Brasilicardins B-D, new tricyclic terpernoids from actinomycete Nocardia brasiliensis

Article abstract of DOI:10.1016/j.bmc.2004.08.007

Three new tricyclic terpenoids, brasilicardins B-D (2-4), were isolated together with brasilicardin A (1), a potent immunosuppressive compound, from the cultured broth of a pathogenic actinomycete Nocardia brasiliensis IFM0406, and the structures and stereochemistry were determined by spectroscopic data and a single crystal X-ray diffraction analysis. The immunosuppressive and cytotoxic activities of 2-4 were examined in the comparison with 1.

Full text of DOI:10.1016/j.bmc.2004.08.007

Bioorganic & Medicinal Chemistry 12 (2004) 5545–5551
Brasilicardins B–D, new tricyclic terpernoids from
actinomycete Nocardia brasiliensis
Kazusei Komatsu,a Masashi Tsuda,a Motoo Shiro,b Yasushi Tanaka,c
Yuzuru Mikamid and JunÕichi Kobayashia,
*
aGraduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
bX-Ray Research Laboratory, Rigaku Corporation, Akishima 196-8666, Japan
cResearch Laboratory, Higeta Shoyu Co., Ltd, Chiba 288-8680, Japan
dResearch Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba 260-0856, Japan
Received 9 July 2004; revised 6 August 2004; accepted 6 August 2004
Available online 8 September 2004
Abstract—Three new tricyclic terpenoids, brasilicardins B–D (2–4), were isolated together with brasilicardin A (1), a potent immuno-
suppressive compound, from the cultured broth of a pathogenic actinomycete Nocardia brasiliensis IFM0406, and the structures and
stereochemistry were determined by spectroscopic data and a single crystal X-ray diffraction analysis. The immunosuppressive and
cytotoxic activities of 2–4 were examined in the comparison with 1.
ꢀ 2004 Elsevier Ltd. All rights reserved.
23
1. Introduction
O
12
11
R
21
H
9'
13
14
O
18
8'
HO
5'
7'
10'
11'
15
O
CO2H
6'
17
9
1'
1
4
16
O
13'
8
2
3
10
During our search for bioactive substances from patho-
genic actinomycetes of the genus Nocardia,1 we have
previously isolated brasilicardin A (1), a novel tricyclic
terpenoid consisting of an anti/syn/anti-perhydrophen-
anthrene skeleton with two sugars and an amino acid
side-chain, from Nocardia brasiliensis IFM-0406.2 This
unique terpenoid moiety was shown to be biosynthe-
sized from glucose via the nonmevalonate pathway.3
Brasilicardin A (1) exhibits potent immunosuppressive
activity in mouse mixed lymphocyte reaction (MLR)
assay and cytotoxic activity against adriamycin-resistant
murine lymphoma cells. Its immunosuppressive potency
is compatible to those of cyclosporin A or ascomycin.4
Further investigation on the extract of this strain
resulted in the isolation of three new congeners, brasili-
cardins B–D (2–4), and the structures and stereochemis-
try were determined by spectroscopic data and a single
crystal X-ray diffraction analysis. In this paper we
describe the isolation and structure elucidation of 2–4
and their immunosuppressive activities.
6" OH
12'
3'
2'
22
4' O
1"
2" NHAc
5
7
4"
NH2
5"
6
O
HO
HO
HO
HO
H
19
3"
20
brasilicardin A (1) : R = OCH3
brasilicardin B (2) : R = H
23
12
R
21H
13
O
18
5'
14
1'
CO2H
17
9
16
O
8
2
3
10
HO
3'
22
4'
5
4
NH2
HO
HO
HO
H
19
20
brasilicardin C (3) : R = OCH3
brasilicardin D (4) : R = H
2. Results and discussion
The supernatant of the fermentation broth (80L) was
subjected to a Diaion HP-20 column (50% MeOH
aq ! MeOH), in which fractions eluted with MeOH
were separated on a silica gel and C18 columns, and cen-
trifugal partition chromatography followed by C18
HPLC (MeOH/H2O and MeOH/H2O/CF3CO2H) to
afford brasilicardins B (2, 6.6mg), C (3, 55mg), and D
(4, 23mg) together with a known related compound,
brasilicardin A (1, 428mg).
Keywords: Terpenoid; Actinomycete; Nocardia brasiliensis; Immuno-
suppressive.
