Unprecedented chemical structure and biomimetic synthesis of erucalexin, a phytoalexin from the wild crucifer Erucastrum gallicum

Article abstract of DOI:10.1039/b515331j

The isolation, structure determination, total synthesis and antifungal activity of erucalexin, a novel phytoalexin produced by the wild crucifer dog mustard are described. Erucalexin is a structurally unique plant alkaloid, representing the first example of a spiro[2H-indole-2,5′(4′H)- thiazol]-3-one, likely derived from a C-3-C-2 carbon migration in a 3-substituted indolyl nucleus. The Royal Society of Chemistry.

Full text of DOI:10.1039/b515331j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Unprecedented chemical structure and biomimetic synthesis of erucalexin, a
phytoalexin from the wild crucifer Erucastrum gallicum†
M. Soledade C. Pedras,* Mojmir Suchy and Pearson W. K. Ahiahonu‡
Received 28th October 2005, Accepted 23rd November 2005
First published as an Advance Article on the web 5th January 2006
DOI: 10.1039/b515331j
The isolation, structure determination, total synthesis and antifungal activity of erucalexin, a novel
phytoalexin produced by the wild crucifer dog mustard are described. Erucalexin is a structurally
unique plant alkaloid, representing the first example of a spiro[2H-indole-2,5ꢀ(4ꢀH)-thiazol]-3-one,
likely derived from a C-3–C-2 carbon migration in a 3-substituted indolyl nucleus.
Introduction
The multifaceted defense systems of plants include an arsenal
of metabolites known as phytoalexins, which are biosynthesized
de novo in response to various sorts of stress such as microbial
attack.1 The phytoalexins produced by crucifer plants comprise
an amazing class of tryptophan-derived alkaloids containing
diverse functional groups that display selective activity against
plant pathogens2 as well as cancer chemopreventive activity.3
Crucifers are economically valuable oilseeds (rapeseed and canola,
Brassica rapa and B. napus), vegetables (cauliflower, B. oleracea
var. italica, broccoli, B. oleracea var. botrytis, rutabaga, B. napus
ssp. rapifera, and Brussels sprouts, B. oleracea var. gemmifera)
and condiments (brown mustard, B. juncea, and wasabi, Eutrema
wasabi).2 Furthermore, the wild crucifer Arabidopsis thaliana is
Results and discussion
the first flowering plant to have its genome sequenced4 and many
wild crucifers5 are sources of desirable agronomic traits of great
interest in plant breeding. For example, dog mustard (Erucastrum
gallicum) is a potential source of genetic resistance to Sclerotinia
sclerotiorum (Lib.) de Bary,6 a fungal plant pathogen causing stem
rot disease in crucifers and other plant families.7,8
Recently, analysis of the chemical response of dog mustard
leaves to the pathogen S. sclerotiorum and to abiotic stress led
to the isolation of three known phytoalexins9 (1–3) and erucalexin
(4), a new phytoalexin which was shown to have an unprecedented
structure by spectroscopic analyses.10 Subsequently, synthesis of
model compounds and synthetic approaches to make this unique
spiro[2H-indole-2,5ꢀ(4ꢀH)-thiazol]-3-one ring system led to the
discovery of a one-pot biomimetic spirocyclization applicable to
both 2- and 3-indolinones. Here we report for the first time the
chemical structure elucidation, synthesis and antifungal activity of
erucalexin (4). The chemical structure of erucalexin (4) represents
the first spiro[2H-indole-2,5ꢀ(4ꢀH)-thiazol]-3-one described to date
and suggests an intriguing rearrangement in its biosynthetic
pathway.
Erucalexin (4) was produced in leaves of dog mustard elicited
with either S. sclerotiorum or CuCl2, and was isolated as reported
in the Experimental section ([a]D +73, c 0.090, MeOH). The
HRMS-EI data of erucalexin (4) indicated it to be an isomer of 1-
methoxyspirobrassinin (3, C12H12N2S2O2, obtained m/z 280.0342,
calc. 280.0340), further corroborated by analysis of its NMR
spectroscopic data. The NMR spectral data of 4 (Table 1) showed
some similarities to that of 1-methoxyspirobrassinin (3), i.e. signals
Table 1 NMR data for erucalexin (4) dissolved in CD3CN
b,c
Position
dH (multiplicity, J in Hz)a
dC
HMBC correlations
2
3
—
—
95.4
193.2
122.1
125.3
125.1
139.4
115.2
161.0
163.6
72.5
3a
4
5
6
7
—
7.68 (d, J = 8)
7.21 (ddd, J = 8, 8, 1)
7.73 (ddd, J = 8, 8, 1)
7.32 (d, J = 8)
—
C-7a, 6, 3
C-7, 3a, 6
C-7a, 4, 5
C-3a, 5, 6
7a
2ꢀ
—
4aꢀ
4bꢀ
OMe
SMe
4.75 (d, J = 16)
4.35 (br d, J = 16)
3.95 (s)
C-2, 2ꢀ
C-2ꢀ, 3
72.5
66.1
16.3
Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon, Saskatchewan, Canada S7N 5C9. E-mail: s.pedras@usask.ca;
Fax: (306) 966-4730; Tel: (306) 966-4772
2.61 (s)
C-2ꢀ, 4aꢀ, 4bꢀ
† Electronic supplementary information (ESI) available: 1H NMR spectra
for compounds 3, 4, 6, 9a, 13, 14, 16, 17, 21, 22, 25–29, 38–40, 43–46. See
DOI: 10.1039/b515331j
a Spectrum recorded at 500 MHz referenced to residual CD2HCN, dH
1.94. Assigned from NOE and 1H NMR decoupling experiments. b Spectra
recorded at 125 MHz and referenced to CD3CN, dC 118.69. c Assigned from
HMQC and HMBC (500 MHz).
‡ Current address: Phenomenome Discoveries Inc., Saskatoon, SK,
Canada.
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due to a methoxy group (dH 3.95, s; dC 66.1) attached to N-1, a
thiomethyl group (dH 2.61, s; dC 16.3), a methylene group, and
four aromatic protons on an ortho-disubstituted benzene ring.
Irradiation of the methoxy protons (dH 3.95) caused an NOE
enhancement of the aromatic proton signal at d 7.32 (1H, d,
J = 8 Hz) allowing its assignment as H-7 (Fig. 1); consecutive
decoupling experiments allowed the assignment of the three
remaining aromatic protons H-4, H-5 and H-6 (Table 1). The
signal at dC 193.2 was suggestive of a 3-indolinone or a 2-
indolethione ring rather than the 2-indolinone system present
in 1-methoxyspirobrassinin (3). This hypothesis was supported
by an HMBC correlation between H-4 and C-3/C-2 of the ring;
additional HMBC correlations observed between the methylene
protons and the carbon at dC 193.2, 95.4, and C-2ꢀ (dC 163.6,
=
S–C N) led to two possible structures 4 or 5 (Fig. 1). Initially,
structure 5 was chosen as a reasonable possibility based on 13C
NMR data and on the biogenetic pathway of this skeletal type of
=
metabolite. Since the (H3C)–S–C N fragment was present in both
4 and 5, the chemical shifts of C-3ꢀ in 4 and in 5 were expected to
be around 165 ppm, whereas the carbon chemical shifts of C-3 in
4 and C-2 in 5 were predicted to be ca. 195 10 ppm. Because
no structures similar to 4 or 5 were known, syntheses of model
compounds 6 and 9 (9a R = CH3) were carried out to determine
the likely structure of erucalexin. Erucalexin was eventually proven
to have structure 4 by total synthesis, as described below.
