Establishment of absolute stereostructure of falcarindiol, algicidal principle against Heterocapsa circularisquama from Notopterygii Rhizoma

Article abstract of DOI:10.1016/j.tetlet.2010.01.047

Falcarindiol (1) was isolated as an algicidal principle against the harmful red tide dinoflagellate, Heterocapsa circularisquama, from Notopterygii Rhizoma through bioassay-guided separation. In order to determine the ambiguous absolute structure of this

Full text of DOI:10.1016/j.tetlet.2010.01.047

Tetrahedron Letters 51 (2010) 1523–1525
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Establishment of absolute stereostructure of falcarindiol, algicidal principle
against Heterocapsa circularisquama from Notopterygii Rhizoma
*
Satoru Tamura, Tomomichi Ohno, Yuuhi Hattori, Nobutoshi Murakami
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Falcarindiol (1) was isolated as an algicidal principle against the harmful red tide dinoflagellate, Hetero-
capsa circularisquama, from Notopterygii Rhizoma through bioassay-guided separation. In order to deter-
mine the ambiguous absolute structure of this active principle, all three stereoisomers as well as
falcarindiol (1) were synthesized. As a result of intensive analysis of their physicochemical properties,
the configuration of 1 was revealed to be 3R,8S. On the other hand, (3S,8S)- and (3S,8R)-isomers were
found to exhibit more potent algicidal activity than (3R,8S)-falcarindiol (1) isolated from Notopterygii
Rhizoma. In addition, the diyne moiety of 1 was established as the crucial structural requirement for algi-
cidal potency.
Received 11 December 2009
Revised 7 January 2010
Accepted 15 January 2010
Available online 20 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
In recent years, harmful algal blooms (HABs) have been re-
ported to occur world-wide affecting public health and fisheries
industries. The HABs frequently caused large-scale red tides and
mass mortalities of cultured fishes and shellfish, resulting in signif-
icant damage to ecosystem. Heterocapsa circularisquama,1 recorded
as a new variety of dinoflagellate in 1995, has been widely recog-
nized to be one of the most troublesome algae causing the noxious
red tide. In particular, this species showed potent toxicity against
bivalves, such as oysters, pearl oysters, and Japanese littlenecks
leading to enormous damage for aquaculture industries.2 However,
there have been few useful and environmentally benign methods
to control HAB of this causative alga so far. Hence, we have been
engaged in exploration of algicidal principles against H. circulari-
squama from medicinal plants. In this Letter, we deal with the
establishment of the absolute structure of falcarindiol (1), isolated
from Notopterygii Rhizoma as an active principle, by the synthesis
of all stereoisomers. Furthermore, we wish to describe the struc-
tural requirement of 1 for algicidal potency.
tent algicidal activity. Bioassay-guided separation of the extract
through successive DIAION HP-20, SiO2, and ODS column chroma-
tography, and ODS HPLC disclosed an active principle (0.066% from
the crude drug) with MIC of 0.5 lg/mL. The gross structure of the
algicidal principle was identified to be that of falcarindiol (1) by
comparison of the spectroscopic features such as the NMR and
FAB-MS data with those described in the literature (Fig. 1).4
Although falcarindiol (1) and its two stereoisomers were iso-
lated from a variety of natural resources, the optical rotation of
falcarindiol (1) from Notopterygii Rhizoma in the present study
was not in accordance with any reported data. In addition, there
seems no consistency between the reported optical rotations and
the absolute stereochemistry (See the Supplementary data). In this
context, the synthesis of falcarindiol (1) and all the stereoisomers
of 1 was conducted to establish the conclusive index resolving
the confusion in stereochemistry of falcarindiols as well as to elu-
cidate the participation of the two chiral centers of 1 in algicidal
activity.
H. circularisquama is a unicellular alga with about 20
l
m of size
Retrosynthetic analysis of 1 exemplified by the 3R,8S diastereo-
mer is depicted in Figure 2. This route enables to synthesize the
four possible stereoisomers of 1 by changing the chirality of the
asymmetric catalysis, namely, the conjugated diyne of 1 would
be constructed by Cadiot–Chodkiewicz reaction5 from two alkynes
i and ii. The chiral centers bearing the hydroxyl groups of i and ii
and whirls briskly in the sphere of life, while dead individuals set-
tle without moving. So, algicidal activity was evaluated by direct
count for the number of whirling and settling H. circularisquama
under microscope observation after co-incubation with each sam-
ple for 48 h in artificial sea water.3
As a result of screening about 400 extracts from medicinal
plants by using this assay, the MeOH extract of Notopterygii Rhi-
zoma (rhizoma of Notopterygium incisum) was found to exhibit po-
* Corresponding author at present address: Kyoto Pharmaceutical University,
Japan. Tel.: +81 75 595 4634; fax: +81 75 595 4768.