*
Corresponding author. Tel.: +81 11 706 4985; fax: +81 11 706
4989; e-mail: jkobay@pharm.hokudai.ac.jp
0968-0896/$ - see front matter ꢀ 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.007

5546
K. Komatsu et al. / Bioorg. Med. Chem. 12 (2004) 5545–5551
Table 1. 1H and 13C NMR data of brasilicardins B–D (2–4) in MeOH-d4
Position
2
HMBC
3
4
dH
m, J (Hz)
dC
44.76
dH
m, J (Hz)
dC
44.78
dH
m, J (Hz)
dC
44.91
_
1 (a)
1.85
1.49
3.76
3.06
m
21
1.82
1.46
3.70
3.03
dd, 12.8, 4.4
m
1.84
1.43
3.70
3.02
dd, 12.5, 3.7
m
_
1 (b)
m
2
m
d, 9.3
80.60
84.15
41.89
45.49
19.62
3
m
d, 9.4
79.83
84.08
41.84
44.99
19.40
m
d, 9.3
80.00
84.24
41.89
45.53
19.66
3
4
3, 5, 19, 20
5
1.67
1.77
1.67
1.90
1.38
m
m
m
m
m
1.66
1.78
1.65
1.78
1.38
m
m
m
m
m
1.64
1.77
1.64m
1.89
1.37
m
m
6 (a)
6 (b)
7 (a)
7 (b)
8
32.25
22
31.98
m
m
32.35
38.89
48.15
38.56
27.71
15b, 22
11, 12, 21
21
39.19
48.02
38.93
48.23
38.55
9
10
1.43
1.94a
5.38
m
m
m
1.31
1.92a
5.38
m
1.43
m
38.20
27.741.93
11 (a)
11 (b)
12
m
27.73
1.89
5.37
m
m
124.05
139.06
12, 22, 23
28.26
11, 23
11, 23
1.61
m
124.22
139.26
1.32
124.06
139.09
13
141.33
15a
15b
16
m
57.50
53.00
m
57.66
1.67
1.46
1.99
1.57
1.46
3.82
3.53b
4.48
32.71
1.64m
1.45
1.97
28.31
m
34.64
dd, 10.8, 3.0
s
d, 3.5
81.26
59.29
55.63
m
––
34.86
OCH3
17
18
3.54
t, 5.6
57.60
175.13
18.19
3.53
t, 5.0
57.60
175.48
18.21
30.15
29.56
24.21
24.16
104.31
73.03
73.32
74.91
70.64
18.68
17
170.88
18.11
30.01
19
20
0.96b
1.04b
1.18b
1.05b
1.72b
5.08
s
3, 20
3, 19
1b, 5, 9
0.95b
1.02b
1.13b
1.08b
1.70b
4.99
m
0.95b
s
s
30.16
29.52
m
1.02b
s
b
21
22
s
m
29.641.18
23.70
23.36
s
s
24.22
24.19
m
1.04b
1.72b
4.99
3.98
3.70
3.42
s
23
10
s
brs
12
s
brs
s
brs
103.79
72.71
80.80
104.18
72.93
73.22
20
4.39
4.13
brd, 3.0
dd, 9.6, 3.0
t, 9.6
3.99
3.70
m
brd, 3.1
dd, 9.3, 3.1
dd, 9.3, 9.3
dq, 9.3, 6.2
d, 6.2
30
10, 20, 100
20, 30,
10, 60
m
t, 9.4
40
5.31
4.06
1.17b
74.83
68.73
18.59
3.42
3.75
1.28b
74.78
70.58
50
dq 9.6, 6.0
d, 6.0
dq, 9.4, 6.4
d, 6.418.62
3.75
b
60
40
1.29
70
168.12
132.9412
118.31
159.69
122.56
131.69
122.849
105.00
57.86
40,0 90, 130
80
90
7.52
d, 1.9
110, 130
90, 120
90, 130
100
110
120
1030 0
1
7.11
7.38
7.59
4.57
3.61
1.52b
dd, 8.1, 1.9
t, 8.1
d, 8.1
0, 110
30, 200
d, 8.1
dd, 8.1, 9.3
s
200
CH3CO
CH3CO
300
23.41
175.00
76.04
72.71
200, CH3CO
3.43
3.36
3.36
3.93
3.74m
dd, 8.1, 9.3
m
200
400
300, 600
a
5000
m
d, 11.8
78.35
63.04
6 (a)
600 (b)
a 2H.
b 3H.