Scheme 1 Retrosynthetic analysis of model compounds 6 and 9.
Fig. 1 Selected HMBC and NOE correlations.
To establish which of the proposed structures represented
erucalexin, compound 6 (the N-methyl analogue of 5) and
compound 9 presented new but accessible targets using potentially
known chemistry (Scheme 1). Compound 6 was synthesized
in five steps from commercially available 1-methylisatin (8), as
summarized in Scheme 2 and described in the Experimental
section. Addition of nitromethane to 8 under basic conditions
followed by catalytic hydrogenation11 of the hydroxynitromethane
product afforded ( )-12 in 79% yield. Subsequent cyclization of
( )-12 with thiophosgene12 gave ( )-13 with the desired spiro-
ring system in 76% yield. Thiation of ( )-13 using phosphorus
pentasulfide in THF, a general procedure for conversion of
carbonyl to thiocarbonyl, afforded ( )-14 in 16% yield.13 The use
Lawesson’s reagent14 for thiation did not improve the yield of ( )-
14. Subsequent methylation of ( )-14 yielded the desired spirooxa-
zoline 6 {( )-2ꢀ-methylsulfanyl-spiro[1-methylindoline-3,5ꢀ-[4ꢀ,5ꢀ]-
dihydrooxazole]-2-thione} in 81% yield. The spectroscopic data
of 6 revealed significant differences from those of the isolated
compound indicating that structure 5 was not erucalexin (4).
Specifically, the UV spectrum of 6 [kmax (log e): 238 (4.1), 287
(3.6), 296 (3.7), 337 (3.8)] and that of 4 [kmax (log e): 234 (4.6), 262
(4.1), 368 (3.4)] were rather different.
Scheme 2 Synthesis of compound 6.
Baeyer–Villiger oxidation of indole-3-carboxaldehydes15 16, 19
and 20 was thought to provide readily accessible starting materials
for the syntheses of model compound 9 (9a R = CH3 or 9b R =
OCH3). Thus, 1-methoxy-2-chloroindole-3-carboxaldehyde [16,
prepared by oxalyl chloride-mediated Vilsmeier formylation of
1-methoxyoxindole (15)] was treated with m-chloroperoxybenzoic
acid (m-CPBA) in CH2Cl2 at room temperature to yield rather
the unstable O-formate 18 in 43% yield, together with a small
amount (10%) of 1-methoxyisatin (17) (Scheme 3).16 Attempts
to hydrolyze 18 or to carry out substitution at C-2 using
sulfides or thiols as nucleophiles (EtSH, MeSNa) yielded only
1-methoxyisatin (17). Similarly, Baeyer–Villiger oxidation fol-
lowed by HCl-mediated hydrolysis of 1-methyl-2-chloroindole-3-
carboxaldehyde (19)17 afforded only 1-methylisatin (8), whereas 1-
methoxyindole-3-carboxaldehyde (20)18 afforded ( )-1-methoxy-
2-hydroxyindolin-3-one (21) (hydrolysis carried out in dioxane)
or ( )-1,2-dimethoxyindolin-3-one (22) (hydrolysis carried out in
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Scheme 4 Synthesis of model compound 9a.
compound ( ) 9a (Scheme 4). As predicted, spectral data of ( )
9a were similar to those of erucalexin (4): specifically, the UV
spectrum of compound 9a [kmax (log e): 235 (4.3), 264 (4.0),
353 (3.0)] and that of 4 [kmax (log e): 234 (4.6), 262 (4.1), 368
(3.4)] were similar, both containing the characteristic indolin-3-
one absorption (>350 nm).
After obtaining a reasonable proof that the spiro structure
4 represented erucalexin, additional strategies to access the 3-
indolinone-2-spirocyclic system of erucalexin (4) were considered.
The most attractive of the various potential routes was designed
based on possible biogenetic pathways to 1-methoxyspirobrassinin
(3) and erucalexin (4) (Scheme 5). Our previous work20 indicates
that 1-methoxybrassinin (30) is a close biosynthetic precursor
of 1-methoxyspirobrassinin (3). Furthermore, the structure of
erucalexin (4), its co-occurrence with 1-methoxyspirobrassinin (3)
in dog mustard leaves and an interesting study by Monde et al.21
suggests that 3 could be a precursor of 4 as well. Nonetheless,
despite the variety of oxidizing reagents and conditions used,
our search for a reagent that transformed 1-methoxybrassinin
(30) into 3 and 4 was unsuccessful, leading mostly to intractable
mixtures of products of no synthetic utility. Unexpectedly, CrO3
in acetic acid oxidized directly 1-methoxybrassinin (30) to ( )-
1-methoxyspirobrassinin (3) (Scheme 6) in 21% yield; however,
no traces of ( )-erucalexin (4) were detected. When the reac-
tion was carried out at 0 ◦C, ( )-1-methoxyspirobrassinol (35)
Scheme 3 Baeyer–Villiger oxidation of indole-3-carboxaldehydes 16, 19,
20 and 23.
MeOH) (Scheme 3). Hence, since none of the aldehydes initially
thought useful starting materials – 16, 19 and 20 – yielded
the desired product, oxidation of the t-Boc protected aldehyde
2319 was attempted. Baeyer–Villiger oxidation of 23 proceeded
smoothly to yield O-formate 24 in 90% yield (Scheme 3). O-
Formate 24 was isolated by flash column chromatography (FCC)
and immediately treated with EtSH in the presence of Et3N to
afford ( )-1-Boc-2-ethylsulfanylindolin-3-one (25) in 59% yield
(based on aldehyde 23) (Scheme 4). Treatment of ( )-25 with
NaH, followed by CH3I yielded ( )-1-Boc-2-ethylsulfanyl-2-
methylindolin-3-one (26) in very good yield (87%), suggesting
that the model compound 9a would be accessible using simi-
lar methodology. Therefore, O-formate 24 was treated with 3-
chloro-1-propanethiol to afford intermediate ( )-27, which upon
intramolecular cyclization mediated by NaH yielded ( )-1-Boc-
spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (28), possessing a
hitherto unknown heterocyclic skeleton (Scheme 4). Finally,
the protecting Boc group was removed using trifluoroacetic
acid (TFA) in CH2Cl2 and the resulting ( )-spiro(indoline-2,2ꢀ-
tetrahydrothiophene)-3-one (29) was methylated to yield model
Scheme 5 Possible biogenetic pathways to erucalexin (4) and 1-methoxy-
spirobrassinin (3).
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Gratifyingly, oxidative cyclization of 1-methoxyisobrassinin
(38) with CrO3 in acetic acid afforded ( )-erucalexin (4) in
moderate yield (30%). Except for the optical rotation, the char-
acterization data of the synthetic material and those of the
natural product were identical in all respects. Additional attempts
to synthesize erucalexin (4) from 38 using different oxidizing
agents (Br2 in dioxane, NBS in CH2Cl2, or pyridinium bromide
perbromide in t-BuOH) and conditions led to formation of the
tricyclic compound 39 (Scheme 8). With 39 in hand, we sought
to carry out an oxidative rearrangement to form a 3-indolinone-
2-spirocyclic compound. However, when 39 was treated with m-
CPBA, an intractable mixture was obtained, whereas reaction of
39 with N-chlorosuccinimide (NCS) afforded 40 in 32% yield
(Scheme 8). Other oxidizing reagents such as H2O2/Na2WO4
yielded 40 in <10% yield and NBS yielded 40 in 21%. Although a
somewhat related rearrangement of a cyclic acetal into a lactone
using m-CPBA/BF3·Et2O has been observed, those conditions did
not work in the case of compound 39.26
Scheme
6
Synthesis of ( )-1-methoxyspirobrassinin (3), ( )-spiro-
brassinin (34) and ( )-1-methoxyspirobrassinol (35).
was obtained in 35% yield (Scheme 6). The yield of ( )-1-
methoxyspirobrassinin (3) could be improved using pyridinium
chlorochromate (PCC) as an oxidizing agent (38% yield). Im-
portantly, oxidation of brassinin (33) by CrO3 in acetic acid and
dioxane afforded ( )-spirobrassinin (34) in 40% yield (Scheme 6).
