Absolute Stereochemistry of Immunosuppressive Macrolide Brasilinolide A and Its New Congener Brasilinolide C

Article abstract of DOI:10.1021/jo035773v

Brasilinolide A (2) is a 32-membered polyhydroxyl macrolide with immunosuppressive activity isolated from a pathogenic actinomycete, Nocardia brasiliensis IFM0406. The absolute configurations at 26 chiral centers of 2 and its new congener, brasilinolide C (1), were determined on the basis of the spectral data of 1 and degradation products derived from 1 and 2.

Full text of DOI:10.1021/jo035773v

Absolu te Ster eoch em istr y of Im m u n osu p p r essive Ma cr olid e
Br a silin olid e A a n d Its New Con gen er Br a silin olid e C
Kazusei Komatsu,† Masashi Tsuda,† Yasushi Tanaka,‡ Yuzuru Mikami,§ and
J un’ichi Kobayashi*,†
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812,
Research Laboratory, Higeta Shoyu Co., Ltd., Chiba 288-8680, and Research Center for Pathogenic Fungi
and Microbial Toxicoses, Chiba University, Chiba 260-0856, J apan
jkobay@pharm.hokudai.ac.jp
Received December 4, 2003
Brasilinolide A (2) is a 32-membered polyhydroxyl macrolide with immunosuppressive activity
isolated from a pathogenic actinomycete, Nocardia brasiliensis IFM0406. The absolute configurations
at 26 chiral centers of 2 and its new congener, brasilinolide C (1), were determined on the basis of
the spectral data of 1 and degradation products derived from 1 and 2.
In tr od u ction
Many macrolides with unique structures as well as
various biological activities have been isolated from
terrestrial actinomycetes.1 Polyhydroxy macrolides such
as notonesomycin A2 or liposidolide A3 are unique com-
pounds having a number of chiral centers with a tetra-
hydropyran ring, an epoxide, and a malonyl or a fatty
acid ester. The gross structures and partial relative
stereochemistry of these macrolides have been elucidated
mainly by means of 2D NMR data, whereas the absolute
configurations remain unsolved. During our search for
bioactive substances from pathogenic actinomycetes of
genus Nocardia,4 we have previously isolated a 32-
membered macrolide, brasilinolide A5 (2), with a sugar
moiety and a malonyl group as well as a tricyclic
metabolite, brasilicardin A,6 containing a rhamnose, an
N-acetylglucosamine, and an amino acid moiety from the
actinomycete Nocardia brasiliensis IFM-0406. Both com-
pounds exhibited potent immunosuppressive activity in
mouse mixed lymphocyte assay. More recently, we have
isolated a new brasilinolide congener, brasilinolide C (1),
from the same strain and determined the absolute
configurations at 26 chiral centers of 1 on the basis of
the spectral data of 1 and its degradation products.
Furthermore, the absolute stereochemistry of 2 was
elucidated by the chemical correlation between 1 and 2.
Here we describe the isolation of 1 and the absolute
stereochemistry of 1 and 2.
Resu lts a n d Discu ssion
Isola tion a n d Str u ctu r e of 1. The supernatant of the
fermentation broth (80 L) of N. brasiliensis IFM-0406 was
subjected to a Diaion HP-20 column (50% MeOH(aq) f
MeOH), in which fractions eluted with MeOH were
separated on silica gel and C18 columns and followed by
C18 HPLC (MeOH/H2O and then MeOH/H2O/CF3CO2H)
to afford 1 (13.4 mg/L of medium) as a colorless solid
together with 25 (0.6 mg/L).
* To whom correspondence should be addressed. Phone and fax: +81
11 706 4985. Fax: +81 11 706 4989.
† Hokkaido University.
‡ Higeta Shoyu Co., Ltd.
The molecular formula of 1 was established to be
C49H88O20 by HRFABMS data [m/z 1019.5760 (M + Na)+,
§ Chiba University.
(1) Shiomi, K.; Ohmura, S. In Macrolide Antibiotics, 2nd ed.;
Ohmura, S., Ed.; Academic Press: San Diego, 2002; pp 1-56.
(2) Sasaki, T.; Furihata, K.; Nakayama, H.; Seto, H.; Otake, N.
Tetrahedron Lett. 1986, 27, 1603-1606.
(3) Kihara, T.; Koshino, H.; Shin, Y.-C.; Yamaguchi, I.; Isono, K. J .
Antibiot. 1995, 48, 1385-1387.
(4) Tsuda, M.; Nemoto, A.; Komaki, H.; Tanaka, Y.; Yazawa, K.;
Mikami Y.; Kobayashi, J . J . Nat. Prod. 1999, 62, 1640-1642 and
references therein.
(5) Shigemori, H.; Tanaka, Y.; Yazawa, K.; Mikami, Y.; Kobayashi,
J . Tetrahedron 1996, 52, 9031-9034.
(6) Shigemori, H.; Komaki, H.; Yazawa, K.; Mikami, Y.; Nemoto,
A.; Tanaka, Y.; Sasaki, T.; In, Y.; Ishida, T.; Kobayashi, J . J . Org.
Chem. 1998, 63, 6900-6904.
1
∆ -0.7 mmu]. H and 13C NMR spectra suggested the
presence of an ester carbonyl, 2 sp2 methines, a quater-
nary carbon due to hemiketal, 25 sp3 methines including
20 oxymethines, 13 sp2 methylenes, and 7 methyl car-
bons, which were similar to those of 25 except for the
absence of a malonyl group and a pentanoyl group.
1
Detailed analysis of 2D NMR data including the H-1H
COSY, TOCSY, HSQC, HSQC-TOCSY, HMBC, and
ROESY spectra revealed gross structures of aglycon and
sugar moieties of 1 (Figure 1). The 1H-1H COSY, TOCSY,
10.1021/jo035773v CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/24/2004
J . Org. Chem. 2004, 69, 1535-1541
1535

Komatsu et al.
F IGURE 1. Selected 2D NMR correlations for 1.
