Halichoblelide, a potent cytotoxic macrolide from a Streptomyces species separated from a marine fish

Article abstract of DOI:10.1016/S0040-4039(02)00102-8

Halichoblelide, a novel macrolide with potent cytotoxicity against tumour cells in culture, has been isolated from a strain of Streptomyces hygroscopicus originally separated from the marine fish Halichoeres bleekeri, and the absolute stereostructure has been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques and some chemical transformations.

Full text of DOI:10.1016/S0040-4039(02)00102-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 1721–1724
Halichoblelide, a potent cytotoxic macrolide from a
Streptomyces species separated from a marine fish
Takeshi Yamada, Katsuhiko Minoura and Atsushi Numata*
Osaka University of Pharmaceutical Sciences, 4-20-1, Nasahara, Takatsuki, Osaka 569-1094, Japan
Received 1 November 2001; revised 27 December 2001; accepted 11 January 2002
Abstract—Halichoblelide, a novel macrolide with potent cytotoxicity against tumour cells in culture, has been isolated from a
strain of Streptomyces hygroscopicus originally separated from the marine fish Halichoeres bleekeri, and the absolute stereostruc-
ture has been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques and some chemical
transformations. © 2002 Elsevier Science Ltd. All rights reserved.
Up until today some of bioactive materials from marine
animals have been isolated from various bacteria.
Recently we have focused our attention on new antitu-
mour materials produced by microorganisms from
marine organisms.1 Previously we have isolated the
structurally unique and potent cytotoxic macrolide,
halichomycin, from a strain of Streptomyces hygro-
scopicus OUPS-N92 originally obtained from the
marine fish Halichoeres bleekeri.2 Further investigation
for metabolites of this fungal strain has now led to the
isolation of one additional new cytotoxic compound
designated as halichoblelide.
employing stepwise combination of Sephadex LH-20
and silica gel column chromatography followed by
reversed-phase HPLC. This sequence of purification
yielded 7.2 mg of halichoblelide (1)3 as colourless
powder.
Based on an [M+Na]+ peak of its methyl acetal deriva-
tive 2 in HRSIMS,3 the molecular formula of hali-
choblelide (1) was deduced as C54H85O19. The UV and
IR spectra of halichoblelide (1) exhibited absorption
bands characteristic for an alcohol and a conjugated
1
carbonyl group. A close inspection of the H and 13C
NMR spectra of 1 (Table 1) by DEPT and 1H–13C
correlation spectroscopy (COSY) experiments revealed
the presence of two primary methyls, ten secondary
methyls, five sp3-hybridised methylenes, 25 sp3-methi-
nes including 14 oxymethines and three anomeric
methines, one anomeric quaternary sp3-carbon, two
1,3-diene moieties, three ester carbonyls, and seven
The microorganism from H. bleekeri fish was cultured
at 27°C for a week in a medium (80 l) containing 0.1%
cone steep liquor and 1% dextrin in artificial seawater
adjusted to pH 7.5. After incubation the AcOEt extract
of the culture filtrate was purified by bioassay-directed
fractionation (cytotoxicities against P388 cell line) with
HO
OH
24'
25'
26'
27'
19'
23'
22'
O
18'
8'
17'
6'
O
16
15
2'
6
H
4'
1'
21
20
12'
O
O
9'
11'
14'
13'
15'
10'
20'
21'
7'
3'
5'
O
11
OH
O
7
O
OH
O
O
14
13
5
3
8
16'
9
O
O
1
10
4
2
12
OH
H
22
17
23
19
18
O
24
25
HO
26
OH
27
1
Keywords: halichoblelide; Streptomyces hygroscopicus; macrolide; marine fish.