23
D
Brasilicardin B {2, ½aŠ +17 (c 1.0, MeOH)} was ob-
nary carbons, one oxymethylene, five methylenes, and
seven methyls, which were similar to those of brasilicar-
din A (1) except for the absence of a methoxy group and
an oxymethine and the presence of an additional meth-
ylene. Detailed analysis of 2D NMR data of 2 implied
the existence of the same tricyclic skeleton (C-1–C-14)
with two sugar moieties (C-10–C-60 and C-100–C-600)
and a 3-hydroxybenzoyl unit (C-70–C-130) as that of 1
(Fig. 1). One (C-10–C-60) of the two sugar moieties in
tained as a colorless amorphous solid. HRFABMS data
[m/z 863.4554, (M+H)+, +1.2mmu] of 2 revealed the
molecular formula to be C44H66N2O15, which was smal-
ler than that of brasilicardin A (1) by 30amu. The 13C
NMR (Table 1) spectrum of 2 disclosed total 44 signals
including one carboxyl, one ester, and one amide carbo-
nyl, three sp2 quaternary carbons, five sp2 methines, two
hemiacetal carbons, nine oxymethines, three sp2 quater-

K. Komatsu et al. / Bioorg. Med. Chem. 12 (2004) 5545–5551
5547
12
23
CD spectrum of 8 showed positive Cotton effect [kext
237nm (De +18.4), 223nm (De ꢀ10.7)], indicating that
the benzoyl groups at C-2 and C-3 should be clock-
wise. Thus, the absolute configurations at C-2 and C-3
were both S. To determine the absolute configura-
tion at C-17, the modified MosherÕ s method6 was
employed.
6'
O
O
21
11
13
22
9'
O
18
5'
4'
1'
7'
14
HO
10'
8'
15
CO2H
17
9
1
4
O
16
3'
2'
8
7
2
3
10
OH
5
13'
11'
NH2
6
12'
HO
O
O
6"
20
19
5"
4"
HO
1"
2"
O
3"
HO
N
H
1H-1H COSY
HMBC
OH
2
Figure 1. Selected 2D NMR correlations for brasilicardin B (2).
1
2 was elucidated to be a-rhamnose by H–1H coupling
constants and ROESY data, while the presence of b-
N-acetylgulcosamine as another sugar unit (C-100–C-600)
was deduced from the 13C chemical shifts and HMBC
correlations for H-200/COCH3 (Fig. 1). The rhamnose
unit was attached to C-2 of aglycone, while N-acetyl-
glucosamine (C-100–C-600) and 3-hydroxybenzoyl unit
(C-70–C-130) were connected at C-30 and C-40 of the
rhamnose, respectively. The relative stereochemistry of
the tricyclic core was implied to be the same as that of
1 by analysis of NOESY data (Fig. 2). The b-aminobu-
tyric acid side chain (C-15–C-18) attached to C-14was
1
revealed by H–1H COSY cross-peaks for H-14/H2-15,
H2-15/H2-16, and H2-16/H-17 and HMBC correlations
for H-12/C-14, H-15/C-8, H-17/C-18, H3-23/C-14. Thus
the structure and relative stereochemistry of brasilicar-
din B were elucidated to be 2.
Hydrolysis of 2 with HCl/MeOH afforded its aglycone 5,
methyl a-rhamnopyranoside (6), and methyl a-glucos-
amine (7). The absolute configurations of the sugar units
6 and 7 were determined as L and D, respectively, on the
Compound 5 was converted into (S)- and (R)-2-meth-
oxy-2-trifluoromethyl-2-phenylacetyl (MTPA) amides
(9a and 9b, respectively). The Dd values [d(9a) ꢀ d(9b)]
suggested the S-configuration at C-17 (Fig. 3).
Therefore, the structure of brasilicardin B (2) was con-
cluded to be the desmethoxy form at C-16 of brasilicar-
din A (1).
22
22
D
basis of its optical rotations (6: ½aŠ ¼ ꢀ39, 7: ½aŠ
D
+35).2 To determine the absolute configuration of the
tricyclic core of 2, the CD exciton chirality method5
was applied for the dihydroxyl group at C-2 and C-3.
The aglycone 5 was treated with p-bromobenzoyl chlo-
ride to afford a 2-O, 3-O, 17-N-trisbenzoate (8), of which
the structure was assigned on the basis of 1H–1H COSY,
NOESY, and HRFABMS data [m/z 742.3689 (M+Na)+,
calcd for C45H53NO7Na, D ꢀ1.4mmu]. The J(H-2,H-3)
value (10.1Hz) indicated that the two benzoyl groups at
C-2 and C-3 were trans diequatrially disposed, and the
The molecular formula, C30H51NO9, of brasilicardin C
23
{3, ½aŠ +65 (c 1.0, MeOH)} was established by HRF-
D
ABMS data [m/z 570.3636 (M+H)+, D ꢀ0.6 mmu]. H
and 13C NMR data (Table 1) showed the signals due
to the same tricyclic aglycone as brasicardin A (1) and
a rhamnose, while signals due to an N-acetylglucos-
amine and a 3-hydroxybenzoate were not observed for
3. The absolute stereochemistry of rhamnose moiety
1
was assigned as L on the basis of the optical rotation
20
D
of methyl a-rhamnopyranoside {6, ½aŠ ꢀ48} obtained
O
by methanolysis of 3. Brasilicardin C (3) was crystallized
from H2O–MeOH as colorless needles, mp 219–221ꢁC.
