Spirocyclization strategy toward indole phytoalexins. The first synthesis of (±)-1-methoxyspirobrassinin, (±)-1-methoxyspirobrassinol, and (±)-1-methoxyspirobrassinol methyl ether

Article abstract of DOI:10.1016/S0040-4039(02)02452-8

The first syntheses of cruciferous indole phytoalexins (±)-1-methoxyspirobrassinin, (±)-1-methoxyspirobrassinol, (±)-1-methoxyspirobrassinol methyl ether as well as a new syntheses of phytoalexins (±)-spirobrassinin and cyclobrassinin were achieved by dioxane dibromide (DDB)-mediated spirocyclization of brassinin and its 1-substituted derivatives.

Full text of DOI:10.1016/S0040-4039(02)02452-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 9489–9492
Spirocyclization strategy toward indole phytoalexins.
The first synthesis of ( )-1-methoxyspirobrassinin,
( )-1-methoxyspirobrassinol, and ( )-1-methoxyspirobrassinol
methyl ether
Peter Kutschy,a,* Mojm´ır Suchy´,a Kenji Monde,b Nobuyuki Harada,c Renata Marusˇkova´,a
a
a
a
a
d
&
Zuzana Curillova´, Milan Dzurilla, Mariana Miklosˇova´, Roman Mezencev and Ja´n Mojzˇisˇ
a
&
Institute of Chemical Sciences, Faculty of Science, P. J. Safa´rik University, Moyzesova 11, 041 67 Kosˇice, Slovak Republic
bDivision of Biological Sciences, Graduate School of Science, Hokkaido University, Kita 10, Nishi 8, Sapporo 060-0810, Japan
cInstitute of Multidisciplinary Research for Advanced Materials, 2-1-1, Katahira, Aoba-ku, Sendai 980-8577, Japan
Institute of Pharmacology, Faculty of Medicine, P. J. Safa´rik University, SNP 1, 040 66 Kosˇice, Slovak Republic
d
&
Received 16 September 2002; revised 22 October 2002; accepted 1 November 2002
Abstract—The first syntheses of cruciferous indole phytoalexins ( )-1-methoxyspirobrassinin, ( )-1-methoxyspirobrassinol, ( )-1-
methoxyspirobrassinol methyl ether as well as a new syntheses of phytoalexins ( )-spirobrassinin and cyclobrassinin were achieved
by dioxane dibromide (DDB)-mediated spirocyclization of brassinin and its 1-substituted derivatives. © 2002 Elsevier Science Ltd.
All rights reserved.
Phytoalexins are defined as antimicrobial low molecular
weight secondary metabolites, produced by plants after
their exposure to physical, biological or chemical
stress.1 About 30 indole phytoalexins were isolated
from the economically and dietary important plants of
the family Cruciferae, cultivated worldwide.2 Selected
examples (except for the synthetic analogs 7, 8 and 11)
are shown in Fig. 1. With respect to their interesting
biological properties, including antitumor activity3–6 it
is important to develop synthetic methods toward cru-
ciferous phytoalexins, since the isolation from plants
does not afford sufficient quantities for biological
screening.
brassinin (3) isolated in 1994 from kohlrabi10 and (−)-1-
methoxyspirobrassinol methyl ether as well as
4
optically inactive methoxyspirobrassinol 5, isolated in
1995 as a minor phytoalexin from Japanese radish11
have not been synthesized to date and their biological
properties are unknown. Compound 5 exists in solution
as a mixture of diastereomers 5a and 5b in a 4:1 ratio,
owing to its unstable hemiaminal structure.11 The abso-
lute stereochemistries of 3–5 remains unknown, formu-
lae 4 and 5 show the diastereomeric structures only, as
determined by NOE experiments.11 Phytoalexins 3–5, 9
and 12 belong to a group of hydroxyindole natural
products.12 Our approach to the target compounds is
based on the finding, that spirobrassinin 1 and cyclo-
brassinin 10 are synthesized in plants from brassinin
6.13 Whereas 10 was prepared by cyclization of 6 with
pyridinium tribromide14 or NBS4 in 34–35% yield and
sinalbin B 12 was obtained by treatment of 1-methoxy-
brassinin 9 with NBS (41%),15 no transformation of
brassinin 6 or its 1-substituted derivatives to spiroindo-
line phytoalexins has been reported to date.