The molecular formula of the macrocyclic aglycon 3 was
revealed to be C43H78O17 by HRFABMS data [m/z 867.5356
(M + H)+, ∆ -1.8 mmu]. 1H and 13C NMR data (Table 1)
of C-1-C-26 and C-38 in 3 were close to those of the
corresponding portion in 1, while 1H and 13C chemical
shifts for the C-27-C-37 and C-39-C-43 portions in 3
differed from those of 1. Chemical shifts for H-28 (δH 3.61)
and H-29 (δH 3.74) in 3 were at lower field than those of
1, suggesting that the epoxide ring in 1 was opened for
3. The HMBC correlation for H-33/C-29 suggested that
a tetrahydropyran ring was formed between C-29 and
C-33 through O-29. Thus, the gross structure of the
aglycon was assigned as 3. The relative stereochemistry
of C-28-C-29 was suggested to be erythro, since the
tetrahydrofuran ring might be produced by an SN2
addition of the oxygen atom at C-33 to the trans-epoxide.
It was difficult to establish the stereochemistry from
F IGURE 2. ROESY correlations and relative stereochemistry
for the (a) C-15-C-19 and (b) C-1′-C-6′ portions in 1. J (Hz)
(H/H): H-16/H-17, 9.3; H-18a/H-19, 10.6; H-18b/H-19, <2; H-1′/
H-2′a, <1; H-1′/H-2′b, 3.2.
and HSQC-TOCSY spectra indicated three partial struc-
tural units of H-2 to H2-14, H-16 to H3-43, and H-1′ to
H3-6′. HMBC correlations for H2-14 (δH 1.99 and 1.83)
and H-16 (δH 3.39) to a hemiketal carbon at C-15 (δC
100.53) revealed the presence of a tetrahydropyran ring
(C-15-C-19). The relative stereochemistry of the tet-
rahydropyran ring was deduced from ROESY correlations
1
1
spectroscopic data such as H-1H and H-13C coupling
1
constants or ROESY data for the aglycon 3, since the H
and 13C NMR data were too complicated. To determine
the absolute stereochemistry of 22 stereogenic centers in
3, the following strategy was planned: (1) fragmentation
through oxidative degradation, (2) determination of rela-
tive and absolute configurations of degradation products,
(3) determination of absolute configurations of parts not
included in the degradation products.
1
and H-1H coupling constants as shown in Figure 2. The
existence of a trans-epoxide at C-28-C-29 was indicated
by a small J (H-28,H-29) value (1.9 Hz). The HMBC
correlation for H-1′ to C-5′ suggested the presence of a
sugar moiety (C-1′-C-6′), which was elucidated to be
1
2-deoxyfucopyranose by ROESY correlations and H-1H
couplings as shown in Figure 2. The HMBC correlation
for H-1′ to C-37 indicated that the sugar was connected
to C-37. The ester linkage between C-1 and C-31 was
deduced from HMBC correlations for H-2 and H-31 to
C-1. Thus, the gross structure of 1 was elucidated to be
a deacyl form of 2, that is, lacking both 23-O-malonyl and
3′-O-pentanoyl groups.
Oxid a tive Degr a d a tion of Aglycon 3 in 1. For
cleavage between C-15 and C-17 and between C-27 and
C-28 in the aglycon 3, oxidative degradation of 3 using
sodium periodate was carried out as follows. Reduction
of the hemiketal at C-15 with sodium borohydride and
then methanolysis of the ester linkage at C-1 with sodium
methoxide were followed by oxidation with sodium per-
iodate to afford a mixture of degradation products, which
was treated with sodium borohydride and then pivaloyl
chloride to give three segments, the 15-O-pivaloyl ester
of the C-1-C-15 segment (7), the 17,27-bis-O-pivaloyl
ester of the C-17-C-27 segment (8), and the 28-O-pivaloyl
ester of the C-28-C-37 segment (9) (Scheme 1). The gross
Absolu te Ster eoch em istr y of 2-Deoxy-r-L-fu co-
p yr a n ose (4) a n d th e Str u ctu r e of Aglycon 3. To
obtain the sugar moiety of 1, it was subjected to hydroly-
sis followed by C18 HPLC separation to afford a macro-
cyclic aglycon (3) and 4, the latter of which was converted
into methyl 2-deoxy-R-L-fucopyranoside (5) by treatment
with hydrogen chloride in methanol (Scheme 1). To apply
the dibenzoate rule8 for 5, the 3,4-bis-O-benzoate 6 was
prepared. The CD spectrum of 6 showed the negative first
and positive second Cotton effects [λext 237 (∆ꢀ -20.5) and
222 (+7.6) nm], thus indicating 3S and 4S configurations
for 5. Therefore, the sugar moiety in 1 was determined
to be 2-deoxy-R-L-fucopyranoside,9 and the absolute
configurations at C-1′, C-3′, C-4′, and C-5′ were assigned
as R, S, S, and S, respectively.
1
structures of 7-9 were assigned by analysis of H-1H
COSY and HMQC spectra as well as HRFABMS data.
Since these degradation products contained three or four
hydroxyl groups adjacent to chiral centers, the relative
and absolute stereochemistries were determined using
these hydroxyl groups.
Absolu te Con figu r a tion s a t C-5, C-7, C-9, a n d C-13
in 1. Since the 15-O-pivaloyl ester of the C-1-C-15
segment (7) contained a 1,3,5-triol portion, acetonization
was performed to obtain its 5,7-isopropylidene derivative
(7) Tanaka, Y.; Komaki, H.; Yazawa, K.; Mikami, Y.; Nemoto, A.;
Tojyo, T.; Kadowaki, K.; Shigemori, H.; Kobayashi, J . J . Antibiot. 1997,
50, 1036-1041.
(8) Harada, N.; Nakanishi, K. Circular Dichroic Spectrometry-
Exciton Coupling in Organic Stereochemistry; University Science
Books: Mill Valley, CA, 1983.
(9) The [R]D value [-140° (c 0.1, H2O)] of 5 corresponded well with
that of methyl 2-deoxy-R-L-fucopyranoside [-160° (c 0.19, H2O)]:
Korytnyk, W.; Sufrin, J . R.; Bernacki, R. J . Carbohydr. Res. 1982, 103,
170-175.