H
H
H
H
Me
H
22
12
H
Me
13 23
2
HO
Me 3
H
5'
1
11
8
H H
H
5
14
6'
20
4
10
9
1'
H
Me
H
O
O
3'
H
21
Me
4'
2'
19Me
OH
H
OH
H
4"
6"
H
5"
O
O
HO
HO
1"
3"
2"
NHAc
H
H
NOESY
H
Figure 2. NOESY correlations and relative stereochemistry for brasili-
cardin B (2). J in hertz (H/H): H-2/H-3: 9.3, H-10H-20: <1, H-20/H-30:
3.0, H-30/H-40: 9.6, H-40/H-50: 9.6, H-100/H-200: 8.1, H-200/H-300: 9.3,
H-300/H-400: 8.1.
Figure 3. Proton chemical differences [Dd (in ppm) = dS ꢀ dR] obtained
for (S)- and (R)-MTPA amides (9a and 9b) of aglycone (5) for
brasilicardin B (2).

5548
K. Komatsu et al. / Bioorg. Med. Chem. 12 (2004) 5545–5551
Table 2. MLR inhibitory and cytotoxic activities of brasilicardins A–
D (1–4)
The stereostructure of brasilicardin C (3) was estab-
lished by a single crystal X-ray diffraction analysis as
shown in Figure 4a. The X-ray analysis revealed the
presence of a tetragonal nitrogen atom [N(1)] and a
carboxylate [C(18)], suggesting that 3 presented as a
zwitter ions. Figure 4b represented association of two
molecules of 3, in which two intermolecular hydrogen
bonds were observed at O(8)–HO(17) and O(17)–
HO(2). A unit cell of crystal was composed of eight
molecules of 3 together with eight molecules of H2O,
and 12 molecules of MeOH, four of which disordered
like the MeOH for C(63)–O25 as shown in Figure 4b.
Compd.
IC50 (lg/mL)
MLR
0.057
L1210
1
1.2
>10
7.8
2
2.5
2.5
3
4
Cyclosporin A
>10
0.15
>10
a
a Not tested.
20
Brasilicardin D {4, ½aŠ +79 (c 0.5, MeOH)} was sug-
of a methoxy signal observed for 3. The structure of 4
including relative stereochemistry was elucidated by de-
tail analysis of 2D NMR data to be the desmethoxy
form of 3. The absolute stereochemistries of the agly-
cone and a rhamnopyranoside in 4 were assigned on
D
gested to possess the molecular formula, C29H49NO8,
by HRFABMS data [m/z 540.3358 (M+H)+,
D
1
+0.2mmu]. H and 13C NMR data (Table 1) were simi-
lar to those of brasilicardin C (3), except for the absence
the basis of spectral data of its aglycone 5 and methyl-
20
D
a-rhamnopyranoside {6, ½aŠ ꢀ46} obtained by meth-
anolysis of 4.
Brasilicardins B–D (2–4) are new congeners of brasili-
cardin A (1) with immunosuppressive activity and cyto-
toxicity. Effects of brasilicardins B–D (2–4) on mouse
MLR were examined in comparison with that of brasili-
cardin A and cyclosporin A. Suppressive activities of
brasilicardins B (2) and C (3) on the proliferative re-
sponse of mouse lymphocytes to alloantigen stimulation
were 50 times less potent than that of 1. On the other
hand, brasilicardin D (4) showed no such suppressive
activity. Cytotoxic activities of 2–4 were tested against
murine lymphoma L1210 cells in vitro. The IC50 value
of brasilicardin C (3) was 7.8lg/mL, which was less po-
tent than that of 1, while 2 and 4 showed no cytotoxicity
(IC50 > 10lg/mL). These results suggest that the pres-
ence of a methoxy group at C-16 as well as a glucos-
amine unit and/or a benzoyl group are important for
the immunosuppressive and cytotoxic activities for bra-
silicardin A (1) (Table 2).
3. Experimental
3.1. General experimental procedure
The 3.35ppm resonance of residual CH3OH and
49.8ppm of CD3OD were used as internal references
1
for H and 13C NMR spectra, respectively. FAB mass
spectra were obtained using nitrobenzylamine as a
matrix.
3.2. Cultivation
The voucher specimen of Nocardia brasiliensis (strain
IFM 0406) was deposited at the Center for Pathogenic
Fungi and Microbial Toxicoses, Chiba University
(deposit No. FERM BP-5498).4 This actinomycete was
grown in the broth [glycerol (2.0%), polypepton
(1.0%), and meat extract (0.5%) in H2O, pH7.0]. Cul-
tures were incubated in a 150L jar fermentor at 32ꢁC
for 4days with stirring at 250rpm and 150L/min aera-
tion rate and were centrifuged.