It is worthy of note that this is the first time that ‘one-pot’
oxidations of 1-methoxybrassinin (30) and brassinin (33) to ( )-
1-methoxyspirobrassinin (3) and ( )-spirobrassinin (34), respec-
tively, are reported. These transformations may be considered
formal biomimetic syntheses of the phytoalexins 3 and 34.
Coincidentally, the absolute configuration of natural (+)-3 was
established recently to be R and ( )-3 was synthesized from 1-
methoxybrassinin (30) in a two-pot process.22
Scheme 8 Oxidation of potential biosynthetic intermediate 38.
After observing that 1-methoxybrassinin (30) could be oxidized
to ( )-1-methoxyspirobrassinin (3) with CrO3 in acetic acid, it
was conceivable that 1-methoxyisobrassinin (38) could likewise
provide access to ( )-erucalexin (4) via oxidative cyclization.
Toward this end, 1-methoxyindole (36)23 appeared to be a suitable
starting material for the synthesis of 1-methoxyisobrassinin (38).
Thus, deprotonation of 1-methoxyindole (36) using t-BuLi,24
followed by quenching with DMF afforded 1-methoxyindole-2-
carboxaldehyde, which was directly converted to oxime 37 in
74% yield. Reduction of 37 with TiCl3 and NaBH3CN25 afforded
the corresponding amine, which was converted in situ to 1-
methoxyisobrassinin (38) by standard treatment with carbon
disulfide and iodomethane (Scheme 7).
To expand the scope of CrO3-mediated oxidation we have also
investigated the oxidative spirocyclization of isobrassinin (43) and
1-Boc-isobrassinin (44). To prepare isobrassinin (43), aldehyde
4127 was converted to the corresponding oxime, which upon
treatment first with NaBH4/NiCl2,28 followed by carbon disulfide
and iodomethane afforded isobrassinin (43) in 30% yield, based
on indole-2-carboxaldehyde (41) (Scheme 9). 1-Boc-Isobrassinin
(44) was prepared similarly, using 1-Boc-indole-2-carboxaldehyde
(42)19 (Scheme 9) in 24% overall yield, based on 1-Boc-indole-2-
carboxaldehyde (42). Oxidation of isobrassinin (43) under various
conditions led to decomposition, as suggested by TLC monitoring
Scheme 7 Synthesis of ( )-erucalexin (4).
Scheme 9 Synthesis of ( )-demethoxyerucalexin (46).
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Table 2 Antifungal activitya of natural 1-methoxyspirobrassinin (3) and erucalexin (4) against the plant pathogens Rhizoctonia solani and Sclerotinia
sclerotiorum
% Inhibitiona
Compound
Concentration/M
R. solani
S. sclerotiorum
(+)-1-Methoxyspirobrassinin (3)
5.0 × 10−4
2.5 × 10−4
5.0 × 10−5
68
14
53
34
10
No inhibition
(+)-Erucalexin (4)
5.0 × 10−4
2.5 × 10−4
5.0 × 10−5
100
48
No inhibition
40
15
No inhibition
a Percent inhibition = 100 − [(growth on medium containing compound/growth on control medium) × 100]; results are the mean of three independent
experiments conducted in triplicate (SD 1).
of the reaction mixture. On the other hand, 1-Boc-isobrassinin (44)
afforded the oxidation product ( )-45 in 26% yield (Scheme 9).
Deprotection of ( )-45 (TFA, CH2Cl2) gave N-demethoxy analog
of erucalexin ( )-46 (Scheme 9).
silica gel grade 60, 230–400 lm. Organic extracts were dried over
Na2SO4 and the solvents were removed using a rotary evaporator.
Analytical HPLC analysis was carried out with a high performance
liquid chromatograph equipped with quaternary pump, automatic
injector, and photodiode array detector (wavelength range 190–
600 nm), degasser, and an ODS column (5 lm particle size silica,
4.6 id × 200 mm) equipped with an in-line filter. Mobile phase:
H2O–CH3CN, 75 : 25 to 100% CH3CN, for 35 min, linear gradient,
and flow rate 1.0 ml min−1. Specific rotations, [a]D were determined
at ambient temperature on a polarimeter using a 1 ml, 10 cm path
length cell; the units are 10−1 deg cm2 g−1 and the concentrations (c)
are reported in g/100 ml. NMR spectra were recorded on 500 MHz
The antifungal activity of (+)-1-methoxyspirobrassinin (3) and
(+)-erucalexin (4) was compared using a mycelia radial growth
bioassay, as described in the Experimental section. These antifun-
gal bioassays established that both (+)-1-methoxyspirobrassinin
(3) and (+)-erucalexin (4) were active against S. sclerotiorum and
Rhizoctonia solani, two of the most important pathogens of oilseed
and vegetable crucifers. Erucalexin (4, 5.0 × 10−4 M) and 1-
methoxyspirobrassinin (3, 5.0 × 10−4 M) were more inhibitory
to R. solani (100 and 68% inhibition, respectively) than to S. scle-
rotiorum (40 and 53% inhibition, after 72 h of incubation, Table 2).
1
spectrometers. For H NMR (500 MHz) the chemical shifts (d)
are reported in parts per million (ppm) relative to TMS. The d
values were referenced to CDCl3 (CHCl3 at 7.27 ppm), CD3CN
(CD2HCN at 1.94 ppm). First-order behavior was assumed in the
Conclusion
1
analysis of H NMR spectra and multiplicities are as indicated
In conclusion, (+)-erucalexin (4) is produced in elicited leaves
of E. gallicum, but is not detectable in non-elicited leaves, and
shows antifungal activity against S. sclerotiorum and R. solani, two
important pathogens of crucifer oilseeds. Therefore, erucalexin (4)
is a novel phytoalexin having a 3-indoxyl system with a spiro ring
at C-2. It is worthy of note that, although E. gallicum produces
phytoalexins active against S. sclerotiorum and R. solani, it appears
that to date only resistance to S. sclerotiorum has been reported.6
Hence, it would be important to evaluate further the resistance of
E. gallicum to other fungi, as it could be a valuable germplasm
source of disease resistance for oilseed improvement.
The first one-pot biomimetic syntheses of both ( )-1-methoxy-
spirobrassinin (3) and ( )-spirobrassinin (34) from 1-methoxy-
brassinin (30) and brassinin (33), respectively, were accomplished.
( )-Erucalexin (4) was synthesized from 1-methoxyindole (36) in
9% overall yield. Further studies addressing the enantioselective
synthesis, resolution and absolute configuration of erucalexin (4)
are underway.
by one or more of the following: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, and br = broad. Spin coupling
constants (J values) are reported to the nearest 0.5 Hz. For 13C
NMR (125.8 MHz) the chemical shifts (d values) were referenced
to CDCl3 (77.23 ppm), CD3CN (118.69 ppm). The multiplicities of
the 13C signals refer to the number of attached protons: s = C, d =
CH, t = CH2, q = CH3, and were determined based on HMQC
correlations and magnitude of J values. Mass spectrometry (MS)
[high resolution (HR), electron impact (EI)] data were obtained
on a mass spectrometer using a solids probe. Fourier trans-
form infrared (FTIR) spectra were recorded on a spectrometer
and spectra were measured by the diffuse reflectance method
on samples dispersed in KBr. Ultraviolet (UV) spectra were
recorded on a spectrophotometer using a 1 cm path length
quartz cell.