E-mail address: murakami@poppy.kyoto-phu.ac.jp (N. Murakami).
Figure 1. Gross structure of falcarindiol (1).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.01.047

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S. Tamura et al. / Tetrahedron Letters 51 (2010) 1523–1525
the optical purities higher than 99% ee of (S)-7, (R)-7, (S)-9, and (R)-
9 were determined by chiral HPLC analysis. Treatment of (S)-9 with
N-bromosuccinic imide (NBS) in the presence of silver nitrate
prompted deprotection of the TMS residue and bromination to give
brominated alkyne (S)-10.9 Cadiot–Chodkiewicz reaction between
(S)-7 and (S)-10 with the aid of cuprous chloride, n-butylamine,
and hydoxylamine hydrochloride afforded diyne (3S,8S)-11. Final-
ly, deprotection of the TBDPS group of 11 by tetra-n-butylammo-
nium fluoride furnished our desired (3S,8S)-110 in 23% overall
yield for 8 steps from 4. In addition, (R)-7 and (R)-10 were prepared
by using (R)-CBS catalyst and the remaining three stereoisomers of
(3S,8S)-110 were synthesized from the corresponding segments (7
and 10) in the same manner.
In comparison with the algicidal falcarindiol (1) from Noto-
pterygii Rhizoma, the synthesized (3R,8S)-1 showed the superim-
posable 1H, 13C NMR, IR, and FAB-MS spectra. In addition, the
optical rotation of the synthesized 1, +232.0° (c 0.33, CHCl3), is
the closest to that of the isolated 1, +232.8° (c 0.33, CHCl3), among
the four synthesized stereoisomers as shown in Table 1. Further-
more, the two diastereomers exhibited the fairly different absolute
values from the isolated and synthesized falcarindiols (1). Thus, the
absolute configuration of 1 from Notopterygii Rhizoma was unam-
biguously concluded to be 3R,8S.
Figure 2. Retrosynthetic analysis of falcarindiol (1).
were provided by asymmetric reduction of corresponding ketones
iii and iv, respectively. The conjugated ketone iii was yielded by
condensation of acrolein 2 and protected acetylene 3, while the ke-
tone iv was also prepared from 3 and aldehyde v in a similar man-
ner. Z-Olefin
v was prepared from 2-decyn-1-ol (4) by
hydrogenation with Lindlar catalysis.
Finally, algicidal activity of the four synthesized stereoisomers
against H. circularisquama was evaluated. As depicted in Table 2,
the (3S,8S)- and (3S,8R)-isomers exhibited more potent activity
than the naturally occurring (3R,8S)-1. Therefore, 3S-configuration
was revealed to enhance algicidal activity with regard to the four
stereoisomers. Moreover to elucidate the participation of the diyne
portion of 1 in bioactivity, both enantiomers [(S)-12 and (R)-12] of
(E)-dodeca-4-ene-1-yne-3-ol, the right half structure of 1, were
prepared from 9. Interestingly, both the compounds exhibited little
The synthesis of falcarindiol (1) is summarized in Scheme 1.
Coupling of acrolein (2) with trimethylsilylacetylene (3) in the
presence of n-BuLi followed by MnO2 oxidation afforded conju-
gated ketone 5. The resulting ketone 5 was subjected to asymmet-
ric reduction by the complex of (S)-2-methyl-CBS-oxazaborolidine
and BH3 [(S)-CBS catalyst]6,7 to furnish chiral alcohol (S)-6. Protec-
tion of the hydroxyl group in (S)-6 as t-butyldiphenylsilyl (TBDPS)
ether and succeeding removal of the trimethylsilyl (TMS) function
provided the left-side segment, alkyne (S)-7, in 50% yield from 3.