Figure 4. (a) Molecular structure of brasilicardin C (3) obtained from
X-ray analysis (ORTEP drawing; ellipsoids are drawn at 30%
probability level) and (b) contact model of two molecules of
brasilicardin C (3).

K. Komatsu et al. / Bioorg. Med. Chem. 12 (2004) 5545–5551
5549
3.3. Extraction and isolation
brasilicardin C (3) under the same condition as 2
22
afforded aglycone 102 {0.34mg; ½aŠ +56 (c 0.06,
D
The supernatant of the fermentation broth (80L) was
passed through a Diaion HP-20 column and washed
with 2M NaCl aq (20L) and H2O (20L) and then eluted
batchwise with MeOH/H2O (1:1, 20L) and MeOH
(20L). The fraction eluted with MeOH was chromato-
graphed on a silica gel column eluted with stepwise gra-
dient of CHCl3/MeOH to yield a fraction (13.7g), which
was separated by a C18 column with stepwise gradient of
MeOH/H2O. The fraction eluted with 50–70% MeOH/
H2O was further separated by C18 HPLC (YMC-Pack
ODS R&D, YMC Co., Ltd, 2 · 25cm; MeOH/H2O,
70:30; flow rate 10mL/min; UV detection at 205nm)
and then C18 HPLC [YMC-Pack ODS R&D; MeOH/
H2O (65:35 ! 67:33) containing CF3CO2H (25ppm);
flow rate 10mL/min; UV detection at 205nm] to afford
brasilicardins B (2, 6.6mg) and C (3, 11.8mg).
MeOH); HRFABMS m/z 437.3135 (M+H)+ (calcd for
C25H44NO5, 438.3219)} and methyl a-L-rhamnopyrano-
20
D
side {6, 0.17mg; ½aŠ ꢀ4 8 (c 0.05, MeOH); HRFABMS
m/z 179.0918 (M+H)+ (calcd for C7H15O5, 179.0920)}
were yielded. Methanolysis of brasilicardin D (4,
25
D
1.2mg) gave an aglycone 5 {0.9mg, ½aŠ +107 (c 0.10,
MeOH); HRFABMS m/z 408.3119 (M+H)+ (calcd for
C24H42NO4, 408.3114)} and methyl a-L-rhamnopyrano-
20
D
side {6, 0.20mg; ½aŠ ꢀ4 6 (c 0.05, MeOH); HRFABMS
m/z 179.0923 (M+H)+ (calcd for C7H15O5, 179.0920)}.
25
D
3.4.1. Compound 5. Colorless amorphous solid; ½aŠ
+107 (c 0.1, MeOH); IR(KBr) mmax 3428, 2924, 1737,
1633, and 1061cmꢀ1; H NMR (CD3OD) d 0.94(3H,
1
s, H3-19), 1.02 (3H, s, H3-20), 1.04(3H, s, H -22), 1.15
3
(3H, s, H3-21), 1.30 (1H, m, H-14), 1.36 (1H, m, H-7),
1.37 (1H, m, H-15), 1.39 (1H, m, H-1), 1.40 (1H, m,
H-9), 1.53 (1H, m, H-15), 1.66 (1H, m, H-5), 1.68 (1H,
m, H-6), 1.69 (3H, s, H3-23), 1.73 (1H, dd, J = 4.3 and
12.5Hz, H-1), 1.78 (1H, m, H-6), 1.80 (1H, m, H-7),
1.83 (2H, m, H2-16), 1.90 (1H, m, H-11), 1.96 (1H, m,
H-11), 2.91 (1H, d, J = 9.5Hz, H-3), 3.53 (1H, t,
J = 6.0Hz, H-17), 3.68 (1H, ddd J = 4.3, 9.5, and
11.4Hz, H-2), 3.78 (3H, s, 18-OCH3), and 5.37 (1H,
m, H-12); 13C NMR (CD3OD) d 18.1 (C-19), 19.1
(C-6), 24.0 (C-23), 24.2 (C-22), 27.7 (C-11), 28.3
(C-15), 29.6 (C-21), 30.1 (C-20), 32.3 (C-7), 37.4
(C-16), 38.6 (C-10), 39.0 (C-8), 41.7 (C-4), 45.6 (C-9),
45.6 (C-1), 48.3 (C-14), 53.3 (COOCH3), 56.2 (C-17),
57.6 (C-5), 71.0 (C-2), 85.3 (C-3), 124.2 (C-12), 139.0
(C-13), and 177.3 (C-18); FABMS m/z 408 (M+H)+;
HRFABMS m/z 408.3128 (M+H)+ (calcd for
C24H42NO4, 408.3114).