Plant material and growth
Experimental
Seeds of dog mustard (Erucastrum gallicum, a wild crucifer) were
obtained from Plant Gene Resources, Agriculture and Agric-
Food Canada Research Station, Saskatoon, SK. The seeds were
sown in commercial potting soil mixture, and plants were grown
in a growth chamber under controlled environmental conditions
(20/18 ◦C with 16/8 day/night cycle) for 2–5 weeks.
General experimental procedures
All solvents were HPLC grade and used as such, except for CH2Cl2
and CHCl3 which were redistilled and THF and Et2O which were
dried over Na and benzophenone. Flash column chromatography:
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Fungal isolates
followed by reverse phase micro flash (2 cm plug of reverse phase
C-18 silica gel in a Pasteur pipette, CH3CN–H2O (60 : 40) to
yield 2.2 mg of (+)-erucalexin (4). This process was repeated
four times to yield sufficient material for chemical characterization
and bioassays (in total, from 200 plants and 6 g of extract ca. 12 mg
of (+)-4 were isolated). [a]D = 73 (c 0.090, MeOH).
Fungal isolates were obtained from Agriculture and Agric-
Food Canada Research Station, Saskatoon, SK. Cultures of
Sclerotinia sclerotiorum clone #33 and Rhizoctonia solani AG-2
were maintained on potato dextrose agar (PDA) cultures.
Elicitation of phytoalexins with Sclerotinia sclerotiorum.
Leaves from five-week-old plants (E. gallicum) were excised with a
sharp blade, the petioles were wrapped with pre-moistened cotton
wool and leaves placed in Petri plates (two leaves per plate). Each
leaf was inoculated with five mycelium plugs placed upside down
(4 mm cut from three-day-old PDA plates of S. sclerotiorum clone
#33) and distributed evenly over the leaf surface. The Petri plates
were sealed and incubated under constant fluorescent light for
seven days. After every 24 h, leaves were frozen in liquid nitrogen,
crushed with a glass rod and extracted with EtOAc by shaking at
120 rpm for 30 min. The EtOAc was filtered, dried over anhydrous
Na2SO4 and the solvent removed under reduced pressure and the
extract was analyzed by HPLC. Two independent experiments
were carried out. The HPLC analysis of the EtOAc extracts
indicated the presence of the novel erucalexin (4, tR = 20.6 min)
and three other compounds with tR = 11.2, 13.1, and 16.9 min, as
previously reported,9 not present in the control samples.
Synthesis
( )-Erucalexin (4). A solution of CrO3 (100 mg, 1.0 mmol) in
water (0.3 ml) was added to a solution of 1-methoxyisobrassinin
(38, 49 mg, 0.18 mmol) in acetic acid (1 ml) in one portion at room
temperature. The mixture was stirred at rt for 5 min, was diluted
with brine (20 ml) and was extracted (EtOAc). The combined
organic extract was washed with 10% K2CO3 solution (20 ml) and
dried. The solvent was concentrated and the residue was subjected
to FCC (hexane–acetone, 5 : 1) to yield erucalexin (4, 16 mg,
30%) as an orange solid, mp 94–97 ◦C. HPLC: tR = 20.6 min. dH
(500 MHz, CDCl3): 7.70 (d, J = 8 Hz, 1H), 7.66 (ddd, J = 8, 8,
1 Hz, 1H), 7.24 (d, J = 8 Hz, 1H), 7.14 (ddd, J = 8, 8, 1 Hz, 1H),
4.76 (d, J = 16 Hz, 1H), 4.49 (d, J = 16 Hz, 1H), 4.00 (s, 3H),
2.68 (s, 3H); dC (125 MHz, CDCl3): 192.0 (s), 165.8 (s), 159.9 (s),
138.0 (d), 124.6 (d), 123.8 (d), 121.1 (s), 114.1 (d), 93.8 (s), 70.9
(t), 65.4 (q), 16.0 (q). HRMS (EI): calc. for C12H12N2O2S2 (M+)
m/z 280.0340, found 280.0342; MS (EI) m/z (% relative int.): 280
[M+] (56), 249 (74), 207 (30), 176 (100), 149 (15), 132 (64), 87
(38). mmax(KBr)/cm−1: 2923, 2850, 1725, 1608, 1576, 1461, 1301,
1106, 1064, 985, 759. kmax (CH3CN)/nm 234 (log e, 4.6), 262 (4.1),
368 (3.4).
Elicitation of phytoalexins with CuCl2. Five-week-old plants
(E. gallicum) were sprayed to the point of run-off with CuCl2
solutions at 24 h intervals for three days. Leaves were excised at
24 h intervals for seven days; control leaves were harvested from
separate plants at the same time and treated in a similar manner
throughout. After various incubation periods, leaves were worked
up as described above and extracts were analyzed by HPLC. Three
independent experiments were carried out.
( )-2ꢀ-Methylsulfanyl-spiro[1-methylindoline-3,5ꢀ-[4ꢀ,5ꢀ]-dihydro-
oxazole]-2-thione (6). To a stirred suspension of powdered
K2CO3 (2.5 mg, 0.018 mmol) in acetone (0.5 ml) were added
spirooxazolidine 14 (4 mg, 0.016 mmol) and CH3I (5 ll,
0.08 mmol). The mixture was stirred for 16 h at rt and was
subjected to TLC (hexane–acetone, 5 : 1) to yield ( )-6 (3.4 mg,
Antifungal bioassays. The antifungal activity of 1-methoxy-
spirobrassinin (3) and erucalexin (4) was determined using the
following mycelia radial growth bioassay. Solutions of each
compound in DMSO (5 × 10−2 M) were used to prepare assay
solutions in minimal media (5 × 10−4 M, 2.5 × 10−4 M, 5 ×
10−5 M) in serial dilution; control solutions contained 1% DMSO
in minimal media. Sterile tissue culture plates (12-well, 24 mm
diameter) containing test solutions and the control solution (1 ml
per well) were inoculated with mycelia plugs placed upside down
on the centre of each plate (5 mm cut from three-day-old and
seven-day-old PDA plates of S. sclerotiorum clone #33 and R.
solani AG 2-1, respectively) and incubated under constant light
for seven days. The radial growth of mycelia the was measured
with a ruler daily for one week. Three independent experiments
were carried out, each one in triplicate.
81%) as a slightly yellow solid, mp 130–132 ◦C. HPLC: tR
=
16.8 min. dH (500 MHz, CD3CN): 7.50 (m, 2H), 7.27 (dd, J =
7, 7 Hz, 1H), 7.18 (d, J = 7 Hz, 1H), 4.27 (d, J = 14 Hz, 1H),
4.15 (d, J = 14 Hz, 1H), 3.56 (s, 3H), 2.55 (s, 3H); dC (125 MHz,
CD3CN): 204.5 (s), 165.4 (s), 146.3 (s), 133.1 (s), 132.4 (d), 126.4
(d), 125.8 (d), 111.9 (d), 92.8 (s), 69.5 (t), 32.4 (q), 15.4 (q). HRMS
(EI): calc. for C12H12N2OS2 (M+) m/z 264.0391, found 264.0391;
MS (EI) m/z (% relative int.): 264 [M+] (58), 217 (81), 190 (35),
175 (100), 89 (14), 71 (20). mmax(KBr)/cm−1: 2926, 1741, 1620,
1466, 1375, 1146. kmax (CH3CN)/nm 238 (log e, 4.1), 287 (3.6),
296 (3.7), 337 (3.8).