In the present paper we report the preparation and
antiproliferative activity of hitherto synthetically
unavailable spiroindoline phytoalexins 3–5 and a new
approach to the compounds 1 and 10. (S)-(−)-Spiro-
brassinin 1 was isolated in 1987 from Japanese radish,7
however, its synthesis by cyclization of dioxibrassinin 28
with SOCl2 or MsCl and absolute stereochemistry were
fully described only recently.9 (−)-1-Methoxyspiro-
Retrosynthetic analysis of 1 and 3–5 revealed a central
role for a spiroindoleninium intermediate 13 (Scheme
1), which can undergo a rearrangement to the cyclo-
brassinin structure, or nucleophilic addition of water
and methanol to a spiroindoline structure.
Keywords: phytoalexins; indoles; dioxane dibromide; spirocyclization;
antitumor activity.
* Corresponding author. Tel.: +421-55-62-22610, ext. 192; fax: +421-
55-62-22124; e-mail: kutschy@kosice.upjs.sk
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02452-8

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P. Kutschy et al. / Tetrahedron Letters 43 (2002) 9489–9492
To verify this prediction, brassinin 614 and 1-methyl-
brassinin 716 with low reactivity of the iminium inter-
mediate versus 1-Boc-brassinin 816 with expected high
reactivity of the corresponding acyliminium ion were
selected as a model compounds. After several unsuc-
cessful experiments with NBS, which seems to react by
a different mechanism, involving bromination at the
indole 3-position, we have found that dioxane dibro-
mide (DDB) in dioxane in the presence of water (0.5%)
is a suitable reagent for generation and rearrangement
or trapping of iminium intermediate 13. The reaction
probably starts at the thiocarbamoyl functionality with
the formation of a sulfenyl bromide in which the elec-
trophilic sulfur attacks the 3-position of indole to
provide an iminium intermediate (Scheme 2). As antici-
pated, the iminium ions with R=H and CH3 afforded
cyclobrassinin 10 (45%) and 1-methylcyclobrassinin 11
(40%) identical with previously described com-
pounds,14,16 whereas the acyliminium ion generated
from the 1-Boc derivative 8 afforded a diastereomeric
mixture of 1-Boc spirobrassinols 14a and 14b in a ratio
4:1 in 47% yield. Individual diastereomers were sepa-
rated by flash chromatography and their structure
determined on the basis of NOE-difference NMR
experiments.17 However, under anhydrous conditions,
1-Boc-brassinin 8 reacted with the formation of cyclo-
brassinin 10 (45%), probably because the HBr liberated
during the reaction deprotected the 9-Boc-cyclo-
brassinin, formed by the rearrangement of Boc-substi-
tuted intermediate 13. Compound 8 was consumed
within 5 min and if, within the next 5 min, 0.5% of
water was added, the spirobrassinols 14a and 14b were
formed. However, when stirring was continued under
anhydrous conditions, slow rearrangement and depro-
tection took place. We have found that the clean reac-
tion with water as a nucleophile proceeds with up to 5%
of water contained in the reaction medium. Therefore
all further experiments were performed in dioxane/
water or dioxane/methanol 95:5 as the solvent.
Figure 1.
A mixture of 14a and 14b was oxidized with CrO3 to
1-Boc-spirobrassinin ( )-15, which after deprotection
with TFA afforded spirobrassinin ( )-1. Treatment of a
mixture of 14a and 14b with TFA proceeded with
deprotection and water elimination toward an iminium
cation with
a trifluoroacetate counterion, which
smoothly rearranged to cyclobrassinin 10 (Scheme 2).
