTIR spectra were recorded on
a Bruker VECTOR 22 spectrometer. NMR spectra were recorded on
Bruker AV500D and DRX400 spectrometers. ESI-TOF mass spectra
were measured with Micromass LCT and Bruker micrOTOF mass
spectrometers.
The 50-keto-derivatives, hamigeromycins F (5) and G (6),
resemble queenslandon (17), which was previously isolated
from Chrysosporium queenslandicum IFM 51121 and proposed to
have the 40R
*
,60S
*
,100S
relative configuration on the basis of
*
NMR spectroscopic data.7 Comparison of the 1H and 13C NMR
spectroscopic data in CDCl3 reported for queenslandon with
those of hamigeromycins showed several marked differences in
chemical shifts of protons and carbons. Significantly, dH 3.95 (H-
100) of queenslandon was not consistent with dH 4.99 (H-100) for
5, whereas H-60 of queenslandon (dH 5.18) was shifted far more
downfield than the corresponding proton of 5 (dH 4.36). The
oxymethine proton (H-100) of compounds 1–7 resonated within
a range of dH 5.36–4.99. Maier and co-workers recently reported
the synthesis of a core structure (18) of queenslandon.8 Selected
chemical shifts of protons for 18 in CDCl3, dH 5.14–5.23 (H-100),
and dH 4.30–4.37 (H-60),8 were reasonably close to those of 5.
Furthermore, the carbon chemical shifts reported for 18, dC 75.1
(C-60), 33.1 (C-70), 20.4 (C-80), 35.3 (C-90), and 72.1 (C-100),8 were
consistent with those of 5 (Table 3), but different from those of
queenslandon, dC 73.0 (C-60), 20.9 (C-70), 34.9 (C-80), 39.5 (C-90),
and 74.2 (C-100).7 We therefore cast some doubts on the pro-
posed structure (17) of queenslandon. Recently, structurally
related nematicidal macrolides caryospomycins A–C were
isolated from Caryospora calliparpa YMF1.01026.9 Hamigero-
mycin C (2) is the 70,80-dihydro analog of caryospomycin C (19),
3.2. Fungal material
H. avellanea Stock & Samson, accession # BCC 17816 was isolated
from a soil sample collected in the Queen Sirikit Botanic Garden,
Chiang Mai province, Thailand, and identified based on morphol-
ogy and ITS rDNA sequence data by Dr. J. Jennifer Luangsa-ard as
described previously.1
3.3. Fermentation and isolation
The fungus BCC 17816 was maintained on potato dextrose agar
at 25 ꢁC, which was then cut into plugs and inoculated in
3ꢂ250 mL Erlenmeyer flasks containing 25 mL of potato dextrose
broth (PDB; potato starch 4.0 g, dextrose 20.0 g, per liter). After
incubation at 25 ꢁC for 6 days on a rotary shaker (200 rpm), each
primary culture was transferred into a 1 L Erlenmeyer flask con-
taining 250 mL of the same liquid medium (PDB), and incubated
for 6 days under identical conditions. These secondary cultures
(700 mL) were transferred into a 10 L bioreactor containing 6.3 L of
which was proposed to possess the 40R
*
,50S
*
,100S
*
relative
configuration.