Parts (50g) of the fraction eluted with MeOH/H2O (1:1)
in the previous HP-20 column were partitioned between
EtOAc/H2O and then n-BuOH/H2O. The n-BuOH-solu-
ble materials were subjected to silica gel column chro-
matography (CHCl3/MeOH/H2O, 7:2:0.2–6:3:0.5) to
yield a fraction (1.1g), which was purified by a C18 col-
umn (MeOH/H2O), centrifuged partition chromatogra-
phy (descending mode, n-BuOH/MeOH/H2O, 4:5:1),
and then C18 HPLC (YMC-Pack ODS R&D,
2 · 25cm; MeOH/H2O 70:30; flow rate 10mL/min; UV
detection at 205nm) to afford brasilicardins C (3,
45mg) and D (4, 23.1mg).
3.3.1. Brasilicardin B (2). Colorless amorphous solid;
23
½aŠ +17 (c 1.0, MeOH); IR (KBr) mmax 3428, 1679,
D
1
and 1633cmꢀ1; H and 13C NMR (Table 1); FABMS
m/z 863 (M+H)+; HRFABMS m/z 863.4554 (M+H)+
(calcd for C44H67N2O15, 863.4542).
3.5. Tris-benzoate (8) of aglycone 5
3.3.2. Brasilicardin C (3). Colorless amorphous solid;
Compound 5 (0.4mg), DMAP (0.23mg) and benzoyl
chloride (1.1mg) in dry pyridine (30lL) was heated at
80ꢁC for 10h. The mixture was diluted with satd NH4Cl
aq and extracted with EtOAc. The organic layer was
washed with water and brine, and then evaporated.
The residue was separated by a silica gel column (hex-
ane/EtOAc, 85:15) and C18 HPLC (Develosil ODS
UG-5, Nomura Chemical Co., Ltd, 10 · 250mm; eluent
MeOH/H2O, 90:10; flow rate, 2.5mL/min; UV detection
at 230nm) to afford a tris-benzoate (8, 0.2mg tR 26min):
UV (MeOH) kmax 228nm (e 39000); CD (MeOH) kext
23
½aŠ +65 (c 1.0, MeOH); IR (KBr) mmax 3433, 1679,
D
1
and 1633cmꢀ1; H and 13C NMR (Table 1); FABMS
m/z 570 (M+H)+; HRFABMS m/z 570.3636 (M+H)+
(calcd for C30H52NO9, 570.3642).
3.3.3. Brasilicardin D (4). Colorless amorphous solid;
20
½aŠ +79 (c 0.5, MeOH); IR (KBr) mmax 3420, and
D
1
1631cmꢀ1; H and 13C NMR (Table 1); FABMS m/z
540 (M+H)+; HRFABMS m/z 540.3358 (M+H)+ (calcd
for C29H50NO8, 540.3356).
1
237 (De + 18.4) and 223nm (ꢀ10.7); H NMR (CDCl3)
3.4. Mathanolysis of brasilicardins B–D (2–4)
d 0.97 (3H, s, H3-20), 1.01 (3H, s, H3-22), 1.13 (3H, s,
H3-19), 1.21 (3H, s, H3-21), 1.35 (1H, m, H-7), 1.39
(1H, m, H-15), 1.40 (1H, m, H-9), 1.60 (3H, s, H2-22),
1.64(1H, m, H-6), 1.71 (1H, m, H-1), 1.73 (1H, m, H-
15), 1.78 (1H, m, H-6), 1.84(1H, m, H-14), 1.86 (2H,
m, H2-11), 1.90 (1H, m, H-16), 1.99 (1H, m, H-1), 2.01
(1H, m, H-7), 2.08 (1H, m, H-16), 2.26 (1H, m, H-5),
3.81 (3H, s, COOCH3), 4.85 (1H, m, H-17), 5.16 (1H,
d, J = 10.1Hz, H-3), 5.29 (1H, s, H-12), 5.47 (1H, m,
H-2), 6.67 (1H, d, J = 6.7Hz, 17-NH), 7.30–7.36 (4H,
m, Ph), 7.44–7.47 (4H, m, Ph), 7.53 (1H, t, J = 7.7Hz,
Ph), 7.81 (2H, d, J = 7.7Hz, Ph), 7.88 (2H, d,
J = 7.7Hz, Ph) and 7.96 (2H, d, J = 7.8Hz, Ph); ESIMS
Generally, each brasilicardins B–D (2–4, 1.0mg each)
was treated with 5% HCl/MeOH (30lL) at 100ꢁC for
16h. After evaporation of the solvent using nitrogen
stream, the residue was subjected to silica gel column
chromatography (CHCl3/MeOH, 9:1). From 2, agly-
cone 5 (0.5mg), methyl a-L-rhamnopyranoside {6,
22
D
0.14mg; ½aŠ ꢀ39 (c 0.02, MeOH), HRFABMS m/z
22
179.0922 (M+H)+ (calcd for C7H15O5, 179.0920)}, and
methyl a-D-glucosamine {7, 0.22mg; ½aŠ +35 (c 0.04,
D
H2O); HRFABMS m/z 193.1032 (M+H)+ (calcd for
C7H16NO5, 193.1029)} were obtained. Methanolysis of

5550
K. Komatsu et al. / Bioorg. Med. Chem. 12 (2004) 5545–5551
m/z 742 (M+Na)+; HRFABMS m/z 742.3689
merged. The linear absorption coefficient, l, for Mo-
Ka radiation was 1.0cmꢀ1. An absorption correction
using the program ABSCOR5 was applied, which re-
sulted in transmission factors ranging from 0.88 to
1.00. The data were corrected for Lorentz and polariza-
tion effects.