( )-1-Methyl-spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (9a).
To a solution of ( )-spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-
one (29) (14 mg, 0.068 mmol) in dry THF (0.5 ml) NaH (60%
in mineral oil, 5 mg, 0.14 mmol) was added and the mixture was
stirred for 5 min at rt, followed by the addition of CH3I (17 ll,
0.27 mmol). The stirring continued for a further 3 h at rt, the
solvent was evaporated and the residue was subjected to FCC
(hexane–acetone, 5 : 1). Evaporation of the eluate afforded ( )-
1-methyl-spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (9a) as a
yellow oil. Yield: 12 mg (80%). HPLC: tR = 19.2 min. dH (500 MHz,
CD3CN): 7.53 (m, 2H), 6.86 (d, J = 8 Hz, 1H), 6.78 (dd, J = 8,
8 Hz, 1H), 3.28 (m, 1H), 3.08 (m, 1H), 3.01 (s, 3H), 2.41 (m, 1H),
Isolation and characterization of (+)-erucalexin (4). Plants (40,
five-week-old) were sprayed with CuCl2 (2 × 10−3 M) solution to
the point of run-off, three times at 24 h intervals and allowed
to stand for three days. Elicited leaves (180 g, fresh weight),
were frozen in liquid nitrogen and crushed with a glass rod, and
extracted with EtOAc in a manner similar to that followed for the
time-course experiment. The EtOAc extract (1.2 g) was subjected
to FCC (gradient elution, CH2Cl2, 100% to CH2Cl2–MeOH, 20 :
80), the fractions containing the HPLC peak at tR = 20.6 min
were combined (284 mg) and further fractionated by reverse phase
FCC (gradient elution, CH3CN–H2O, 20 : 80) to CH3CN, 100%)
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2.31 (m, 1H), 2.14 (m, 2H); dC (125 MHz, CD3CN): 202.5 (s),
161.0 (s), 139.0 (d), 125.8 (d), 119.9 (s), 119.1 (d), 110.3 (d), 86.1
(s), 37.8 (t), 35.7 (t), 32.6 (t), 28.9 (q). HRMS (EI): calc. for
C12H13NOS (M+) m/z 219.0718, found 219.0716; MS (EI) m/z
(% relative int.): 219 [M+] (100), 190 (58), 163 (80), 130 (15), 77
(17). mmax(KBr)/cm−1: 2927, 1707, 1615, 1483, 1369, 1318, 1146,
950, 745. kmax (CH3CN)/nm 235 (log e, 4.3), 264 (4.0), 353 (3.0).
1-Methoxy-2-chloroindole-3-carboxaldehyde (16). Dry DMF
(4 ml) was cooled to 0 ◦C and oxalyl chloride (540 ll, 6.1 mmol) was
added dropwise. 1-Methoxyoxindole (15)29 (400 mg, 2.45 mmol)
was added and the mixture was stirred for 1 h at 75 ◦C. After
cooling to rt, the mixture was poured into 100 ml of water and
extracted with Et2O. The combined organic extract was dried, the
solvent was evaporated and the residue subjected to FCC (hexane–
acetone, 5 : 1). Crystallization from CH2Cl2–hexane afforded
1-methoxy-2-chloroindole-3-carboxaldehyde (16, 365 mg, 71%)
as colorless crystals, mp 82–83 ◦C. HPLC: tR = 17.8 min. dH
(500 MHz, CDCl3): 10.09 (s, 1H), 8.31 (d, J = 8 Hz, 1H), 7.46
(d, J = 8 Hz, 1H), 7.39 (dd, J = 8, 8 Hz, 1H), 7.35 (dd, J = 8,
8 Hz, 1H), 4.21 (s, 3H); dC (125 MHz, CDCl3): 183.7 (s), 132.5
(s), 131.5 (s), 124.9 (d), 124.2 (d), 121.7 (d), 121.0 (s), 109.8 (s),
108.4 (d), 66.5 (q). HRMS (EI): calc. for C10H8ClNO2 (M+) m/z
209.0244, found 209.0243; MS (EI) m/z (% relative int.): 209 [M+]
(100), 166 (41), 114 (16). mmax(KBr)/cm−1: 2952, 1656, 1507, 1386,
1243, 1167, 1043, 952, 741.
( )-Spiro[1-methyl-3,5ꢀ-oxazolidin]-2-one-2ꢀ-thione (13). Et2NH
(21 ll, 0.20 mmol) was added to 1-methylisatin (8, 322 mg,
2.0 mmol) suspended in a solution of CH3NO2 11 (325 ll, 5.8 mmol)
and EtOH (1 ml) at 0 ◦C. The reaction mixture was kept at 0 ◦C for
10 min, and the solvent was removed under reduced pressure. The
residue was dissolved in a solution of MeOH (6 ml) and acetic
acid (0.5 ml), 10% Pd/C (50 mg) was added and mechanically
agitated at 3 atm of H2 for 16 h. The reaction mixture was
filtered, the solvent evaporated and the residue subjected to FCC
(CH2Cl2–MeOH–30% aqueous NH3, 80 : 20 : 1) to yield ( )-12
(304 mg, 79%) as a colorless oil. A solution of amine 12 (50 mg,
0.26 mmol, in CH2Cl2, 2 ml) was added dropwise (over a 10 min
period) to a vigorously stirred mixture of 5% Na2CO3 and CSCl2
(22 ll, 0.29 mmol in CH2Cl2, 4 ml) at rt. After 2 h, the aqueous
layer was separated, extracted with CH2Cl2, the organic phases
combined and, after removal of the solvent, the crude product was
crystallized using acetone–hexane to yield ( )-spi◦rooxazolidine 13
Baeyer–Villiger oxidation of aldehydes 16 and 19. m-CPBA
(43 mg, 0.25 mmol) was added to separate solutions of aldehydes
16 and 19 (0.125 mmol) in CH2Cl2 (1 ml), and the mixtures were
stirred for 2 h at rt. Unstable O-formate 18 was separated by
FCC (hexane–Et2O, 10 : 1) to yield 12 mg (43%) of 1-methoxy-2-
chloroindole-3-yl formate (18) containing a small amount (10%) of
1-methoxyisatin (17). When aldehyde 19 was used, 1-methylisatin
(8) was isolated as the sole product (12 mg, 60%). HCl (one
drop of 1 M) was added to a solution of O-formate 18 (17 mg,
0.08 mmol) in MeOH (1 ml) and the mixture was stirred for 1 h
at rt. The solvent was evaporated and the residue was subjected
to FCC (CH2Cl2). Evaporation of the eluate afforded 7 mg
(52%) of 1-methoxyisatin (17) as a red solid, mp 105–107 ◦C
(lit.16 110–113 ◦C).