1536 J . Org. Chem., Vol. 69, No. 5, 2004

Absolute Stereochemistry of Brasilinolides A and C
SCHEME 1
10 as a single product. The position of the acetonide ring
was assigned by NOESY correlations from H-5 (δH 3.58)
and H-7 (δH 3.94) to R-methyl (δH 1.20) of the isopropyl-
idene group. Since R- and â-methyl groups of the isopro-
pylidene group resonated at δC 19.3 and 30.1, respec-
tively, the 1,3-diol at C-5 and C-7 was assigned as a syn
relation according to the Rychnovsky rule.10 The relative
stereochemistry of C-7 and C-9 remained undefined, since
the 7,9-isopropylidene derivative of 7 was not obtained.
However, the carbon chemical shift of C-7 (δC 68.9) in
the 13C NMR spectrum of 3 in methanol-d4 corresponded
well to that of C-5 (δC 68.4) for the 3,7-anti form of
decane-1,3,5,7-tetraol,11 indicating a C-5/C-9-anti relation
for 3.
acetonide were observed for 12. Two methyl carbons of
the acetonide in 11 resonated at δC 24.7 and 24.8,
suggesting the 19,21-anti relation.9 The 21,23-anti rela-
tion in 12 was deduced from the 13C chemical shifts of
two methyls (δC 24.8 and 25.0) of the acetonide. The
absolute configuration at C-19 in 12 was elucidated to
be S on the basis of application of the modified Mosher
method11 (Figure 4).
Therefore, the absolute configurations of the C-15-
C-23 portion including the tetrahydropyran ring in 1
were assigned as 15S, 16R, 17S, 19R, 21S, and 23R.
Absolu te Con figu r a tion s of C-28-C-37 in 1. Treat-
ment of the C-28-C-37 segment (9) with 2,2-dimethoxy-
propane gave the 35,37-isopropylidene derivative 13. The
relative stereochemistry of the tetrahydropyran portion
in 13 was implied to be 29,30-anti, 30,31-syn, 31,32-anti,
and 32,33-anti relations by NOESY correlations for H-28/
H3-39, H-29/H-34, and H-30/H3-40 and J (H-29,H-30),
J (H-30,H-31), J (H-31,H-32), and J (H-32,H-33) values
(Figure 5). The 35,37-syn relation was confirmed by 13C
chemical shifts of two acetonide methyl groups (R-CH3,
δC 30.7; â-CH3, δC 19.8) obtained by the HMQC spectrum
of 13.11 NOESY correlations for H-35 (δH 3.70) and H-37
Compound 10 was converted into the 9,13-bis-(S)- and
-(R)-MTPA esters 14a and 14b, respectively, through
MTPA esterification and then deprotection of the ac-
1
etonide. ∆δ values obtained from the H NMR spectra of
14a and 14b suggested that the absolute configurations
at C-9 and C-13 were S and R, respectively12 (Figure 3).
Therefore, the absolute configurations at C-5, C-7, C-9,
and C-13 in 1 were concluded to be S, R, S, and R,
respectively. Since the stereochemical relationship be-
tween C-12 and C-13 was not derived from the NMR
data, the relative stereochemistry was elucidated as
described later.
Absolu te Con figu r a tion s a t C-15, C-16, C-17, C-19,
C-21, a n d C-23 in 1. The 17,27-bis-O-pivaloyl ester of
the C-17-C-27 segment (8) was converted into 19,21- and
21,23-isopropylidene derivatives 11 and 12, respectively.
The NOESY spectrum of 11 showed correlations for H-19
(δH 3.89)/R-CH3 (δH 1.31) of the acetonide and H-21
(δH 4.11)/â-CH3 (δH 1.36) of the acetonide, while NOESY
correlations for H-21 (δH 4.14)/â-CH3 (δH 1.38) of the
acetonide and H-23 (δH 3.68)/R-CH3 (δH 1.32) of the
1
(δH 3.56) to â-CH3 (δH 1.52) in the acetonide and H-1H
coupling constants for H-35/H-36 (9.0 Hz) and H-36/
H-37 (9.8 Hz) suggested that the relative configurations
of H-35/H-36 and H-36/H-37 were both trans-diaxial
orientations. On the other hand, the relative configura-
tions at C-33/C-34 and C-34/C-35 were implied to be both
erythro by NOESY correlations for H-34/H3-42, H-36/
H3-41, and H-35/H-32 and J (H-33/H-34) (9.0 Hz) and
J (H-34/H-35) (<2 Hz) values.
1
∆δ values obtained from H NMR data of the (S)- and
(R)-MTPA esters 16a and 16b, respectively, of 13 indi-
cated the 31R configuration in 13 (Figure 6). Therefore,
the absolute configurations of the C-28-C-37 portion
including the trans-epoxide ring in 1 were concluded to
be 28S, 29S, 30S, 31R, 32S, 33S, 34R, 35R, 36R, and 37S.
Absolu te Con figu r a tion s a t C-12 a n d C-27 in 1.
From the experiments described above, absolute configu-
rations at 24 of 26 chiral centers in 1 have been
(10) (a) Rychnovsky, S. D.; Skalitzky, D. J . Tetrahedron Lett. 1990,
31, 945-948. (b) Rychnovsky, S. D.; Rogers, B.; Yang, G. J . Org. Chem.
1993, 58, 3511-3515.
(11) Kobayashi, Y.; Tan, C.-H.; Kishi, Y. Helv. Chim. Acta 2000, 83,
2562-2571.
(12) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am.
Chem. Soc. 1991, 113, 4092-4096.
J . Org. Chem, Vol. 69, No. 5, 2004 1537

Komatsu et al.
TABLE 1. 1H a n d 13C NMR Da ta of 1 a n d Its Aglycon 3
in CD3OD
1
3
position
δC
δH (m, J (Hz))
δC
δH (m, J (Hz))
1
2
3
4
169.26
125.34 5.99 (d, 15.6)
148.98 7.07 (dt, 15.6, 7.7) 140.90 7.07 (dt, 15.7, 7.6)
41.69 2.50 (dt, 14.6, 7.4) 41.44 2.55 (m)
168.27
125.55 5.97 (d, 15.7)
2.40 (m)
70.40 3.97 (m)
45.66 1.67 (m)
68.81 4.04 (m)
46.24 1.55 (m)
70.15 3.79 (m)
36.94 1.52 (m)
31.64 1.62 (m)
1.19 (m)
2.47 (m)
70.51 4.00 (m)
45.31 1.62 (m)
68.91 4.06 (m)
45.99 1.59 (m)
70.23 3.84 (m)
37.96 1.53 (m)
31.06 1.67 (m)
1.22 (m)
F IGURE 3. ∆δ values [∆δH (ppm) ) δS - δR] obtained for
the 9,13-bis-(S)- and (R)-MTPA esters 14a and 14b, respec-
tively, of the C-1-C-15 segment (7) in 1.