M. Isaka et al. / Tetrahedron 66 (2010) 955–961
959
peptone yeast glucose medium (PYGM: bacteriological peptone
5.0 g, yeast extract 20.0 g, glucose 10.0 g, KH2PO4 1.0 g,
MgSO4$7H2O 0.5 g, per liter), and final fermentation was carried
out at 25 ꢁC for 7 days. The culture was filtered to separate the
residue (mycelium) and the filtrate (broth). The broth was
extracted with EtOAc (3ꢂ5.5 L) and concentrated to yield a yellow
solid (1.30 g; extract A). The mycelium was macerated in MeOH
(1.5 L, room temperature, 2 days) and filtered. This extraction was
repeated one more time. The filtrate was defatted with hexane
(2.1 L), and the MeOH phase was evaporated. The residue was
diluted with EtOAc (3 L), washed with H2O (300 mL), concentrated
under reduced pressure to give a pale brown gum (1.06 g, extract
B). Extract A was subjected to column chromatography (CC) on Si
gel (3.0ꢂ21 cm) using CH2Cl2/MeOH as eluent to obtain 14 frac-
tions (1–14). Fraction 3 (44 mg) was fractionated by CC on Si gel
(1.5ꢂ20 cm, step gradient elution with CH2Cl2/MeOH) to obtain
eight fractions (3-1–3-8). Fractions 3-3 (6.5 mg) and 3-4 (3.3 mg)
were combined and further purified by HPLC using a reverse phase
column (Phenomenex Luna 10u C18(2) 100A, 21.2ꢂ250 mm; mo-
bile phase MeCN/H2O 27:73, flow rate 15 mL/min) to furnish 6
(1.8 mg, tR 25 min) and 5 (3.7 mg, tR 38 min). Fraction 10 (300 mg)
was subjected to HPLC (MeCN/H2O 27:73) to obtain 3 (26 mg, tR
21 min), 4 (37 mg, tR 27 min), and a mixture of 2 and 1 corre-
sponding to the broad peak around tR 43 min (165 mg). This
mixture was separated by CC on Si gel (CH2Cl2/EtOAc) to furnish
pure compounds 2 (30 mg) and 1 (70 mg). Fraction 11 (20 mg) was
also purified by HPLC (MeCN/H2O 27:73) to afford 3 (1.9 mg, tR
21 min) and 1 (7.0 mg, tR 35 min). Fraction 10 (240 mg) was trit-
urated in CH2Cl2 (3 mL) and MeOH (3 mL) to leave uracil (70 mg)
as a residual colorless solid. The mycelial extract (extract B) was
also subjected to the NMR-guided chromatographic fractionation,
however, no new compound was found.
(125 MHz, CDCl3) data, see Table 3; HRMS (ESI-TOF) m/z 401.1568
[MþNa]þ (calcd for C20H26O7Na, 401.1576).
3.4. Preparation of the acetonide derivatives
To a solution of hamigeromycin C (2; 1.3 mg) in 2,2-dimethoxy-
propane (0.3 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 1 M NaHCO3. The organic
layer was dried over MgSO4 and concentrated in vacuo to obtain
a colorless solid, which was purified by HPLC using a reverse phase
column (MeCN/H2O 50:50) to afford compound 10 (1.1 mg) as
a colorless solid. Using the same procedure, compounds 9,11, and 13
were obtained as major reaction products, respectively, from 1, 3,
and 4.
3.4.1. Acetonide of hamigeromycin
solid; IR (CHCl3) nmax 1726, 1645, 1597, 1247, 1222, 1059, 755 cmꢀ1
1H NMR (500 MHz, CDCl3)
A (9). Colorless amorphous
;
d
11.85 (1H, s, 2-OH), 6.67 (1H, dd, J¼16.1,
2.2 Hz, H-10), 6.45 (1H, s, H-3), 6.01 (1H, ddd, J¼16.1, 9.4, 3.0 Hz, H-
20), 5.26 (1H, m, H-100), 4.59 (1H, d, J¼6.3 Hz, H-50), 4.55 (1H, m, H-
40), 3.89 (3H, s, 4-OCH3), 3.57 (3H, s, 5-OCH3), 3.13 (1H, ddd, J¼19.4,
8.6, 4.3 Hz, Ha-70), 3.05 (1H, dq, J¼16.3, 3.0 Hz, Ha-30), 2.38 (1H, m,
Hb-70), 2.35 (1H, m, Hb-30), 2.06 (1H, m, Ha-80), 1.83 (1H, m, Ha-90),
1.80 (1H, m, Hb-90), 1.67 (3H, s, H3-100), 1.49 (1H, m, Hb-80), 1.40 (3H,
s, H3-300), 1.34 (3H, d, J¼6.3 Hz, H3-110); 13C NMR (125 MHz, CDCl3)
d
208.5 (C, C-60), 171.1 (C, -COO-), 161.3 (C, C-2), 158.8 (C, C-4), 140.3
(C, C-5), 132.7 (C, C-6), 130.0 (CH, C-20), 127.0 (CH, C-10), 110.0 (C, C-
200), 103.6 (C, C-1), 99.9 (CH, C-3), 80.7 (CH, C-50), 77.5 (CH, C-40),
74.7 (CH, C-100), 60.2 (CH3, 5-OCH3), 55.9 (CH3, 4-OCH3), 41.6 (CH2,
C-70), 33.8 (CH2, C-30), 32.7 (CH2, C-90), 26.5 (CH3, C-100), 25.9 (CH3,
C-300), 20.2 (CH3, C-110), 18.6 (CH2, C-80); HRMS (ESI-TOF) m/z
457.1843 [MþNa]þ (calcd for C23H30O8Na, 457.1833).