To a solution of 1-methoxyoxindole (15, 41 mg, 0.25 mmol)
in acetic acid (2 ml), a solution of CrO3 (125 mg, 1.25 mmol) in
water (0.5 ml) was added and the mixture was stirred at rt. After
1 h the reaction mixture was diluted with brine (20 ml), extracted
with EtOAc, and the combined extract was washed with 10%
K2CO3 solution (20 ml), dried and concentrated. The residue was
subjected to FCC (CH2Cl2–MeOH, 98 : 2) to yield 1-methoxyisatin
(17, 33 mg, 75%). HPLC: tR = 8.2 min. dH (500 MHz, CD3CN):
7.70 (dd, J = 7, 7 Hz, 1H), 7.58 (d, J = 7 Hz, 1H), 7.20 (dd,
J = 7, 7 Hz, 1H), 7.15 (d, J = 7 Hz, 1H), 4.04 (s, 3H); dC
(125 MHz, CD3CN): 182.5 (s), 155.0 (s), 149.0 (s), 139.9 (d), 125.7
(d), 125.5 (d), 118.1 (s), 110.3 (d), 65.2 (q). HRMS (EI): calc.
for C9H7NO3 (M+) m/z 177.0426, found 177.0428; MS (EI) m/z
(% relative int.): 177 [M+] (15), 149 (85), 104 (23), 91 (100), 78
(47), 63 (13). mmax(KBr)/cm−1: 2948, 1743, 1614, 1460, 1315, 1191,
1043, 935, 754.
(46 mg, 76%) as colorless crystals, mp 180–182 C. HPLC: tR
=
6.8 min. dH (500 MHz, CD3CN): 8.04 (br s, D2O exchange, 1H),
7.54 (d, J = 7.5 Hz, 1H), 7.49 (ddd, J = 7.5, 7.5, 1 Hz, 1H), 7.18
(ddd, J = 7.5, 7.5, 1 Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H), 4.01 (d,
J = 11 Hz, 1H), 3.96 (d, J = 11 Hz, 1H), 3.17 (s, 3H); dC (125 MHz,
CD3CN): 189.8 (s), 173.3 (s), 146.1 (s), 133.3 (d), 126.5 (s), 126.1
(d), 124.8 (d), 110.7 (d), 86.1 (s), 52.2 (t), 27.4 (q). HRMS (EI):
calc. for C11H10N2O2S (M+) m/z 234.0463, found 234.0463; MS
(EI) m/z (% relative int.): 234 [M+] (44), 178 (58), 162 (100), 77
(12). mmax(KBr)/cm−1: 3300, 2925, 1730, 1616, 1524, 1471, 1376,
1241, 1165, 983, 755. kmax (CH3CN)/nm 211 (log e, 4.0), 249 (3.5),
296 (2.9).
( )-Spiro[1-methylindolin-3,5ꢀ-oxazolidine]-2,2ꢀ-dithione (14).
To a solution of ( )-spirooxazoline 13 (23 mg, 0.1 mmol) in
dry THF (4 ml) were added P4S10 (222 mg, 0.50 mmol)13 and
NaHCO3 (84 mg, 1.0 mmol) and the reaction mixture was stirred
for 24 h under reflux (condenser equipped with a CaCl2 trap).
The solvent was evaporated, the residue was dissolved in water
(20 ml) and extracted with CH2Cl2. The combined organic
extract was dried, concentrated and the residue was subjected
to FCC (hexane–EtOAc, 2 : 1) to yield starting material (5 mg,
22%) and dithione 14 (4 mg, 16%) as a slightly yellow solid, mp
155–157 ◦C. HPLC: tR = 12.8 min. dH (500 MHz, CD3CN): 7.98
(br s, D2O exchange, 1H), 7.63 (d, J = 7 Hz, 1H), 7.54 (ddd, J =
7, 7, 1 Hz, 1H), 7.30 (ddd, J = 7, 7, 1 Hz, 1H), 7.20 (d, J = 7 Hz,
1H), 4.09 (d, J = 11 Hz, 1H), 4.01 (d, J = 11 Hz, 1H), 3.56 (s,
3H); dC (125 MHz, CD3CN): 201.9 (s), 189.2 (s), 146.6 (s), 133.2
(d), 131.4 (s), 126.6 (d), 126.5 (d), 112.1 (d), 92.6 (s), 55.5 (t),
32.6 (q). HRMS (EI): calc. for C11H10N2OS2 (M+) m/z 250.0235,
found 250.0231; MS (EI) m/z (% relative int.): 250 [M+] (10), 217
(50), 175 (40), 97 (50). mmax(KBr)/cm−1: 3267, 2925, 1701, 1614,
1527, 1467, 1376, 1164, 1098, 752. kmax (CH3CN)/nm 243 (log e,
3.9), 287 (3.4), 337 (3.5).
Baeyer–Villiger oxidation of 1-methoxyindole-3-carboxaldehyde
(20). m-CPBA (142 mg, 0.83 mmol) was added to a solution
of 1-methoxyindole-3-carboxaldehyde (20,18 44 mg, 0.25 mmol)
in CH2Cl2 (2 ml) and the mixture was stirred at rt for 16 h.
The reaction mixture was diluted with CH2Cl2 (20 ml), washed
with 10% K2CO3 solution (10 ml), the aqueous layer was re-
extracted with CH2Cl2, and the combined organic extract was
dried and concentrated. After chromatography of the residue
(hexane–acetone, 5 : 1), the product was dissolved in dioxane
(1.5 ml), HCl (1 M, 0.6 ml) was added, and the reaction mixture
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was stirred at rt. After 16 h, the reaction mixture was diluted
with brine (20 ml), extracted with EtOAc, and the combined
organic extract was dried and concentrated. The residue was
subjected to FCC (hexane–acetone, 2 : 1). Evaporation of the
eluate afforded ( )-1-methoxy-2-hydroxyindolin-3-one (21) as a
C16H20ClNO3S (M+) m/z 341.0852, found 341.0843; MS (EI) m/z
(% relative int.): 341 [M+] (14), 286 (15), 268 (60), 233 (26), 177
(37), 132 (39). mmax(KBr)/cm−1: 2984, 1716, 1607, 1468, 1367, 1279,
1157, 1060, 757.
( )-1-Boc-2-Ethylsulfanyl-2-methylindolin-3-one (26). To
a
colorless oil (18 mg, 40%, based on aldehyde 20). HPLC: tR
=
solution of 1-Boc-2-ethylsulfanylindolin-3-one (25, 18 mg,
0.037 mmol) NaH, (60% in mineral oil, 6 mg, 0.15 mmol) in dry
THF (0.5 ml) was added and the mixture was stirred for 10 min at
rt followed by the addition of CH3I (10 ll, 0.15 mmol). The stirring
continued for 1 h at rt, the solvent was evaporated and the residue
subjected to FCC (hexane–Et2O, 10 : 1). Evaporation of the eluate
afforded ( )-1-Boc-2-ethylsulfanyl-2-methylindolin-3-one (26) as
a colorless oil. Yield: 10 mg (87%). HPLC: tR = 32.8 min. dH
(500 MHz, CDCl3): 8.21 (d, J = 8 Hz, 1H), 7.79 (d, J = 8 Hz,
1H), 7.66 (dd, J = 8, 8 Hz, 1H), 7.17 (dd, J = 8, 8 Hz, 1H), 2.38
(m, 2H), 1.84 (s, 3H), 1.65 (s, 9H), 1.07 (t, J = 7 Hz, 3H); dC
(125 MHz, CDCl3): 197.9 (s), 152.3 (s), 151.0 (s), 137.9 (d), 124.7
(d), 123.7 (d), 122.3 (s), 117.0 (d), 83.5 (s), 72.9 (s), 28.7 (q), 23.7
(3 × q), 23.4 (t), 14.4 (q). HRMS (EI): calc. for C16H21NO3S (M+)
m/z 307.1242, found 307.1242; MS (EI) m/z (% relative int.): 307
[M+] (100), 292 (55), 262 (29). mmax(KBr)/cm−1: 2935, 1712, 1608,
1465, 1350, 1259, 1162, 963, 754.