5
6
7
8
9
10
11
12
13
14
41.15 1.52 (m)
71.91 3.80 (m)
45.52 1.99 (m)
1.83 (m)
41.44 1.50 (m)
72.07 4.03 (m)
44.49 1.89 (br d, 5.1)
15
16
17
18
100.53
100.87
F IGURE 4. ∆δ values [∆δH (ppm) ) δS - δR] obtained for
the 19-(S)- and (R)-MTPA esters 15a and 15b, respectively,
of the C-17-C-27 segment (8) in 1.
77.83 3.39 (m)
70.40 3.89 (m)
41.93 1.93 (m)
1.34 (m)
78.72 3.31 (m)
70.36 3.91 (m)
41.89 1.97 (br d, 10.8)
1.38 (m)
19
20
21
22
23
24
66.75 4.17 (br t, 10.4)
45.86 1.58 (m)
69.83 3.79 (m)
46.66 1.49 (m)
66.85 4.06 (m)
39.69 1.47 (m)
1.41 (m)
67.34 4.20 (br t, 9.2)
45.00 1.66 (m)
70.03 3.88 (m)
45.92 1.62 (m)
67.57 4.01 (m)
39.18 1.56 (m)
25
26
23.40 1.62 (m)
1.19 (m)
34.63 1.45 (m)
1.34 (m)
23.40 1.72 (m)
1.47 (m)
34.02 1.82 (m)
1.46 (m)
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
1′
71.70 3.45 (br d, 8.6)
64.60 2.79 (dd, 3.8, 1.9)
61.17 2.74 (dd, 8.4, 1.9)
41.04 1.52 (m)
75.95 5.33 (br d, 10.5)
39.23 1.95 (m)
78.67 3.36 (br d, 9.3)
36.67 1.79 (m)
80.34 3.45 (br d, 8.6)
41.15 1.97 (m)
74.95 4.04 (m)
15.44 0.92 (d, 6.6)
16.38 1.04 (d, 7.0)
10.17 0.86 (d, 6.8)
5.74 0.89 (d, 6.8)
10.89 0.79 (d, 6.8)
15.23 1.08 (d, 6.5)
98.49 4.95 (d, 3.2)
34.63 1.89 (m)
1.68 (m)
74.13 3.61 (m)
78.78 3.74 (dd, 4.5, 8.7)
78.64 3.88 (m)
32.85 2.42 (m)
78.72 5.14 (dd, 3.9, 6.7)
34.07 2.17 (m)
83.34 3.83 (m)
36.87 2.15 (m)
79.74 3.55 (br d, 9.4)
44.19 1.80 (m)
70.99 4.03 (m)
15.04 0.93 (d, 6.7)
15.44 1.09 (d, 6.9)
16.20 1.02 (d, 6.9)
7.79 0.99 (d, 6.9)
12.09 0.80 (d, 6.9)
16.20 1.13 (d, 6.7)
F IGURE 5. NOESY correlations and relative stereochemistry
for the 35,37-O-isopropyridene C-28-C-37 segment (13) in 2.
J (Hz) (H/H): 29/30, 7.8; 30/31, <2; 31/32, <2; 32/33, <2;
33/34, 9.0; 34/35, <2; 35/36, 9.0; 36/37, 9.8.
2′
F IGURE 6. ∆δ values [∆δH (ppm) ) δS - δR] obtained for
the 31-(S)- and (R)-MTPA esters 16a and 16b, respectively,
of the 35,37-O-isopropylidene C-28-C-37 segment (13) in 1.
3′
4′
5′
6′
67.73 3.95 (m)
73.09 3.56 (br s)
68.37 3.95 (m)
17.93 1.19 (d, 6.5)
sugar unit, and then subjected to acetonidation with 2,2-
dimethoxypropane to afford three degradation products,
17-19.
determined, while those of C-12 and C-27 remain un-
solved. To obtain the C-1-C-15 segment possessing an
acetal ring at C-9-C-13 and the C-17-C-37 segment with
a 1,2-diol at C-27-C-28, another oxidative degradation
of 1 was carried out as shown in Scheme 2. Compound 1
was treated with sodium borohydride, sodium methoxide,
sodium periodate, and sodium borohydride in turn to
yield a mixture of C-1-C-15 and C-17-C-37 segments,
the latter of which possessed a sugar unit at C-37. The
mixture was treated with HCl/methanol to remove the
The molecular formula of compound 19 was elucidated
to be C38H68O10 by HRESIMS data [m/z 707.4714 (M +
Na)+, ∆ -0.4 mmu]. The 1H NMR spectrum of 19
disclosed eight singlet methyl signals due to four isopro-
pyridene acetals and five doublet methyls. Analysis of
1
the H-1H COSY and TOCSY spectra revealed that the
carbon chain of 19 corresponded to the C-17-C-37 part
of 1. The NOESY spectrum showed the correlation for
H-28 (δH 4.45)/H-33 (δH 3.42), indicating the presence of
a tetrahydropyran ring at C-29-C-33. Three six-mem-
1538 J . Org. Chem., Vol. 69, No. 5, 2004

Absolute Stereochemistry of Brasilinolides A and C
SCHEME 2
bered acetonide rings were revealed to be formed at
C-17-C-19, C-21-C-23, and C-35-C-37 by NOESY cor-
relations for H-17 (δH 3.69) and H-19 (δH 4.08) to a methyl
signal at δH 1.39, H-21 (δH 4.28) to a methyl at δH 1.47,
H-23 (δH 3.91) to a methyl at δH 1.49, and H-35 (δH 3.70)
and H-37 (δH 3.53) to a methyl at δH 1.43. The residual
acetonide was implied to be a five-membered ring formed
at C-27-C-28 by NOESY correlations for H-27 (δH 4.32)
and H-28 (δH 4.45) to one (δH 1.38) of the acetonide
methyls, suggesting an H-27/H-28-syn relation (27,28-
erthro). Thus, the absolute configuration at C-27 in 19
was elucidated to be S.