* Corresponding author. Tel./fax: +81-728-90-1080.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00102-8

1722
T. Yamada et al. / Tetrahedron Letters 43 (2002) 1721–1724
Table 1. NMR spectral data of halichoblelide A (1) in CDCl3
a
a
Position
lH
J/Hz
lC
Position
lH
J/Hz
lC
1
2
3
4
5
6
7
8
9
10
11
12a
12b
13
14
15
16
17
18
19
20A
20B
21
168.10 (q)b
120.95 (t)
145.05 (t)
132.02 (t)
144.19 (t)
40.90 (t)
77.88 (t)
35.87 (t)
70.65 (t)
41.60 (t)
99.05 (q)
38.93 (s)
1%
2%
3%
4%
5%
6%
7%
8%
170.02 (q)b
121.42 (t)
145.10 (t)
131.35 (t)
144.77 (t)
41.66 (t)
76.63 (t)
35.76 (t)
72.98 (t)
40.08 (t)
158.2 (q)
95.13 (t)
68.67 (t)
44.01 (t)
74.23 (t)
18.67 (p)
15.49 (p)
9.51 (p)
5.69 d
15.8
5.66 d
15.8
6.98 dd
6.12 dd
5.67 dd
2.54 tq
4.77 dd
1.96 dqd
4.12 ddd
1.72 qd
15.8, 11.2
14.5, 11.2
14.5, 10.3
10.3, 7.0
10.3, 1.5
10.3, 7.1, 1.5
10.3, 0.8, 2.1
7.3, 0.8
6.96 dd
6.06 dd
5.61 dd
2.49 tq
5.12 dd
1.91 dqd
3.52 br d
2.32 qd
15.8, 11.2
14.5, 11.2
14.5, 10.2
10.3, 7.0
10.3, 1.6
10.3, 7.1, 1.6
10.3
9%
10%
11%
12%
13%
14%
15%
16%
17%
18%
19%
20%A
20%B
21%
22%
23%a
23%b
24%
25%
26%
27%
9%-OH
24%-OH
25%-OH
7.0, 3.6
2.38 dd
1.03 t
3.97 td
1.19 tt
3.91 dq
1.11 d
1.02 d
0.81 d
1.01 d
1.44 dqd
1.62 dqd
0.86 t
10.2, 4.7
10.2
10.2, 4.7
10.2, 3.8
10.2, 6.4
6.4
7.0
7.1
7.3
4.78 d
4.07 dd
1.56 tt
3.96 quintet
1.29 d
1.05 d
0.93 d
1.05 d
1.42 dqd
1.50 dqd
0.89 t
3.0
6.4, 3.0
6.4, 5.0
6.4
6.4
7.0
7.1
7.0
70.26 (t)
48.42 (t)
66.55 (t)
19.12 (p)
14.90 (p)
8.75 (p)
7.07 (p)
19.39 (s)
10.63 (p)
21.65 (s)
12.5, 7.5, 5.0
12.5, 7.5, 5.0
7.5
12.9, 7.5, 3.8
12.9, 7.5, 3.8
7.5
9.75 (p)
93.22 (t)
33.33 (s)
9.10 (p)
94.25 (t)
33.58 (s)
5.10 t
2.8
22
5.05 t
2.1
1.82 dt
1.83 ddd
3.98 br d
3.64 br s
4.00 qd
1.26 d
2.63 d
2.04 br s
1.95 br s
12.3, 2.8
12.3, 10.8, 2.8
10.8
23a
23b
24
25
26
1.79 dt
1.81 ddd
3.97 br d
3.64 br s
3.99 qd
1.24 d
3.98 br s
5.22 d
2.03 br s
1.95 br s
12.3, 2.1
12.3, 11.1, 2.1
11.1
66.02 (t)
71.38 (t)
65.92 (t)
16.77 (p)
66.07 (t)
71.43 (t)
65.88 (t)
16.74 (p)
7.0, 1.8
7.0
3.7
7.0, 1.8
7.0
27
9-OH
11-OH
24-OH
25-OH
2.0
\
a 1H chemical shift values (l ppm from SiMe4) followed by multiplicity and then the coupling constants (J/Hz).
b Letters, p, s, t and q in parentheses indicate respectively primary, secondary, tertiary and quaternary carbons, assigned by DEPT.