28
3.3.1. Hamigeromycin C (2). Colorless solid; mp 102–105 ꢁC; [
ꢀ11 (c 0.12, MeOH); UV (MeOH) lmax (log
321 (3.71) nm; IR (KBr) nmax 3446,1710,1644,1596,1359,1317,1247,
1053, 1020, 755 cmꢀ1 1H NMR (500 MHz, CDCl3) and 13C NMR
a]
D
3) 230 (4.37), 267 (3.90),
3.4.2. Acetonide of hamigeromycin C (10). Colorless amorphous
solid; IR (CHCl3) nmax 1651, 1599, 1249, 756 cmꢀ1
;
1H NMR
;
(500 MHz, CDCl3)
d
11.77 (1H, s, 2-OH), 6.74 (1H, dd, J¼16.1, 2.0 Hz,
(125 MHz, CDCl3) data, see Tables 1 and 2; HRMS (ESI-TOF) m/z
H-10), 6.42 (1H, s, H-3), 6.13 (1H, ddd, J¼16.1, 9.6, 3.3 Hz, H-20), 4.94
(1H, m, H-100), 4.20 (1H, dt, J¼9.2, 3.2 Hz, H-40), 3.97 (1H, d,
J¼9.2 Hz, H-50), 3.88 (3H, s, 4-OCH3), 3.58 (3H, s, 5-OCH3), 3.46 (3H,
s, 60-OCH3), 3.28 (3H, s, 60-OCH3), 3.05 (1H, m, Ha-30), 2.42 (1H, ddd,
J¼16.7, 9.6, 2.9 Hz, Hb-30), 1.93 (1H, m, Ha-70), 1.72 (1H, m, Ha-80),
1.68 (1H, m, Ha-90), 1.57 (1H, m, Hb-90), 1.46 (3H, s, H3-100), 1.39 (3H,
d, J¼6.1 Hz, H3-110), 1.35 (3H, s, H3-300), 1.30 (1H, m, Hb-80), 1.28 (1H,
395.1707 [MþH]þ (calcd for C20H27O8, 395.1706).
27
3.3.2. Hamigeromycin D (3). Colorless solid; mp 115–117 ꢁC; [
þ24 (c 0.10, MeOH); UV (MeOH) lmax (log
(3.88), 268 sh (3.78), 309 (3.61) nm; IR (KBr) nmax 3489, 1713, 1645,
1595, 1248, 1029 cmꢀ1 1H NMR (500 MHz, CDCl3) and 13C NMR
a]
D
3
) 221 (4.37), 259 sh
;
(125 MHz, CDCl3) data, see Tables 1 and 2; HRMS (ESI-TOF) m/z
m, Hb-70); 13C NMR (125 MHz, CDCl3)
d 171.4 (C, –COO–),161.0 (C, C-
395.1716 [MþH]þ (calcd for C20H27O8, 395.1706).
2), 158.8 (C, C-4), 140.4 (C, C-5), 133.8 (C, C-6), 129.5 (CH, C-20), 126.9
(CH, C-10), 107.6 (C, C-200), 104.0 (C, C-1), 100.0 (C, C-60), 99.5 (CH, C-
3), 80.1 (CH, C-50), 75.9 (CH, C-40), 74.0 (CH, C-100), 60.4 (CH3, 5-
OCH3), 55.8 (CH3, 4-OCH3), 50.8 (CH3, 60-OCH3), 49.1 (CH3, 60-OCH3),
33.6 (CH2, C-70), 35.1 (CH2, C-30), 36.6 (CH2, C-90), 27.0 (CH3, C-100),
27.0 (CH3, C-300), 21.0 (CH3, C-110), 20.5 (CH2, C-80); HRMS (ESI-TOF)
m/z 503.2256 [MþNa]þ (calcd for C25H36O9Na, 503.2252).
28
3.3.3. Hamigeromycin E (4). Colorless solid; mp 139–142 ꢁC; [
þ44 (c 0.10, MeOH); UV (MeOH) lmax (log
(3.99), 269 sh (3.93), 315 (3.73) nm; IR (KBr) nmax 3423, 1710, 1656,
a]
D
3
) 231 (4.43), 260 sh
1599, 1360, 1314, 1249, 1225, 1020 cmꢀ1
;
1H NMR (500 MHz,
CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Tables 1 and 2;
HRMS (ESI-TOF) m/z 395.1703 [MþH]þ (calcd for C20H27O8,
395.1706).