[(M+Na)+, calcd for C45H53NO7Na, 742.3703].
3.6. (S)- and (R)-MTPA amide (9a and 9b) of aglycone 5
A solution of compound 5 (0.2mg) in CH2Cl2 (20lL)
were added to DCC (0.13mg) and (S)-(+)-MTPA
(0.13mg), and the mixture was stirred at room tempera-
ture for 1h. The residue was passed through a silica gel
column (hexane/EtOAc, 1:1) and purified by C18 HPLC
(Develosil ODS UG-5, 10 · 250mm; eluent MeOH/
H2O, 82:12; flow rate, 2.5mL/min; UV detection at
230nm) to give the (S)-MTPA amide (9a, 0.1mg, tR
The structure was solved by direct methods
(SHELXS86)7 and expanded using the Fourier tech-
nique (DIRDIF94).8 The nonhydrogen atoms were re-
fined anisotropically. Hydrogen atoms were included
but not refined. The final cycle of full matrix least-
squares refinement was based on 10,630 observed reflec-
tions (I > ꢀ3.00r(I), 2h < 60.07) and 828 variable
parameters and converged with unweighted and
weighted agreement factors of R1 = 0.0825, Rw =
0.2016. The standard deviation of an observation of unit
weight was 1.02. The weighting scheme was based on
counting statistics and included a factor (p = 0.062) to
downweight the intense reflections. Plots of
Rx(Fo2 ꢀ Fc2)2 versus Fo2 reflection order in data col-
lection, sin h/k and various classes of indices showed
no unusual trends. The maximum and minimum peaks
on the final difference Fourier map corresponded to
1
17min) of compound 5: H NMR (CDCl3) d 0.84(3H,
s), 0.87 (3H, s), 0.92 (3H, s), 0.95 (3H, s), 1.21 (1H, m,
H-9), 1.23 (1H, m, H-14), 1.25 (1H, m, H-7), 1.32 (1H,
m, H-1), 1.41 (3H, s, H3-22), 1.45 (1H, m, H-5), 1.54
(1H, m, H-7), 1.54(1H, m, H-6), 1.67 (1H, m, H-6),
1.71 (1H, m, H-1), 1.79 (1H, m, H-11), 1.81 (2H, m,
H2-16), 1.86 (1H, m, H-11), 1.95 (2H, m, H2-15), 2.89
(1H, d, J = 10.0Hz, H-3), 3.46 (3H, s, OCH3 of MTPA),
3.70 (1H, m, H-2), 3.71 (3H, s, 18-OCH3), 4.60 (1H, m,
H-17), 5.24(1H, m, H-12), 7.13 (1H, br s, 17-NH), 7.32
(3H, m, Ph), and 7.49 (2H, m, Ph); HRESIMS m/z
646.3326 (M+Na)+ (calcd for C34H48NO6F3Na,
646.3332).
ꢀ
3
˚
1.37 and ꢀ0.71 e /A , respectively. All calculations were
performed using the teXsan crystallographic software
package of Molecular Structure Corporation. Crystallo-
graphic data for brasilicardin C (3) have been deposited
at the Cambridge Crystallographic Data Center (deposi-
tion number CCDC 244063).
The (R)-MTPA amide (9b, 0.1mg) of compound 5 was
prepared from the same procedure as described above.