1
hydroxy groups. The H–1H COSY analysis of 1 led to
five partial structural units (A, B, C×2 and D) as shown
by bold-faced lines in Fig. 1. These results were supported
by HMBC correlations. The geometrical configuration of
both the conjugated diene moieties (C-2–C-5 and C-2%–C-
5%) was deduced as trans-s-trans from the coupling
constants of the olefinic protons and NOEs (H-3/H-5 and
H-3%/H-5%). The connection of these five units and the
remaining functional groups was determined on the basis
of the key HMBC correlations summarised in Fig. 1, and
the planar structure of 1 was elucidated.
The relative stereochemistry for 1 was established by a
combination of observed coupling constants and
unit C
HO
OH
O
unit D
unit B
O
O
O
O
OH
O
O
OH
O
O
O
O
HO
O
1H-1H COSY correlations
HMBC correlations
unit A
HO
OH
unit C
Figure 1. Typical 2D NMR correlations in halichoblelide (1).

T. Yamada et al. / Tetrahedron Letters 43 (2002) 1721–1724
1723
Figure 2. The relative configuration and conformation of the partial structural units and derivative 3, and observed NOEs.
NOESY data of 1 and the products which were
obtained by the degradation of 1 involving several
reaction steps shown in Scheme 1. The observation of
the coupling constants (J9,10=0.8 Hz and J8,9=10.3 Hz)
and NOEs for H-10/H-18, H-9/H-18 and H-8/H-19 in
unit A (Fig. 2) suggested that H-9 and H-10, and H-8
and H-9 are in gauche and anti arrangements, respec-
tively. Similarly, the J7,8 and J6,7 values (1.5 and 10.3
Hz, respectively) and NOEs for H-6/H-18 suggested
that H-7 and H-8, and H-6 and H-7 are located in
gauche and anti arrangements, respectively. NOEs for
H-12b/H-14 and H-13/11-OH implied that the pyranose
CH3O
OCH3
O
O
O
O
d
p -Br-Bz
H
O
OCH3
p -Br-Bz
c
O
OH
O
H
O
OH O
7 (derivative from unit B)
HO
O
CH3O
Bz-p -Br
Bz-p -Br
3
O
O
O
O
CH3O
OCH3
O
O
OH
O-Bz-p -Br
CH3O
a
d
d
1
b
8 (derivative from unit A)
OH
O-Bz-p -Br
4
9 (derivative from unit D)
O
OCH3
O
OCH3
O
7
O
OCH3
12
6
CH3O
10
8
O
9
HO
p -Br-Bz-O
O-Bz-p -Br
OH
OH
6 (derivative from unit A)
10 (derivative from unit C)
5
Scheme 1. Reagents and conditions: (a) Pd–C/H2, rt; (b) p-TsOH/MeOH, rt; (c) conc. H2SO4/MeOH, rt; (d) p-BrBzCl/pyridine,
rt.

1724
T. Yamada et al. / Tetrahedron Letters 43 (2002) 1721–1724
ring exists in a chair conformation with H-13 and
11-OH in coaxial arrangements. Furthermore, the J13,14
and J14,15 values (both 10.2 Hz) suggested that H-13,
H-14 and H-15 are oriented trans-axially the one to
another. This stereochemistry of unit A was supported
by stereochemical analysis of degraded product 6 of
halichoblelide (1) (Scheme 1). The observation of the
coupling constant (J6,7=10.1 Hz) and NOEs for H-10/
H-18, H-9/H-7, H-9/H-12a, H-17/H-8, H-17/H-18 and
H-6/H-18 in 6 allowed assignment of the relative
configuration of 6 as shown in Scheme 1.
1-OCH3. This measurements evidently lead to the rela-
tive configuration of unit C.
The absolute configuration of halichoblelide (1) was
elucidated by application of the modified Mosher’s
method for degraded product 6 of 1 and CD spectra of
1
the other products. The H chemical-shift differences
between the (R)- and (S)-MTPA esters of 6 showed
that C-9 has an R-configuration. Bis-p-bromobezoate 8
with the same absolute configuration as 6 exhibited a
positive Cotton effect in the CD spectrum. Based on
this evidence, a positive Cotton effect observed in the
CD spectrum of bis-p-bromobezoate 7 allowed assign-
ment of the absolute configuration of 7 as shown in
Scheme 1. In addition, compound 10 exhibited a nega-
tive cotton effect while compound 9 showed a positive
Cotton effect, thus allowing the assignments of their
absolute configuration represented in Scheme 1. The
absolute configuration of 6, 7, 9 and 10 led to deter-
mine the structures of four units A, B, D and C,
respectively, and consequently the absolute stereostruc-
ture for halichoblelide (1) was elucidated.