Next, with the experimental conditions in hand for the
preparation of 1-Boc-spirobrassinol 14, we attempted
the cyclization of 1-methoxybrassinin 9.15,18,19 It
appeared that the methoxyiminium intermediate
formed is a highly reactive species, probably because of
the activating effect of the electronegative oxygen atom.
Consequently, the water present in the reaction mixture
smoothly added to the carbon atom of the iminium
intermediate to produce methoxyspirobrassinol 5 in
90% yield (Scheme 3).
Scheme 1.
The preferred pathway should be dependent on the
reactivity of iminium intermediate 13, towards nucle-
ophiles. A low reactivity is expected to produce cyclo-
brassinin, and on the other hand its high reactivity
should result in nucleophilic trapping resulting in the
formation of spiroindolines.
Oxidation of
5 with CrO3 afforded racemic 1-
methoxyspirobrassinin ( )-3 in 40% yield. If methanol
was added as a nucleophile, instead of water, the
methoxyiminium intermediate reacted with the forma-
tion of a mixture of the natural 4a and unnatural 4b

P. Kutschy et al. / Tetrahedron Letters 43 (2002) 9489–9492
9491
Scheme 2.
Scheme 3.
diastereomers of methoxyspirobrassinol methyl ether in
a ratio of 1:2 which separated by flash chromatography
(ether/hexane, 1:2). Spectroscopic data of the synthe-
sized phytoalexins 1, 3–5 and 10 are identical with those
of the natural products.7,10,11,14
oration of the solvent subjected to flash chromatogra-
phy on 5 g of silica gel (cyclohexane/ethyl acetate, 5:1),
affording 60 mg (90%) of 5 as a semi-solid colourless
material with all the spectral data fully identical with
the described natural product.11 ( )-1-Methoxyspiro-
brassinin 3: To a stirred solution of 5 (91 mg, 0.32
mmol) in 98% acetic acid (9 mL) was added CrO3 (160
mg, 1.6 mmol) and the mixture stirred for 1 h at room
temperature. After pouring into water (40 mL), the
product was extracted with diethyl ether (2×30 mL), the
extract washed with 10% NaOH (2×15 mL) and brine
(15 mL), dried over Na2SO4 and the residue obtained
after evaporation of the solvent subjected to flash chro-
matography on 30 g of silica gel (diethyl ether/light
petroleum,1:2) to afford 36 mg (40%) of 3 as colourless
needles, mp 129–131°C (ethyl acetate/hexane). The
spectral data was fully identical with those of the
natural product.10
Experimental procedure for 1-methoxyspirobrassinin:
( )-Methoxyspirobrassinol 5: To a stirred solution of
1-methoxybrassinin 9 (63 mg, 0.24 mmol) in a mixture
of dioxane/water (95:5, 5 mL) was added a freshly
prepared solution of DDB (2 mL, 0.26 mmol; the stock
solution was obtained by dissolving of 0.04 mL of
bromine in 6 mL of dioxane). The reaction mixture was
stirred for 10 min at room temperature, then triethyl-
amine (0.26 mmol) was added, the mixture poured into
water (50 mL), the product extracted with diethyl ether
(2×10 mL), the extract washed with brine (20 mL), then
dried over Na2SO4 and the residue obtained after evap-

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P. Kutschy et al. / Tetrahedron Letters 43 (2002) 9489–9492
The antiproliferative activity of the synthesized com-
pounds was examined by MTT (thiazolyl blue) test,20
using the selected human cancer cell lines (MDA-MB-
231, MCF-7, U-87 MG and CACO-2). The highest
activity was found with the natural diastereomer of
methoxyspirobrassinol methyl ether 4a which at con-
centration of 0.2 mmol L−1 inhibited the growth of
CACO-2 (colon adenocarcinoma) cell line to 38% of
solvent control after 72 h incubation. This finding is
consistent with the recommendation of consuming bras-
sica vegetables as a protection against cancer.21
10. Gross, D.; Porzel, A.; Schmidt, J. Z. Naturforsch., Sect.
C 1994, 49, 281–285.
11. Monde, K.; Takasugi, M.; Shirata, A. Phytochemistry
1995, 39, 581–586.