F IGURE 7. NOESY correlations and relative stereochemistry
for the C-9-C-13 portion of the 9,13-acetal derivative 20 of
the C-1-C-15 segment (18) in 1. J (Hz) (H/H): 10a/11a, 4.7;
10a/11b, 2.4; 10b/11a, 13.8; 10b/11b, 4.6; 12/13, 2.7.
HRESIMS data of compounds 17 [m/z 411.2363 (M +
Na)+, ∆ +0.5 mmu] and 18 [m/z 411.2360 (M + Na)+,
∆ +0.2 mmu] suggested the common molecular formula
C-15 with acetic acid in methanol yielded compound 20
having the ketal ring at C-9-C-13, which was indicated
by the NOESY correlation for H-13/9-OCH3 (δH 3.14)
(Figure 7). The 12,13-erythro relation was deduced from
NOESY correlations for H-10â (δH 1.62)/H3-38 (δH 0.85)
and H-11R (δH 2.10)/H-13 (δH 4.36) and J (H-11R/H-12)
(<3 Hz), J (H-11â/H-12) (5.0 Hz), and J (H-12/H-13) (2.7
Hz) values. Thus, the absolute configuration at C-12 in
1 was concluded to be R.
From the results described above, the absolute con-
figurations at 26 chiral centers in 1 were determined to
be 5S, 7R, 9S, 12R, 13R, 15S, 16R, 17S, 19R, 21S, 23R,
27S, 28S, 29S, 30S, 31R, 32S, 33S, 34R, 35R, 36R, 37S,
1′R, 3′S, 4′S, and 5S.
1
C20H36O7. In the H NMR spectrum of 17 in pyridine-d5,
oxymethine (δH 4.76, H-3) and methylene (δH 2.54 and
2.68, H2-2) proton signals were observed. The NOESY
correlations for H-13 (δH 3.67) and H-15R (δH 3.80) to
R-CH3 (δH 4.76) of the acetonide suggested that the
isopropylidene acetal was attached to C-13 and C-15.
Since the lower field shifts of H-5 and H-9 were observed
by esterification of 17 with (MTPA)Cl, C-5 and C-9 were
elucidated to be adjacent to a hydroxyl group. The
presence of a tetrahydropyran ring with a 3,7-syn relation
at C-3-C-7 in 17 was indicated by the NOESY correla-
tion for H-3/H-7. The structure of 17 was assigned as the
3,7-syn-3,7-oxa-13,15-isopropylidene derivative of the
C-1-C-15 segment. Analysis of the 1H-1H COSY and
TOCSY spectra revealed that compound 18 had the same
gross structure as 17. The NOESY correlation for H-2/
H-7 was suggestive of a 3,7-trans relation for 18. The
tetrahydropyran ring might be generated from Michael
addition of the 7-hydroxyl group to the C-2-C-3 double
bond.
Absolu te Ster eoch em istr y of 2. 2 was subjected to
1
the hydrolysis in water to give an aglycon (3) and 4. H
and 13C NMR data as well as the [R]D value of the aglycon
3 obtained from 2 were completely identical with those
of 3 derived from 1. Therefore, the absolute configura-
tions of 2 were concluded as 5S, 7R, 9S, 12R, 13R, 15S,
16R, 17S, 19R, 21R, 23R, 27S, 28S, 29S, 30S, 31R, 32S,
33S, 34R, 35R, 36R, 37S, 1′R, 3′S, 4′R, and 5S.
To determine the relative stereochemistry at C-12-
C-13, formation of a ketal ring at C-9-C-13 was per-
formed as follows. Oxidation of two hydroxyl groups at
C-5 and C-9 in compound 18 using Dess-Martin perio-
dinane and then deprotection of the acetonide at C-13-
Biologica l Activity of 2 a n d C 1. 2 exhibited sup-
pressive activity on mouse mixed lymphocyte reaction
(MLR) with an IC50 value of 0.625 µg/mL. Although the
immunosuppressive activity of 2 was less effective than
J . Org. Chem, Vol. 69, No. 5, 2004 1539

Komatsu et al.
H-2′), 2.33 (1H, dt, J ) 12.4 and 3.3 Hz, H-2′), 3.47 (3H, s,
OMe), 4.32 (1H, q, J ) 6.6 Hz, H-5′), 5.06 (1H, d, J ) 3.3 Hz,
H-1′), 5.58 (1H, br, H-4′), 5.63 (1H, ddd, J ) 3.1, 5.1, and 12.4
Hz, H-3′), 7.37 (2H, d, J ) 7.9 Hz, Ph), 7.57 (3H, t, J ) 7.6 Hz,
Ph), 7.70 (1H, tt, J ) 7.5 and 1.3 Hz, Ph), 7.83 (2H, d, J ) 8.2
and 1.2 Hz, Ph), and 8.11 (2H, dd, J ) 8.2 and 1.2 Hz, Ph);
ESIMS m/z 393 (M + Na)+; HRESIMS m/z 393.1308 (M +
Na)+, calcd for C21H22O6Na 393.1314.
those of cyclosporin A (IC50 0.016 µg/mL) and ascomycin
(IC50 0.040 µg/mL), 2 showed no toxicity in a dose of 500
mg/kg in mice. Therefore, 2 may be a favorable immuno-
suppressive reagent. On the other hand, 1 showed no
suppressive activity on mouse MLR at 20 µg/mL. These
results suggested that 23-O-malonyl and 3′-O-pentanoyl
groups were important for the immunosuppressive activ-
ity of 2. Furthermore, 1 showed cytotoxicity against
murine lymphoma P388 cells (IC50 8.3 µg/mL), while 2
was not cytotoxic at 100 µg/mL.