1
Compound 9b: H NMR (CDCl3) d 0.92 (3H, s), 0.97
(3H, s), 1.00 (3H, s), 1.09 (3H, s), 1.31 (1H, m, H-9),
1.31 (1H, m, H-7), 1.34(1H, m, H-1), 1.41 (1H, m, H-
14), 1.53 (1H, m, H-5), 1.62 (1H, m, H-6), 1.62 (3H, s,
H3-22), 1.66 (1H, m, H-7), 1.74(1H, m, H-1), 1.82
(1H, m, H-11), 1.83 (1H, m, H-6), 1.85 (2H, m, H2-
16), 1.91 (1H, m, H-11), 2.03 (2H, m, H2-15), 2.97
(1H, d, J = 10.0Hz, H-16), 3.37 (3H, s, OCH3 of
MTPA), 3.76 (3H, s, 18-OCH3), 4.63 (1H, m, H-17),
5.33 (1H, m, H-12), 7.41 (3H, m, Ph), 7.46 (1H, brs,
17-NH), and 7.54(2H, m, Ph); HRESIMS 664.3328
(M+Na)+ (calcd for C34H48NO6F3Na, 646.3331).
3.8. Mouse MLR assay
Mouse MLR assay was performed as described by Hat-
anaka et al.9 The spleens obtained from BALB/C and
C57BL/6 mice (female, 6–7 weeks old) were homoge-
nized into single cell suspensions. Ammonium chloride
buffer (0.15M KHCO3, 0.1mM Na2 EDTA, pH7.2)
was added to the cell suspension in order to lyse erythro-
cytes followed by washing three times with RPMI1640
medium. The erythrocyte-free cell preparation was then
resuspended in RPMI1640 complete medium (supple-
mented with 10% FCS and 50lM 2-mercaptoethanol).
The mouse MLR was performed in a 96-well round-bot-
tom microtest plate with each well containing 5 · 105
C57BL/6 spleen cells (responder cells, H-2b) and
5 · 105 mitomycin C-treated (25lg/mL of mitomycin
C at 37ꢁC for 30min and washed three times with
RPMI1640 medium) BALB/C spleen cells (stimulator
cells, H-2d), and various amounts of test compound in
0.2mL RPMI1640 complete medium. The cells were
incubated at 37ꢁC in a humidified atmosphere of 5%
CO2–95% air. After 92h of cultivation, cells were
pulse-labeled with 0.5lCi of [3H]thymidine for 4h at
37ꢁC and harvested using a multiple cell harvester.
The radioactivity incorporated into the cells was meas-
ured with a liquid scintillation counter. Results were ex-
pressed as IC50 values.
3.7. X-ray crystallography of brasilicardin C (3)
Brasilicardin C (3) was crystallized from H2O–MeOH as
colorless needles, mp 219–221ꢁC. Crystal data:
C31.5H61NO12.5 [C30H51NO9, 1.5(CH4O), 2(H2O), Mr =
653.83, crystal dimensions 0.400 · 0.100 · 0.050mm,
C-centered monoclinic, space group C2 (no. 5), a =
˚
˚
˚
32.27(1)A,
b = 8.243(4)A,
c = 27.83(1)A,
b =
111.18(3)ꢁ, V = 6873(4)A , Z = 8, Dcalc = 1.264g/cmꢀ1
All measurements were made on Rigaku RAXIS-
RAPID Imaging Plate diffractometer with graphite
.
3
˚
˚
monochromated Mo-Ka radiation (k = 0.71075A) at a
temperature of ꢀ175 1ꢁC to a maximum 2h value of
60.1ꢁ. A total of 70 images, corresponding to 210.0ꢁ
oscillation angles, were collected with two different gonio-
meter settings. Exposure time was 5.50min per degree.
The camera radius was 127.40mm. Readout was per-
formed in the 0.100mm pixel mode. Data were pro-
cessed by the PROCESS-AUTO program package. Of the
37,433 reflections, which were collected, 10,630 were
unique (Rint = 7.7%); equivalent reflections were
3.9. Cytotoxicity assay
Cytotoxicity assay was performed in 96-well flat-bottom
microtest plate; each well contained 104cells and a var-

K. Komatsu et al. / Bioorg. Med. Chem. 12 (2004) 5545–5551
5551
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4. Komaki, H.; Nemoto, A.; Tanaka, Y.; Takagi, H.;
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University Science Books: Mill Valley, CA, 1983.
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Acknowledgements
We thank Ms. S. Oka and Miss. M. Kiuchi, Center for
Instrumental Analysis, Hokkaido University, for
ESIMS and FABMS measurements. This work was
partly supported by a Grant-in-Aid from the Takeda
Science Foundation and a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
References and notes
1. Tsuda, M.; Nemoto, A.; Komaki, H.; Tanaka, Y.;
Yazawa, K.; Mikami, Y.; Kobayashi, J. J. Nat. Prod.
1999, 62, 1640, and references cited therein.
10. Hansen, M. B.; Nielsen, S. E.; Berg, B. J. Immunol. Meth.
1989, 119, 203.