12. For a review on 1-hydroxyindoles see: Somei, M. Hetero-
cycles 1999, 50, 1157–1211.
13. Monde, K.; Takasugi, M.; Ohnishi, T. J. Am. Chem. Soc.
1994, 116, 6650–6657.
14. Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Bull.
Chem. Soc. Jpn. 1988, 61, 285–289.
15. Pedras, M. S. C.; Zaharia, I. L. Phytochemistry 2000, 55,
213–216.
16. Kutschy, P.; Dzurilla, M.; Takasugi, M.; To¨ro¨k, M.;
Achbergerova´, I.; Homzova´, R.; Ra´cova´, M. Tetrahedron
1998, 54, 3549–3566.
Acknowledgements
17. Data for 14a: White solid, mp 72–74°C (CHCl3/light
petroleum); Rf (benzene/acetone, 19:1) 0.21; [Found: C,
54.4; H, 5.5; N, 8.2. C16H20N2O3S2 requires C, 54.52; H,
5,72; N, 7.95]; wmax (CHCl3) 3607, 2993, 2940, 2880, 1687,
1570, 1480, 1370, 1163 cm−1; lH (400 MHz, DMSO-d6)
7.59 (1H, d, J 8.1 Hz, H-7), 7.42 (1H, dd, J 7.6, 0.7 Hz,
H-4), 7.28 (1H, ddd, J 7.9, 7.9, 1.4 Hz, H-6), 7.04 (1H,
ddd, J 7.4, 7.4, 1.2 Hz, H-5), 6.95 (1H, d, J 6.3 Hz,
OH–D2O exchangeable), 5.63 (1H, d, J 6.4 Hz, CH), 4.83
(1H, d, J 15.6 Hz, Hb), 4.38 (1H, d, J 15.9 Hz, Ha), 2.54
(3H, s, SCH3), 1.53 (9H, s, C(CH3)3); lC (100 MHz,
DMSO-d6) 161.6 (C), 150.9 (C), 140.6 (C), 130.0 (C),
129.2 (CH), 123.6 (CH), 122.8 (CH), 114.2 (CH), 90.8
(CH), 81.0 (C), 70.6 (C), 65.9 (CH2), 27.6 (CH3), 14.3
(SCH3); m/z (EIMS) 352 (15, M+), 296 (72), 252 (20), 234
(17), 161 (32), 145 (71), 117 (23), 87 (20), 57 (100). Data
for 14b: Colourless plates, mp 126–128 °C (CH2Cl2/light
petroleum); Rf (benzene/acetone, 19:1) 0.29; [Found: C,
54.3; H, 5.9; N, 8.1. C16H20N2O3S2 requires C, 54.52; H,
5,72; N, 7.95]; wmax (CHCl3) 3587, 3013, 2986, 2940, 1683,
1560, 1480, 1366, 1163 cm−1; lH (400 MHz, DMSO-d6)
7.61 (1H, d, J 7.8 Hz, H-7), 7.29–7.24 (2H, m, H-4, H-6),
7.05–7.00 (2H, m, H-5, OH-D2O exchangeable), 5.49
(1H, d, J 6.0 Hz, CH), 4.36 (1H, d, J 15.4 Hz, Hb), 3.82
(1H, d, J 15.1 Hz, Ha), 2.49 (3H, s, SCH3), 1.54 (9H, s,
C(CH3)3); lC (100 MHz, DMSO-d6) 164.1 (C), 151.0 (C),
139.2 (C), 132.1 (C), 128.8 (CH), 132.2 (CH), 122.6 (CH),
114.1 (CH), 87.6 (CH), 80.9 (C), 74.4 (CH2), 73.7 (C),
27.6 (CH3), 14.2 (SCH3); EIMS identical with 14a.
18. Kawasaki, T.; Somei, M. Heterocycles 1990, 31, 1605–
1608.
We thank the Slovak Ministry of Education (MVTS
project Jap/Slov/UPJS) for financial support of this
work.
&
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