3.4.3. Acetonide of hamigeromycin D (11). Colorless amorphous
solid; IR (CHCl3) nmax 1646, 1597, 1246 cmꢀ1 1H NMR (500 MHz,
;
26
3.3.4. Hamigeromycin F (5). Colorless amorphous solid; [
(c 0.13, MeOH); UV (MeOH) lmax (log
268 sh (3.75), 307 (3.61) nm; IR (KBr) nmax 3455, 1710, 1646, 1597,
1247, 1023, 756 cmꢀ1 1H NMR (500 MHz, CDCl3) and 13C NMR
a
]
þ41
CDCl3)
d
11.66 (1H, s, 2-OH), 6.49 (1H, d, J¼16.1 Hz, H-10), 6.41 (1H, s,
D
3
) 219 (4.41), 257 sh (3.89),
H-3), 6.38 (1H, dt, J¼16.1, 6.9 Hz, H-20), 5.28 (1H, m, H-100), 4.03
(1H, ddd, J¼9.1, 5.9, 2.8 Hz, H-40), 3.95 (1H, d, J¼9.1 Hz, H-50), 3.88
(3H, s, 4-OCH3), 3.59 (3H, s, 5-OCH3), 3.42 (3H, s, 60-OCH3), 3.31 (3H,
s, 60-OCH3), 2.95 (1H, m, Ha-30), 2.42 (1H, dt, J¼15.9, 6.3 Hz, Hb-30),
1.88 (1H, m, Ha-70), 1.71 (1H, m, Ha-90), 1.67 (1H, m, Hb-70), 1.62 (1H,
m, Hb-90), 1.60 (1H, m, Ha-80), 1.53 (1H, m, Hb-80), 1.46 (3H, s, H3-
100), 1.42 (3H, s, H3-300), 1.35 (3H, d, J¼6.4 Hz, H3-110); 13C NMR
;
(125 MHz, CDCl3) data, see Table 3; HRMS (ESI-TOF) m/z 401.1570
[MþNa]þ (calcd for C20H26O7Na, 401.1576).
26
3.3.5. Hamigeromycin G (6). Colorless amorphous solid; [
(c 0.08, MeOH); UV (MeOH) lmax (log
267 sh (3.58), 305 (3.45) nm; IR (CHCl3) nmax 3451, 1711, 1643, 1594,
1245, 1023, 755 cmꢀ1 1H NMR (500 MHz, CDCl3) and 13C NMR
a
]
þ17
D
3
) 219 (4.24), 258 sh (3.69),
(125 MHz, CDCl3) d 171.0 (C, –COO–), 160.8 (C, C-2), 158.7 (C, C-4),
140.5 (C, C-5), 132.8 (C, C-6), 130.4 (CH, C-20), 126.6 (CH, C-10), 107.8
(C, C-200), 104.2 (C, C-1), 100.6 (C, C-60), 99.5 (CH, C-3), 80.3 (CH, C-
;

960
M. Isaka et al. / Tetrahedron 66 (2010) 955–961
50), 76.9 (CH, C-40), 72.4 (CH, C-100), 60.1 (CH3, 5-OCH3), 55.8 (CH3,
4-OCH3), 50.0 (CH3, 60-OCH3), 49.1 (CH3, 60-OCH3), 36.1 (CH2, C-30),
34.9 (CH2, C-90), 32.8 (CH2, C-70), 27.1 (CH3, C-100), 27.1 (CH3, C-300),
18.1 (CH3, C-110), 17.0 (CH2, C-80); HRMS (ESI-TOF) m/z 503.2255
[MþNa]þ (calcd for C25H36O9Na, 503.2252).
(1H, m, Ha-70), 2.66 (1H, m, Hb-70), 2.48 (1H, m, Hb-30), 1.85 (1H, m,
Ha-80), 1.75 (1H, m, Hb-80), 1.72 (1H, m, Ha-90), 1.64 (1H, m, Hb-90),
1.00 (3H, d, J¼6.1 Hz, H3-110); HRMS (ESI-TOF) m/z 858.3579
[MþNa]þ (calcd for C47H53N3O11Na, 858.3572).