Oxid a tive Degr a d a tion of th e Aglycon 3 of 1. The
aglycon 3 (9.6 mg) of 1 was treated with NaBH4 (1.2 mg) in
MeOH (500 µL) at room temperature for 40 min. After addition
of 5% aqueous AcOH (200 µL) and then evaporation of the
organic solvent, the residual aqueous solution was subjected
to an HP-20 column (H2O and then MeOH). The fraction eluted
with MeOH was evaporated in vacuo to give a residue (9.9
mg). To a solution of the residue in MeOH (300 µL) was added
a 28% solution of NaOMe in MeOH (18.5 µL), and the mixture
was stirred at room temperature for 6 h. After addition of
saturated aqueous NH4Cl, evaporation of the organic solvent,
and chromatography on an HP-20 column (H2O and then
MeOH), the MeOH-eluted portion (9.3 mg) was treated with
NaIO4 (4.9 mg) in MeOH/H2O (1:1; 600 µL) at room temper-
ature for 30 min. To this reaction mixture was added NaBH4
(2.2 mg), and stirring was continued at room temperature for
30 min. After evaporation of the organic solvent, and chroma-
tography on an HP-20 column (H2O and then MeOH), the
MeOH-eluted fraction (7.7 mg) was treated with pivaloyl
chloride (16 mg) and pyridine (400 µL) at room temperature
for 30 min. The reaction mixture was partitioned between
EtOAc and saturated aqueous NaHCO3, and the organic phase
was washed with saturated aqueous NH4Cl, H2O, and then
brine and evaporated in vacuo. The residue was subjected to
C18 HPLC [Develosil ODS-UG-5, Nomura Chemical Co., Ltd.,
10 × 250 mm; eluent CH3CN/H2O, 45:55 (∼5 min) to 50:50
(∼15 min); flow rate 2.5 mL/min; UV detection at 210 nm] to
afford the 15-pivaloyl ester of the C-1-C-15 segment (7; 0.4
mg, tR 11 min), the 17,27-bis-pivaloyl ester of the C-17-C-27
segment (8; 1.3 mg, tR 24 min), and the 28-pivaloyl ester of
the C-28-C-37 segment (9; 1.6 mg, tR 15 min).
Exp er im en ta l Section
Cu ltiva tion . The voucher specimen of N. brasiliensis
(strain IFM 0406) was deposited at the Center for Pathogenic
Fungi and Microbial Toxicoses, Chiba University (deposition
no. FERM BP-5498).5 This actinomycete was grown in broth
[glycerol (2.0%), polypepton (1.0%), and meat extract (0.5%)
in H2O, pH 7.0]. Cultures were incubated in a 150 L jar
fermentor at 32 °C for 4 days with stirring at 250 rpm and a
150 L/min aeration rate and were centrifuged.
Extr a ction a n d Sep a r a tion . The supernatant of the
fermentation broth (80 L) was passed through a Diaion HP-
20 column, washed with 2 M NaCl(aq) (20 L) and H2O (20 L),
and then eluted batchwise with MeOH/H2O (1:1, 20 L) and
MeOH (20 L). The fraction eluted with MeOH was chromato-
graphed on a silica gel column eluted with a stepwise gradient
of CHCl3/MeOH to yield a fraction (13.7 g) which was sepa-
rated by a C18 column with a 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 × 25 cm; MeOH/H2O, 70:30; flow rate 10 mL/min; UV
detection at 205 nm) and then C18 HPLC again [YMC-Pack
ODS R&D; MeOH/H2O, 65:35 f 67:33, containing CF3CO2H
(25 ppm); flow rate 10 mL/min; UV detection at 205 nm] to
afford 1 (285 mg) and 2 (50 mg). Data for 1: colorless solid;
[R]D22 -21° (c 1.0, MeOH); IR (KBr) νmax 3402, 1703, and 1651
cm-1; UV (MeOH) λmax 210 nm (ꢀ 15400); 1H and 13C NMR
(Table 1); FABMS m/z 1019 (M + Na)+; HRFABMS m/z
1019.5760 (M + Na)+, calcd for C49H88O20Na 1019.5767.
Hyd r olysis of 1. 1 (50 mg) was dissolved in H2O (500 µL)
and heated at 110 °C for 9 h. The mixture was purified by C18
HPLC to afford an aglycon (3; 9.6 mg) and 4 (1.0 mg). Data
5,7-Isop r op yr id en e Aceta l of 7 (10). To a solution of the
15-pivaloyl ester of the C-1-C-15 segment (7, 0.4 mg) in MeOH
(10 µL) were added 2,2-dimethoxypropane (40 µL) and TsOH‚
H2O (0.02 mg), and the mixture was stirred at room temper-
ature for 40 min. After addition of saturated aqueous NaHCO3,
the mixture was extracted with EtOAc, and the organic layer
was washed with H2O and brine and evaporated in vacuo. The
residue was separated by preparative TLC (hexane/EtOAc, 1:1;
for 3: colorless solid; [R]D +22° (c 1.0, MeOH); H and 13C
NMR (Table 1); FABMS m/z 867 (M + H)+ and 889 (M + Na)+;
HRFABMS m/z 867.5356 (M + H)+, calcd for C43H79O17
867.5374.
21
1
1
four times) to afford compound 10 (0.4 mg): colorless oil; H
NMR (C6D6) δ 0.90 (3H, d, J ) 6.8 Hz, H3-38), 0.92 (1H, m,
H-6â), 1.14 (1H, m, H-6R), 1.18 (1H, m, H-11), 1.18 (1H, m,
H-10), 1.20 (9H, s, Piv), 1.25 (3H, s, â-CH3 of acetonide), 1.38
(1H, m, H-12), 1.42 (3H, s, R-CH3 of acetonide), 1.43 (1H, m,
H-8), 1.44 (1H, m, H-11), 1.52 (1H, m, H-8), 1.60 (2H, m, H2-
14), 1.70 (1H, m, H-10), 1.99 (1H, J ) 7.0 and 14.3 Hz, H-4â),
2.21 (1H, dd, J ) 7.4 and 14.3 Hz, H-4R), 3.45 (3H, s,1-OCH3),
3.51 (1H, m, H-13), 3.58 (1H, m, H-5), 4.88 (1H, m, H-9), 3.94
(1H, m, H-7), 4.15 (1H, dt, J ) 10.9 and 5.6 Hz, H-15), 4.32
(1H, dt, J ) 10.9 and 7.3 Hz, H-15), 5.96 (1H, d, J ) 15.4 Hz,
H-2), and 7.12 (1H, dt, J ) 15.4 and 7.6 Hz, H-3); ESIMS m/z
495 (M + Na)+; HRESIMS m/z 495.2927 (M + Na)+, calcd for
A solution of 4 (1.0 mg) in MeOH (200 µL) was treated with
6 M HCl(aq) (5 µL) at 100 °C for 30 min. After dilution with
H2O and then evaporation of organic solvent, the residue was
subjected to an HP-20 column (H2O and then MeOH). The
fraction eluted with MeOH was purified on a silica gel column
(acetone/hexane) to afford 5 (0.5 mg): [R]D -140° (c 0.1, H2O);
1H NMR (CDCl3) δ 1.28 (3H, d, J ) 6.7 Hz, H3-6′), 1.77 (1H,
dt, 3.9 and 13.1 Hz, H-2), 1.91 (1H, dd, 5.4 and 13.1 Hz, H-2),
3.32 (3H, s, OMe), 3.63 (1H, m, H-4′), 3.90 (1H, q, J ) 6.7 Hz,
H-5′), 3.99 (1H, m, H-3′), and 4.78 (1H, d, J ) 3.6 Hz, H-1′);
ESIMS m/z 185 (M + Na)+; HRESIMS m/z 185.0775 (M +
Na)+, calcd for C7H14O4Na 185.0790.