The measured coupling constants and NOEs in unit B
(Fig. 2) closely resembled those of unit A. The J9%,10%
and J8%,9% values (3.6 and 10.3 Hz, respectively) and
NOEs for H-10%/H-18%, H-9%/H-18% and H-8%/H-19%
implied that H-9% and H-10%, and H-8% and H-9% are
located in gauche and anti arrangements, respectively.
Similarly, the J7%,8% and J6%,7% values (1.6 and 10.3 Hz,
respectively) and NOEs for H-6%/H-18%, H-8%/H-17% and
H-7%/H-17% suggested that H-7%/H-8% and H-6%/H-7% are
in gauche and anti arrangements, respectively.
Unit D and degraded product 9 of halichoblelide (1)
(Scheme 1) exhibited similar coupling constants and
NOEs. The observation of an NOE between H-13% and
H-15% in unit D implied that H-13% and H-15% are in a
co-pseudoaxial arrangement. In addition, NOEs from
H-20%, but not from H-14%, to H-13% and H-15% indicated
and H-20% is arranged cis to H-13% and H-15%. The
observation of the coupling constants (J12%,13%=3.0 Hz in
unit D and J1,2=3.8 Hz in 9) indicated that H-12% and
H-13% in unit D (H-1 and H-2 in 9) are orientated cis on
the basis of comparison of coupling constants with a-
and b-galactofuranoses.4 This evidence allowed assign-
ment of the stereochemistry of unit D as shown in Fig.
2.
The biological activity tests of halichoblelide (1)
showed that this new product exhibited potent cyto-
toxic activity against the murine P388 cell line (ED50
0.63) and the 39 human cancer cell lines (BSY-1, NCI-
H522, MKN74 and the other. The tested mean value of
log GI50 over all cell lines appeared to be −5.25).
References
1. Amagata, T.; Doi, M.; Tohgo, M.; Minoura, K.; Numata,
A. Chem. Commun. 1999, 1321–1322.
2. Takahashi, C.; Takada, T.; Yamada, T.; Minoura, K.;
Uchida, K.; Matsumura, E.; Numata, A. Tetrahedron Lett.
1994, 35, 5013–5014.
3. Halichoblelide A (1): colourless powder, mp 137–139°C,
[h]D −16 (c 0.16, EtOH); UV umax (EtOH)/nm: 252 (log m
4.56); IR wmax (KBr)/cm−1: 3425, 1722, 1692, 1645, 1638;
HRSIMS m/z: 1029.5035 [M−2CH3−H+Na]+ (calcd for
C52H78O19Na: 1029.5036). Methyl acetal derivative 2:
colourless powder, mp 143–145°C, [h]D −38.6 (c 0.12,
EtOH); HRSIMS m/z: 1074.2154 [M+Na]+ (calcd for
C55H87NaO19: 1074.2156).
The stereochemistry of unit C was elucidated by analy-
sis of coupling constants and NOE signals in degraded
product 10 of halichoblelide (1) (Scheme 1). The same
stereochemistry of the anomers in 10 and unit C was
deduced from the fact that both the J values from the
anomeric proton to the two vicinal protons were small
(1.8–3.0 Hz) in 10 and unit C. The J values (J1,2b=1.8
Hz, J1,2a=3.0 Hz and J3,4=J4,5=2.2 Hz) and NOEs
from H-5 to H-3 and 1-OCH3, and from H-4 to H-5
and 5-CH3 in 10 implied that 3-OH, 4-OH and 5-CH3
in 10 are orientated cis to one another and opposite to
4. York, W.-S.; Hantus, S.; Albersheim, P.; Darvill, A. G.
Carbohydr. Res. 1997, 199–206.