3.6. Preparation of bis-MTPA esters
3.4.4. Acetonide of hamigeromycin
E
(12). Colorless solid; IR
1H NMR (500 MHz,
(CHCl3) nmax 1723, 1648, 1598, 1247, 756 cmꢀ1
;
Hamigeromycin F (5, 0.4 mg) was treated with (þ)-(S)-MTPACl
(2 mg)inpyridine(0.1 mL)atroomtemperaturefor16 h. Themixture
was diluted with EtOAc and washed with H2O and 1 M NaHCO3, and
the organic layerwasconcentrated invacuo. The residuewaspurified
by preparative HPLC (MeCN/H2O) to furnish the bis-(R)-MTPA ester
15b (1.2 mg). Similarly, bis-(S)-MTPA ester 15a was prepared from
5 and (ꢀ)-(R)-MTPACl. In the same fashion, bis-(S)- and bis-(R)-MTPA
esters, 16a and 16b, were synthesized from hamigeromycin G (6).
The assignments of protons are based on the COSY data.
CDCl3)
d
11.73 (1H, s, 2-OH), 6.48 (1H, d, J¼16.3 Hz, H-10), 6.43 (1H,
s, H-3), 6.19 (1H, ddd, J¼16.3, 8.6, 4.5 Hz, H-20), 5.19 (1H, m, H-100),
4.65 (1H, dt, J¼1.1, 7.6 Hz, H-40), 4.61 (1H, d, J¼7.6 Hz, H-50), 3.88
(3H, s, 4-OCH3), 3.55 (3H, s, 5-OCH3), 2.99 (1H, ddd, J¼19.9, 9.9,
4.5 Hz, Ha-70), 2.77 (1H, ddd, J¼19.9, 5.6, 4.5 Hz, Hb-70), 2.65 (1H, m,
Ha-30), 2.54 (1H, dd, J¼16.6, 8.6 Hz, Hb-30), 2.05 (1H, m, Ha-90), 1.93
(1H, m, Ha-80), 1.81 (1H, m, Hb-90), 1.56 (3H, s, H3-100), 1.53 (1H, m,
Hb-80), 1.36 (3H, s, H3-300), 1.34 (3H, d, J¼6.3 Hz, H3-110); 13C NMR
(125 MHz, CDCl3)
d
210.4 (C, C-60), 171.3 (C, –COO–), 160.8 (C, C-2),
158.7 (C, C-4), 140.7 (C, C-5), 133.6 (C, C-6), 130.9 (CH, C-20), 125.0
(CH, C-10), 109.5 (C, C-200), 103.9 (C, C-1), 99.7 (CH, C-3), 82.2 (CH, C-
50), 75.9 (CH, C-40), 75.2 (CH, C-100), 60.1 (CH3, 5-OCH3), 55.9 (CH3,
4-OCH3), 39.0 (CH2, C-70), 34.0 (CH2, C-30), 32.7 (CH2, C-90), 26.2
(CH3, C-100), 24.8 (CH3, C-300), 20.7 (CH3, C-110), 19.6 (CH2, C-80);
HRMS (ESI-TOF) m/z 457.1839 [MþNa]þ (calcd for C23H30O8Na,
457.1833).
3.6.1. Bis-(S)-MTPA ester 15a. Colorless gum; 1H NMR (400 MHz,
CDCl3)
d
7.60–7.40 (10H, m, phenyl of MTPA ꢂ2), 6.576 (1H, s, H-3),
6.398 (1H, d, J¼16.5 Hz, H-10), 6.177 (1H, m, H-20), 5.225 (1H, dd,
J¼6.0, 4.6 Hz, H-60), 4.736 (1H, m, H-100), 3.846 (3H, s, 4-OCH3),
3.737 (3H, s, 5-OCH3), 3.669 (3H, br s, OCH3 of MTPA), 3.591 (3H, br
s, OCH3 of MTPA), 2.738 (1H, m, Ha-30), 2.600 (1H, m, Ha-40), 2.460
(1H, m, Hb-30), 2.445 (1H, m, Hb-40), 1.850 (1H, m, Ha-70), 1.813 (1H,
m, Hb-70), 1.65–1.10 (4H, m, Ha-80, Hb-80, Ha-90, and Hb-90), 0.88þ2
(3H, d, J¼6.1 Hz, H3-110); HRMS (ESI-TOF) m/z 833.2369 [MþNa]
(calcd for C40H40O11F6Na, 833.2367).