5.8 min. dH (500 MHz, CDCl3): 7.67 (dd, J = 8, 8 Hz, 1H), 7.64
(d, J = 8 Hz, 1H), 7.31 (d, J = 8 Hz, 1H), 7.17 (dd, J = 8, 8 Hz,
1H), 5.15 (s, 1 H), 4.10 (s, 3H), 3.27 (br s, D2O exchange, 1H); dC
(125 MHz, CDCl3): 194.5 (s), 159.0 (s), 137.9 (d), 124.2 (d), 124.0
(d), 120.9 (s), 115.4 (d), 93.4 (d), 64.9 (q). HRMS (EI): calc. for
C9H9NO3 (M+) m/z 179.0582, found 179.0586; MS (EI) m/z (%
relative int.): 179 [M+] (27), 148 (100), 130 (10), 92 (24), 77 (21),
65 (37). mmax(KBr)/cm−1: 3355, 2935, 1726, 1612, 1461, 1208, 1150,
984, 811, 760.
When the acid-catalyzed (1 M HCl, 0.3 ml) hydrolysis was
carried out in MeOH (2 ml) instead of dioxane (2 h at rt), followed
by the work-up described above, ( )-1,2-dimethoxyindolin-3-one
(22) was obtained (colorless oil, 18 mg, 37%, based on aldehyde
20). HPLC: tR = 13.6 min. dH (500 MHz, CDCl3): 7.66 (m, 2H),
7.33 (d, J = 8 Hz, 1H), 7.15 (dd, J = 8, 8 Hz, 1H), 5.02 (s, 1 H),
4.08 (s, 3H), 3.52 (s, 3H); dC (125 MHz, CDCl3): 193.2 (s), 159.5
(s), 137.8 (d), 124.1 (d), 123.8 (d), 122.0 (s), 115.4 (d), 98.0 (d),
64.5 (q), 54.2 (q). HRMS (EI): calc. for C10H11NO3 (M+) m/z
193.0739, found 193.0741; MS (EI) m/z (% relative int.): 193 [M+]
(20), 162 (100), 146 (43), 130 (98), 92 (15). mmax(KBr)/cm−1: 2941,
1729, 1610, 1463, 1216, 1146, 1032, 763.
( )-1-Boc-Spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (28).
1-Boc-2-(3-Chloropropyl)sulfanylindolin-3-one (27, 50 mg,
0.15 mmol) was dissolved in dry DMF (1 ml) and NaH (60%
in mineral oil, 20 mg, 0.58 mmol) was added. The mixture was
stirred for 15 min at 60 ◦C, before being cooled to 0 ◦C and diluted
with brine (20 ml). The product was extracted with EtOAc, and the
combined extract was washed with brine (2 × 25 ml), then dried
and concentrated. The residue was subjected to FCC (hexane–
Et2O, 10 : 1). Evaporation of the eluate afforded ( )-1-Boc-
spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (28) as a colorless
Reaction of O-formate 24 with EtSH and MeSNa. To a
solution of aldehyde 2319 (70 mg, 0.25 mmol) in CH2Cl2 (2 ml),
m-CPBA (86 mg, 0.5 mmol) was added and the mixture was
stirred for 2 h at rt. O-Formate 24 was separated by FCC (hexane–
EtOAc, 8 : 1), affording 68 mg (90%) of 1-Boc-2-chloroindole-3-yl
formate (24). O-Formate 24 (68 mg, 0.24 mmol) was dissolved in
THF (2 ml), EtSH (72 ll, 0.96 mmol) or 3-chloro-1-propanethiol
(45 ll, 0.46 mmol) were added, followed by addition of Et3N
(130 ll, 0.92 mmol). The mixtures were stirred for 2 h at rt, before
being concentrated and the residues subjected to FCC (hexane–
Et2O, 10 : 1). Evaporation of the eluates afforded compounds
( )-25 and ( )-27, respectively, as colorless oils. ( )-1-Boc-2-
Ethylsulfanylindolin-3-one (25). Yield 40 mg, 59% (based on
aldehyde 23). HPLC: tR = 29.7 min. dH (500 MHz, CDCl3): 8.09
(br s, 1H), 7.75 (d, J = 8 Hz, 1H), 7.65 (dd, J = 8, 8 Hz, 1H), 7.17
(dd, J = 8, 8 Hz, 1H), 5.15 (s, 1H), 2.68 (m, 1H), 2.59 (m, 1H), 1.63
(s, 9H), 1.22 (t, J = 7 Hz, 3H); dC (125 MHz, CDCl3): 195.4 (s),
152.6 (s), 150.5 (s), 137.6 (d), 124.5 (d), 123.6 (d), 123.5 (s), 116.7
(d), 83.4 (s), 65.3 (d), 28.5 (3 × q), 23.5 (t), 14.8 (q). HRMS (EI):
calc. for C15H18NO3S (M − H+) m/z 292.1007, found 292.1011;
MS (EI) m/z (% relative int.): 292 [M − H+] (6), 233 (88), 193 (26),
177 (100), 148 (16), 132 (94), 92 (26). mmax(KBr)/cm−1: 2978, 1714,
1604, 1467, 1681, 1365, 1280, 1153, 754.
◦
solid. Yield: 40 mg (89%); mp 86–88 C. HPLC: tR = 31.3 min.
dH (500 MHz, CDCl3): 8.09 (br s, 1H), 7.77 (d, J = 8 Hz, 1H),
7.64 (dd, J = 8, 8 Hz, 1H), 7.16 (dd, J = 8, 8 Hz, 1H), 3.44
(m, 1H), 3.21 (m, 1H), 2.70 (m, 1H), 2.53 (m, 2H), 2.23 (m, 1H),
1.66 (s, 9H); dC (125 MHz, CDCl3): 200.3 (s), 152.5 (s), 150.6 (s),
137.4 (d), 124.9 (d), 123.5 (d), 121.4 (s), 117.2 (d), 83.5 (s), 82.0
(s), 37.7 (t), 36.0 (t), 32.5 (t), 28.8 (3 × q). HRMS (EI): calc. for
C16H19NO3S (M+) m/z 305.1086, found 305.1084; MS (EI) m/z
(% relative int.): 305 [M+] (79), 249 (41), 232 (11), 205 (100), 177
(23), 149 (82). mmax(KBr)/cm−1: 2930, 1714, 1610, 1463, 1353, 1253,
1156, 979, 756.