Hydrogenation to 2-decyn-1-ol (4) by Lindlar catalyst under a
hydrogen atmosphere gave Z-olefin, which was submitted to
MnO2 oxidation and subsequent condensation with 3 to afford
alcohol 8. After conversion of alcohol 8 to the corresponding
ketone, asymmetric reduction of the ketone by (S)-CBS catalyst
provided chiral secondary alcohol (S)-9. The predictable configura-
tions of both (S)-6 and (S)-9 were confirmed to be S by application
of modified Mosher’s method8 (See the Supplementary data), while
algicidal activity at the concentration of 4 lM. Consequently, the
conjugated diyne moiety of falcarindiol (1) was clarified to be
essential for potent algicidal activity against H. circularisquama.
In conclusion, we disclosed falcarindiol (1) as the algicidal prin-
ciple against H. circularisquama from Notopterygii Rhizoma
through bioassay-guided separation. By intensive comparison with
the optical rotations among the four synthesized stereoisomers, 1
was securely elucidated to possess 3R,8S-configuration. In addi-
tion, the conjugated diyne portion was established as the crucial
Scheme 1. Synthesis of falcarindiol and stereoisomers. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 °C to rt, 95%; (b) MnO2, CH2Cl2, rt, 82%; (c) (S)-2-methyl-CBS-
oxazaborolidine, BH3ꢁTHF, THF, ꢀ40 °C; (d) TBDPSCl, imidazole, CH2Cl2, rt; (e) KOH, MeOH, THF, rt, S: 64%, R: 65% for 7 from 5, S: 94%, R: quant. for 12 from 9; (f) H2, Pd–CaCO3,
MeOH, rt, 98%; (g) MnO2, CH2Cl2, rt, 87%; (h) 3, n-BuLi, THF, ꢀ78 °C to rt, 82%; (i) MnO2, CH2Cl2, rt, 75%; (j) (S)-2-methyl-CBS-oxazaborolidine, BH3ꢁTHF, THF, ꢀ40 °C, S: 85%, R:
82%; (k) NBS, AgNO3, acetone, rt, S: 87%, R: 86%; (l) (S)-7, CuCl, n-BuNH2, NH2OHꢁHCl, MeOH, 0 °C; (m) n-Bu4NF, THF, rt, 3S,8S: 60%, 3S,8R: 61%, 3R,8S: 64%, 3R,8R: 62% from 10.

S. Tamura et al. / Tetrahedron Letters 51 (2010) 1523–1525
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Table 1
values in the modified Mosher’s method for (S)-6 and (S)-9 are
available) associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2010.01.047.
Optical rotations of falcarindiol and stereoisomers
½ ꢃ (c 0.33)
a 2D4
Synthetic product
Natural product
3R,8S
3R,8R
3S,8S
3S,8R
3R,8S
+232.0°
ꢀ187.3°
+188.1°
ꢀ233.2°
+232.8°
References and notes
1. Horiguchi, T. Phycol. Res. 1995, 43, 129.
2. Nakashima, T.; Niwano, Y.; Takeshita, S. Aquac. Res. 2008, 39, 1346.
3. In 96-well microculture plate, each sample solution dissolved with 75 lL of 2%
DMSO in artificial sea water was inoculated to H. circularisquama (4.0 ꢂ 104
cells) in 75 lL of artificial sea water [DAIGO artificial sea water (Wako) 36 g,
DAIGO IMK medium (Wako) 0.252 g, distilled water 1 L]. After 48 h cultivation
at 25 °C under 2000 lux of illumination with a 12 h light/12 h dark cycle, the
number of whirling and settling cells was counted up to a hundred individuals
with the microscope in triplicate.
Table 2
Algicidal activity of falcarindiol and stereoisomers
4. Nitz, S.; Spraul, M. H.; Drawert, F. J. Agric. Food Chem. 1990, 38, 1445.
5. Cadiot, P.; Chodkiewicz, W. In Chemistry of Acetylenes; Viehe, H. G., Ed.; Marcel
Dekker: New York, 1969; pp 597–648.
6. Corey, E. J.; Bakshi, R. K.; Shibata, S.; Chen, C.-P.; Singh, V. K. J. Am. Chem. Soc.
1987, 109, 7925.
7. McDonald, E. F.; Reddy, K. S.; Diaz, Y. J. Am. Chem. Soc. 2000, 122, 4304.
8. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092.
Algicidal activity
4
+
lM
2
l
M
1 lM
Synthetic compound
Natural product
3R,8S
3R,8R
3S,8S
3S,8R
3R,8S
ꢀ
ꢀ
+
+
+
+
+
9. Nishikawa, T.; Shibuya, S.; Hosokawa, S.; Isobe, M. Synlett 1994, 485.
ꢀ
10. (3S,8S)-1: colorless oil. IR (KBr): 3250, 2155, 1646 cmꢀ1 1H NMR (CDCl3): d
.