3.5. Preparation of tris-p-dimethylaminobenzoate derivatives
Hamigeromycin C (2, 2.0 mg) was treated with p-dimethylamino-
benzoyl chloride (15 mg) and 4-N,N-dimethylaminopyridine
(DMAP, 2.5 mg) in 0.3 mL pyridine/CH2Cl21:2 at room temperature for
16 h. The mixture was diluted with EtOAc and washed with H2O
and 1 M NaHCO3, and the organic layer was concentrated in vacuo to
obtain a colorless solid (10 mg), which was purified by CC on Si gel
(EtOAc/CH2Cl2 5:95) to furnish the tris-p-dimethylaminobenzoate
derivative 13 (2.3 mg). Similarly, tris-p-dimethylaminobenzoate 14
(2.5 mg) was prepared from hamigeromycin D (3, 2.0 mg).
3.6.2. Bis-(R)-MTPA ester 15b. Colorless gum; 1H NMR (400 MHz,
CDCl3)
d
7.60–7.40 (10H, m, phenyl of MTPA ꢂ2), 6.699 (1H, s, H-3),
6.381 (1H, d, J¼16.2 Hz, H-10), 6.155 (1H, m, H-20), 5.240 (1H, dd,
J¼6.7, 4.2 Hz, H-60), 4.938 (1H, m, H-100), 3.852 (3H, s, 4-OCH3),
3.730 (3H, s, 5-OCH3), 3.584 (3H, br s, OCH3 of MTPA), 3.548 (3H, br
s, OCH3 of MTPA), 2.634 (1H, m, Ha-30), 2.573 (1H, m, Ha-40), 2.310
(1H, m, Hb-40), 2.279 (1H, m, Hb-30), 1.880 (1H, m, Ha-70), 1.855 (1H,
m, Hb-70), 1.65–1.10 (4H, m, Ha-80, Hb-80, Ha-90, and Hb-90), 0.89þ4
(3H, d, J¼6.3 Hz, H3-110); HRMS (ESI-TOF) m/z 833.2361 [MþNa]
(calcd for C40H40O11F6Na, 833.2367).
3.5.1. Compound 13. Colorless gum; UV (MeOH) lmax 228 (3.96),
319 (4.88) nm; CD (MeOH) D3 330 (ꢀ16.0), 307 (þ22.4) nm; 1H
NMR (400 MHz, CDCl3)
d
7.98 (2H, d, J¼9.0 Hz, p-dimethylamino-
3.6.3. Bis-(S)-MTPA ester 16a. Colorless gum; 1H NMR (400 MHz,
benzoyl), 7.97 (2H, d, J¼9.1 Hz, p-dimethylaminobenzoyl), 7.80 (2H,
d, J¼9.1 Hz, p-dimethylaminobenzoyl), 6.88 (1H, s, H-3), 6.78 (2H, d,
J¼9.1 Hz, p-dimethylaminobenzoyl), 6.74 (2H, d, J¼9.0 Hz, p-
dimethylaminobenzoyl), 6.63 (2H, d, J¼9.1 Hz, p-dimethylamino-
benzoyl), 6.54 (1H, d, J¼16.0 Hz, H-10), 6.28 (1H, ddd, J¼16.0, 8.8,
6.3 Hz, H-20), 5.51 (1H, d, J¼4.5 Hz, H-40), 5.46 (1H, m, H-50), 5.12
(1H, m, H-100), 3.88 (3H, s, 4-OCH3), 3.69 (3H, s, 5-OCH3), 3.09 (6H,
s, p-dimethylaminobenzoyl), 3.06 (6H, s, p-dimethylamino-
benzoyl), 3.04 (6H, s, p-dimethylaminobenzoyl), 2.85–2.79 (2H, m,
Ha-30 and Hb-30), 2.77 (1H, m, Ha-70), 2.55 (1H, m, Hb-70), 1.78–1.72
(3H, m, Ha-80, Hb-80, and Ha-90), 1.54 (1H, m, Hb-90), 0.97 (3H, d,
J¼6.2 Hz, H3-110); HRMS (ESI-TOF) m/z 858.3577 [MþNa]þ (calcd
for C47H53N3O11Na, 858.3572).