C
25H44O8Na 495.2934.
To a solution of 2-deoxyfucopyranoside 5 (0.5 mg) in pyridine
(100 µL) were added 4-(dimethylamino)pyridine (1 mg), tri-
ethylamine (1 µL), and benzoyl chloride (2 µL), and the mixture
was stirred at 37 °C for 8 h and then at 80 °C for 2 h. After
addition of saturated aqueous NH4Cl, the reaction mixture was
extracted with EtOAc. The organic layer was washed with
saturated NaHCO3, H2O, and brine. The organic phase was
evaporated, and the residue was separated on a silica gel
column (hexane/EtOAc, 95:5 and 90:10) to afford methyl 3,4-
bis-O-benzoyl-2-deoxy-R-L-fucopyranose (6; 0.055 mg): λext 237
9,13-Bis-(S)-MTP A E st er of t h e C-1-C-15 Segm en t
(14a ). To a solution of 10 (0.1 mg) in pyridine (50 µL) were
added DMAP (0.1 mg) and (R)-(-)-(MTPA)Cl (0.2 µL), and
stirring was continued at 37 °C for 9 h. N,N-Dimethyl-1,3-
propanediamine (1 µL) and saturated aqueous NaHCO3 were
added, and the reaction mixture was extracted with EtOAc.
The organic phase was washed with H2O and brine and dried
by a N2 stream. The residue was purified by preparative TLC
(hexane/EtOAc, 7:3) to afford a 9,13-bis-(S)-MTPA ester of 10
(0.1 mg) as a colorless oil. This MTPA ester was treated with
AcOH/MeOH/H2O (1:8:1; 50 µL) at 50 °C for 1 h. The solvent
1
(∆ꢀ -20.5) and 222 (+7.6) nm; H NMR (CD3OD) δ 1.26 (3H,
d, J ) 6.6 Hz, H3-6′), 2.14 (1H, ddd, J ) 12.4, 5.1, and 1.2 Hz,
1540 J . Org. Chem., Vol. 69, No. 5, 2004

Absolute Stereochemistry of Brasilinolides A and C
was removed by a N2 stream to afford 14a (0.1 mg) as a
colorless oil: 1H NMR (CDCl3) δ 0.82 (3H, d, J ) 6.9 Hz, H3-
38), 0.91 (1H, m, H-11), 1.20 (9H, s, Piv), 1.20 (1H, m, H-11),
1.49 (2H, m, H2-6), 1.53 (2H, m, H2-10), 1.59 (2H, m, H2-8),
1.65 (1H, m, H-12), 1.83 (2H, m, H2-14), 2.32 (1H, m, H-4),
2.39 (1H, m, H-4), 3.49 (3H, s, OCH3 of MTPA), 3.51 (3H, s,
OCH3 of MTPA), 3.65 (1H, m, H-7), 3.79 (3H, s, 1-OCH3), 3.89
(1H, m, H-5), 3.90 (1H, m, H-15), 4.11 (1H, m, H-15), 5.09 (1H,
m, H-13), 5.15 (1H, m, H-9), 5.90 (1H, d, J ) 15.1 Hz, H-2),
6.96 (1H, dd, J ) 7.7 and 15.1 Hz, H-3), 7.43-7.39 (6H, m,
Ph), 7.50 (2H, m, Ph), and 7.53 (2H, m, Ph); ESIMS m/z 887
(M + Na)+; HRESIMS m/z 887.3436 (M + Na)+, calcd for
b was treated with 6 M HCl (0.16 µL) in MeOH (200 µL) at
100 °C for 4 h. After concentration, the residue was treated
with 2,2-dimethoxypropane (50 µL) and TsOH‚H2O (0.1 mg)
in MeOH (50 µL) at room temperature for 1 h. After addition
of saturated aqueous NaHCO3, the reaction mixture was
extracted with EtOAc. The organic layer was washed with H2O
and brine and evaporated in vacuo. The residue was subjected
to a silica gel column (hexane/EtOAc, 3:1) to afford compound
19 (0.1 mg).