Algicidal activity; +: 100%, 100%> =50%, ꢀ <50%.
5.94 (1H, ddd, J = 16.8, 10.2, 5.3 Hz, 2-H), 5.60 (1H, dt, J = 10.4, 7.3 Hz, 10-H),
5.51 (1H, dd, J = 10.4, 8.0 Hz, 9-H), 5.47 (1H, d, J = 16.8 Hz, 1-Ha), 5.26 (1H, d,
J = 10.2 Hz, 1-Hb), 5.20 (1H, br d, J = 8.0 Hz, 8-H), 4.94 (1H, br d, J = 5.3 Hz, 3-H),
2.11 (2H, dt, J = 7.3, 7.1 Hz, 11-H), 1.98 (2H, br s, 3, 8-OH), 1.38 (2H, quint,
J = 7.1 Hz, 12-H), 1.27 (8H, m, 13, 14, 15, 16-H), 0.88 (3H, t, J = 7.1 Hz, 17-H).
FAB-MS m/z: 261 [M+H]+. FAB-HRMS m/z: calcd for C17H25O2: 261.1854, found:
261.1855.
structural requirement for algicidal potency of 1 on the basis of
structure activity relationship of the synthesized congeners of 1.
On the other hand, it should be noted that the (3S,8S)- and
(3S,8R)-stereoisomers showed twofold potent algicidal activity in
comparison with falcarindiol (1). So far, synthetic hydroxy fatty
acid amides11 and polyunsaturated fatty acids12 were only found
out as low-molecule algicides against H. circularisquama. Among
(3R,8S)-1: colorless oil. IR (KBr): 3252, 2155, 1645 cmꢀ1 1H NMR (CDCl3): d
.
5.95 (1H, ddd, J = 16.5, 10.5, 5.5 Hz, 2-H), 5.60 (1H, dt, J = 11.0, 7.5 Hz, 10-H),
5.52 (1H, dd, J = 11.0, 7.5 Hz, 9-H), 5.47 (1H, d, J = 16.5 Hz, 1-Ha), 5.26 (1H, d,
J = 10.5 Hz, 1-Hb), 5.20 (1H, dd, J = 7.5, 5.1 Hz, 8-H), 4.95 (1H, dd, J = 6.5, 5.5 Hz,
3-H), 2.12 (2H, dt, J = 7.5, 7.0 Hz, 11-H), 2.02 (1H, d, J = 6.5 Hz, 3-OH), 1.92 (1H,
d, J = 5.1 Hz, 8-OH), 1.39 (2H, quint, J = 7.0 Hz, 12-H), 1.28 (8H, m, 13, 14, 15,
16-H), 0.89 (3H, t, J = 7.0 Hz, 17-H). FAB-MS m/z: 261 [M+H]+. FAB-HRMS m/z:
calcd for C17H25O2: 261.1854, found: 261.1850.
the former, the amide containing
0.3 g/mL, while the latter displayed growth inhibition ranging
from 30% to 69% at the concentration of 25 g/mL. Taking these
L-aspartic acid showed MIC of
l
(3S,8R)-1: colorless oil. FAB-MS m/z: 261 [M+H]+. FAB-HRMS m/z: calcd for
C17H25O2: 261.1854, found: 261.1854. The IR and 1H NMR spectra were
superimposable on those of (3R,8S)-1.
l
findings into account, falcarindiol (1) as well as the (3S,8S)- and
(3S,8R)-isomers should be regarded as significantly potent algicidal
principles against H. circularisquama.
(3R,8R)-1: colorless oil. FAB-MS m/z: 261 [M+H]+. FAB-HRMS m/z: calcd for
C17H25O2: 261.1854, found: 261.1853. The IR and 1H NMR spectra were
superimposable on those of (3S,8S)-1.
11. Kawabata, Y.; Shizusato, Y.; Tomosawa, T.; Kawamata, M.; Ueno, S. Jpn. Kokai
Tokkyo Koho JP2002348264, 2002.
12. Alamsjah, M. A.; Hirao, S.; Ishibashi, F.; Oda, T.; Fujita, Y. J. Appl. Phycol. 2008,
20, 713.
Supplementary data
Supplementary data (the previously reported absolute configu-
rations and optical rotations of falcarindiol, and distributions of
Dd