CDCl3)
d
7.60–7.40 (10H, m, phenyl of MTPA ꢂ2), 6.595 (1H, s, H-3),
6.504 (1H, d, J¼16.2 Hz, H-10), 6.096 (1H, m, H-20), 5.312 (1H, dd,
J¼6.1, 3.5 Hz, H-60), 4.810 (1H, m, H-100), 3.847 (3H, s, 4-OCH3),
3.746 (3H, s, 5-OCH3), 3.661 (3H, br s, OCH3 of MTPA), 3.524 (3H, br
s, OCH3 of MTPA), 2.833 (1H, m, Ha-30), 2.740 (1H, m, Ha-40), 2.615
(1H, m, Hb-40), 2.360 (1H, m, Hb-30), 2.105 (1H, m, Ha-70), 1.810 (1H,
m, Hb-70), 1.65–1.10 (4H, m, Ha-80, Hb-80, Ha-90, and Hb-90), 0.88þ7
(3H, d, J¼6.2 Hz, H3-110); HRMS (ESI-TOF) m/z 833.2370 [MþNa]
(calcd for C40H40O11F6Na, 833.2367).
3.6.4. Bis-(R)-MTPA ester 16b. Colorless gum; 1H NMR (400 MHz,
CDCl3)
d
7.65–7.40 (10H, m, phenyl of MTPA ꢂ2), 6.729 (1H, s, H-3),
6.530 (1H, d, J¼16.2 Hz, H-10), 6.120 (1H, m, H-20), 5.273 (1H, dd,
J¼5.7, 3.3 Hz, H-60), 4.939 (1H, m, H-100), 3.858 (3H, s, 4-OCH3),
3.756 (3H, s, 5-OCH3), 3.632 (3H, br s, OCH3 of MTPA), 3.555 (3H, br
s, OCH3 of MTPA), 2.894 (1H, m, Ha-30), 2.806 (1H, m, Ha-40), 2.638
(1H, m, Hb-40), 2.360 (1H, m, Hb-30), 2.070 (1H, m, Ha-70), 1.728 (1H,
m, Hb-70), 1.65–1.10 (4H, m, Ha-80, Hb-80, Ha-90, and Hb-90), 0.86þ0
(3H, d, J¼6.2 Hz, H3-110); HRMS (ESI-TOF) m/z 833.2369 [MþNa]
(calcd for C40H40O11F6Na, 833.2367).
3.5.2. Compound 14. Colorless gum; UV (MeOH) lmax (log
(3.92), 317 (4.89) nm; CD (MeOH) D3 327 (þ26.1), 304 (ꢀ8.5) nm;
1H NMR (400 MHz, CDCl3)
7.97 (2H, d, J¼9.1 Hz, p-dimethylami-
3) 226
d
nobenzoyl), 7.87 (2H, d, J¼9.1 Hz, p-dimethylaminobenzoyl), 7.84
(2H, d, J¼9.1 Hz, p-dimethylaminobenzoyl), 6.88 (1H, s, H-3), 6.78
(2H, d, J¼9.1 Hz, p-dimethylaminobenzoyl), 6.72 (2H, d, J¼9.1 Hz, p-
dimethylaminobenzoyl), 6.69 (2H, d, J¼9.1 Hz, p-dimethylamino-
benzoyl), 6.63 (1H, d, J¼16.3 Hz, H-10), 6.40 (1H, dt, J¼16.3, 7.0 Hz,
H-20), 5.63 (1H, m, H-40), 5.59 (1H, d, J¼4.8 Hz, H-50), 5.18 (1H, m, H-
100), 3.88 (3H, s, 4-OCH3), 3.71 (3H, s, 5-OCH3), 3.09 (6H, s, p-
dimethylaminobenzoyl), 3.05 (6H, s, p-dimethylaminobenzoyl),
3.04 (6H, s, p-dimethylaminobenzoyl), 2.85 (1H, m, Ha-30), 2.81
3.7. Biological assays
Cytotoxicity against KB cells (oral human epidermoid carci-
noma), MCF-7 cells (human breast cancer), NCI-H187 cells (human
small-cell lung cancer), and Vero cells (African green monkey

M. Isaka et al. / Tetrahedron 66 (2010) 955–961
961
kidney fibroblasts) were evaluated using the resazurin microplate
References and notes
assay.10 Assay for activity against P. falciparum (K1, multi-drug re-
sistant strain) was performed using the microculture radioisotope
technique described by Desjardins et al.11
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Acknowledgements
Financial support from the Bioresources Research Network,
National Center for Genetic Engineering and Biotechnology (BIO-
TEC), is gratefully acknowledged.
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Supplementary data
1H and 13C NMR spectra of 1–6, and CD and UV spectra of 13 and
14. Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.11.101.