9,13-Aceta l Der iva tive of 18 (20). To a solution of 18 (0.77
mg) in CH2Cl2 (50 µL) were added Dess-Martin periodinane
(4.0 mg) and NaHCO3 (1.2 mg) at room temperature, and the
mixture was stirred in a shield tube at 37 °C for 16 h. After
addition of saturated aqueous NaHCO3, the mixture was
extracted with EtOAc. The organic phase was washed with
H2O and brine and evaporated. The residue was treated with
AcOH (10 µL) in MeOH (80 µL) at 40 °C for 210 min. After
the solvent was removed under a N2 stream, the residue was
separated by preparative TLC (hexane/acetone, 6:4) and
afforded 20 (0.15 mg) as a colorless oil: 1H NMR (C6D6) δ 0.85
(3H, d, J ) 7.0 Hz, H3-38), 1.19 (1H, m, H-14), 1.23 (1H, dt,
J ) 13.1 and 5.0 Hz, H-11â), 1.31 (1H, m, H-12), 1.40 (1H,
ddt, J ) 13.8, 4.7, and 2.4 Hz, H-10R), 1.57 (1H, dd, J ) 5.0
and 14.8 Hz, H-8), 1.62 (1H, dt, J ) 4.6 and 13.8 Hz, H-10â),
1.69 (1H, m, H-14), 1.92 (1H, dd, J ) 6.0 and 14.7 Hz, H-8),
1.96 (1H, dd, J ) 7.2 and 14.4 Hz, H-4R), 2.05 (1H, m, H-2),
2.05 (1H, m, H-6R), 2.10 (1H, m, H-11R), 2.17 (1H, bd, J ) 4.9
and 14.4 Hz, H-4â), 2.24 (1H, dd, J ) 5.0 and 14.2 Hz, H-6â),
2.43 (1H, dd, J ) 8.2 and 15.3 Hz, H-2), 3.14 (3H, s, 9-OCH3),
3.35 (3H, s, 1-OCH3), 3.60 (1H, m, H-15), 3.63 (1H, m, H-15),
3.83 (1H, dt, J ) 10.0 and 2.7 Hz, H-13), 4.20 (1H, m, H-7),
4.36 (1H, m, H-3); ESIMS m/z 381 (M + Na)+; HRESIMS m/z
381.1892 (M + Na)+, calcd for C18H30O7Na 381.1889.
C
42H54F6O12Na 887.3417.
31-(S)-MTP A Ester of 13 (16a ). To a solution of 13 (0.3
mg) in pyridine (100 µL) were added DMAP (0.06 mg) and (R)-
(-)-MTPACl (0.4 µL), and stirring was continued at 37 °C for
9 h. N,N-Dimethyl-1,3-propanediamine (1 µL) and saturated
aqueous NaHCO3 were added, and the reaction mixture was
extracted with EtOAc. The organic phase was washed with
H2O and brine and dried by a N2 stream. The residue was
purified on a silica gel column (hexane/EtOAc, 92:8) to afford
the 31-(S)-MTPA ester of 13 (16a ; 0.1 mg) as a colorless oil:
1H NMR (CDCl3) δ 0.63 (3H, d, J ) 6.9 Hz, H3-42), 0.86 (3H,
d, J ) 6.9 Hz, H3-39), 0.96 (3H, d, J ) 6.9 Hz, H3-41), 1.13
(3H, d, J ) 7.2 Hz, H3-40), 1.15 (3H, d, J ) 6.1 Hz, H3-43),
1.22 (9H, s, Piv), 1.31 (3H, s, CH3 of acetonide), 1.36 (3H, s,
CH3 of acetonide), 1.39 (1H, m, H-36), 2.08 (1H, m, H-32), 2.12
(1H, m, H-30), 2.15 (1H, m, H-34), 3.46 (3H, s, OCH3 of MTPA),
3.47 (1H, m, H-35), 3.52 (1H, dd, J ) 6.3 and 9.9 Hz, H-37),
3.56 (1H, dd, J ) 3.9 and 8.5 Hz, H-33), 3.64 (1H, m, H-29),
4.13 (1H, dd, J ) 5.8 and 11.8 Hz, H-28), 4.18 (1H, dd, J ) 3.0
and 11.8 Hz, H-28), 5.14 (1H, t, J ) 3.0 Hz, H-31), 7.43 (3H,
m, Ph), and 7.54 (2H, m, Ph); ESIMS m/z 653 (M + Na)+;
HRESIMS m/z 653.3279 (M + Na)+, calcd for C33H49F3O8Na
653.3277.
Oxid a tive Degr a d a tion of 1. To a solution of 1 (20 mg)
in MeOH (1 mL) was added NaBH4 (4.0 mg), and the mixture
was stirred at room temperature for 1 h. The reaction mixture
was diluted with H2O, concentrated in vacuo, and subjected
to an HP-20 column (H2O and then MeOH). The MeOH-soluble
materials (19.2 mg) were treated with NaOMe (25 mg) in
MeOH (750 µL) at room temperature for 2 h. After addition of
saturated aqueous NH4Cl and then concentration of the
solvent, the reaction mixture was subjected to an HP-20
column (H2O and then MeOH) to give a residue (17.3 mg). To
a solution of the residue in MeOH/H2O (2:1; 1 mL) was added
NaIO4 (4.4 mg), and the mixture was stirred for 20 min at
room temperature. After addtion of NaBH4 (2.5 mg), stirring
was continued at room temperature for 30 min. After evapora-
tion of the solvent, the reaction mixture was subjected to a
HP-20 column (H2O and then MeOH) and then a silica gel
column (CHCl3/MeOH, 90:10) to give two fractions, a (1.6 mg)
and b (1.6 mg). To a solution of fraction a in MeOH (50 µL)
were added 2,2-dimethoxypropane (50 µL) and TsOH‚H2O (0.1
mg), and the mixture was stirred at room temperature for 10
min. After addition of saturated aqueous NaHCO3, the reaction
mixture was extracted with EtOAc. The organic layer was
washed with H2O and brine and evaporated in vacuo. The
residue was purified by preparative TLC (hexane/acetone, 6:4)
to afford compounds 17 (0.49 mg) and 18 (0.32 mg). Fraction
Hyd r olysis of 2. 2 (30 mg) was dissolved in H2O (500 µL),
and the mixture was heated at 110 °C for 9 h. The mixture
was purified by C18 HPLC to afford an aglycon (3; 3.2 mg) and
23
4 (1.0 mg). Data for 3: colorless solid; [R]D +18° (c 0.56,
MeOH); 1H and 13C NMR (Table 1); FABMS m/z 867 (M + H)+
and 889 (M + Na)+; HRFABMS m/z 867.5356 (M + H)+, calcd
for C43H79O17 867.5374.
Ack n ow led gm en t. We thank Dr. E. Fukushi and
Prof. J . Kawabata, Graduate School of Agriculture,
Hokkaido University, for measurements of HETLOC
spectra and 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, Sci-
ence, and Technology of J apan.
Su p p or tin g In for m a tion Ava ila ble: Data of 7, 8, 9, 11,
12, 13, 14b, 15a , 15b, 16b, 17, 18, 19, and the MTPA esters
of 17 and NMR spectra of 1, 3, 10-13, 19, and 20. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O035773V
J . Org. Chem, Vol. 69, No. 5, 2004 1541