( )-Spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (29). 1-Boc-
Spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (28, 28 mg,
0.092 mmol) was dissolved in CH2Cl2 (600 ll) and TFA (140 ll,
1.83 mmol) was added dropwise. The mixture was stirred for
1 h at rt, the solvent was evaporated and the residue subjected
to FCC (hexane–acetone, 5 : 1). Evaporation of the eluate
afforded ( )-spiro(indoline-2,2ꢀ-tetrahydrothiophene)-3-one (29)
as a yellow oil. Yield: 15 mg (80%). HPLC: tR = 12.3 min. dH
(500 MHz, CDCl3): 7.66 (d, J = 8 Hz, 1H), 7.48 (dd, J = 8, 8 Hz,
1H), 6.89 (dd, J = 8, 8 Hz, 1H), 6.83 (d, J = 8 Hz, 1H), 4.82 (br s,
D2O exchange, 1H), 3.26 (m, 1H), 3.19 (m, 1H), 2.51 (m, 1H),
2.25 (m, 1H), 2.15 (m, 2H); dC (125 MHz, CDCl3): 201.3 (s), 159.3
(s), 137.7 (d), 125.4 (d), 120.2 (s), 120.0 (d), 112.6 (d), 80.3 (s),
42.0 (t), 35.0 (t), 31.2 (t). HRMS (EI): calc. for C11H11NOS (M+)
( )-1-Boc-2-(3-Chloropropyl)sulfanylindolin-3-one (27). Yield
52 mg, 61% (based on aldehyde 23). HPLC: tR = 32.2 min. dH
(500 MHz, CDCl3): 8.06 (br s, 1H), 7.75 (d, J = 8 Hz, 1H), 7.66
(dd, J = 8, 8 Hz, 1H), 7.18 (dd, J = 8, 8 Hz, 1H), 5.13 (s, 1H),
3.62 (m, 2H), 2.85 (m, 1H), 2.76 (m, 1H), 2.00 (m, 2H), 1.63 (s,
9H); dC (125 MHz, CDCl3): 195.2 (s), 152.5 (s), 150.5 (s), 137.8
(d), 124.6 (d), 123.7 (d), 123.4 (s), 116.9 (d), 83.6 (s), 65.2 (d),
43.3 (t), 32.5 (t), 28.5 (3 × q), 26.7 (t). HRMS (EI): calc. for
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m/z 205.0561, found 205.0560; MS (EI) m/z (% relative int.): 205
[M+] (75), 176 (25), 130 (10). mmax(KBr)/cm−1: 3333, 2930, 1690,
1617, 1467, 1321, 1061, 894, 752.
was evaporated and the crude amine was used directly in the next
step.◦To a solution of this amine dissolved in pyridine (1 ml) cooled
to 0 C, CS2 (30 ll, 0.5 mmol) and Et3N (70 ll, 0.5 mmol) were
added. After 1 h at 0 ◦C, CH3I (30 ll, 0.5 mmol) was added and the
mixture was kept at 3 ◦C for 16 h. The reaction mixture was then
diluted with 1.5 M H2SO4 (15 ml) and extracted with Et2O. The
combined organic extract was dried, the solvent was evaporated
and the residue was subjected to FCC (hexane–EtOAc, 8 : 1) to
yield 1-methoxyisobrassinin (38, 55 mg, 41%, slightly yellow oil).
HPLC: tR = 24.4 min. dH (500 MHz, CDCl3): 7.57 (d, J = 8 Hz,
1H), 7.44 (d, J = 8 Hz, 1H), 7.28 (dd, J = 8, 8 Hz, 1H), 7.15 (m,
D2O exchange, 2H), 6.39 (s, 1 H), 5.15 (d, J = 4 Hz, 2H), 4.12 (s,
3H), 2.68 (s, 3H); dC (125 MHz, CDCl3): 199.4 (s), 133.1 (d), 131.3
(s), 123.7 (s), 123.1 (s), 121.4 (d), 120.8 (d), 108.5 (d), 98.9 (d), 65.9
(q), 42.3 (t), 18.5 (q). HRMS (EI): calc. for C12H14N2OS2 (M+)
m/z 266.0548, found 266.0550; MS (EI) m/z (% relative int.): 266
[M+] (10), 235 (100), 187 (35), 160 (62), 129 (83). mmax(KBr)/cm−1:
3326, 2935, 1502, 1306, 1084, 917, 745.
CrO3-mediated oxidation of 1-methoxybrassinin (30) and
brassinin (33). To a solution of 1-methoxybrassinin25 (30, 16 mg,
0.06 mmol) in acetic acid (0.5 ml), a solution of CrO3 (30 mg,
0.3 mmol) in water (0.2 ml) was added in one portion at rt.
The mixture was stirred at rt for 20 min, was diluted with brine
(10 ml) and then extracted (EtOAc). The combined organic extract
was washed with 10% K2CO3 solution (10 ml) and was dried. The
solvent was concentrated and the residue subjected to preparative
TLC (multiple development, hexane–EtOAc, 4 : 1) to yield ( )-
1-methoxyspirobrassinin (3), 4 mg (24%); when the reaction
◦
mixture was stirred at 0 C, ( )-1-methoxyspirobrassinol (35)30
was obtained, 6 mg (35%) instead of ( )-3.
To a solution of 1-methoxybrassinin (30, 19 mg, 0.07 mmol)
in CH2Cl2 (1 ml), PCC (108 mg, 0.5 mmol) was added and
the reaction mixture was stirred for 2 h at rt. The reaction
mixture was diluted with CH2Cl2 (10 ml), silica gel was added
and the solvent was evaporated. The residue was subjected to
FCC (hexane–EtOAc, 4 : 1). Evaporation of the eluate afforded
( )-1-methoxyspirobrassinin (3),9 8 mg (38%).
9-Methoxy-3-methylsulfanyl-1,3-thiazino[5,6-b]indole (39).
A
solution of dioxane dibromide (150 ll, 0.11 mmol, prepared
by dissolving 40 ll of Br2 in 1 ml dioxane) was added to 1-
methoxyisobrassinin (38, 25 mg, 0.09 mmol) dissolved in dioxane
(1 ml), and the mixture was stirred for 30 min at rt. The reaction
mixture was diluted with brine (10 ml) and then extracted with
EtOAc. The combined organic extract was dried, the solvent was
evaporated and the residue was subjected to FCC (hexane–EtOAc,
8 : 1) to afford 39 (22 mg, 88%) as an orange solid, mp 45–
47 ◦C. HPLC: tR = 31.7 min. dH (500 MHz, CDCl3): 7.45 (d,
J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H), 7.28 (dd, J = 8, 8 Hz,
1H), 7.16 (dd, J = 8, 8 Hz, 1H), 5.08 (s, 2H), 4.08 (s, 3H), 2.57
(s, 3H); dC (125 MHz, CDCl3): 165.0 (s), 133.5 (s), 124.1 (s), 123.3
(d), 121.0 (d + s), 118.3 (d), 109.1 (d), 95.8 (s), 66.1 (q), 46.6 (t),
15.8 (q). HRMS (EI): calc. for C12H12N2OS2 (M+) m/z 264.0391,
found 264.0392; MS (EI) m/z (% relative int.): 264 [M+] (32), 191
(100), 176 (22), 160 (53), 120 (32), 89 (12). mmax(KBr)/cm−1: 2935,
1599, 1447, 1301, 1232, 1134, 948, 740.
To a solution of brassinin2 (33, 19 mg, 0.07 mmol) in acetic
acid (0.7 ml) and dioxane (0.3 ml), a solution of CrO3 (50 mg,
0.5 mmol) in water (0.9 ml) was added in one portion at rt. The
mixture was stirred at rt for 30 min, before being diluted with brine
(20 ml) and extracted (EtOAc). The combined organic extract was
washed with 10% K2CO3 solution (10 ml) and dried. The solvent
was concentrated and the residue was subjected to FCC (hexane–
acetone, 5 : 1) to yield ( )-spirobrassinin (34),12 10 mg (40%).
1-Methoxyisobrassinin (38). t-BuLi in pentane (3.7 ml,
4.8 mmol) was added dropwise to a solution of 1-methoxyindole23
(36, 589 mg, 4 mmol) in dry Et2O (10 ml, under an Ar atmosphere),
cooled to −20 ◦C (ice–NaCl).24 The mixture was stirred for 15 min
at −20 ◦C, DMF (0.75 ml, 4.8 mmol) was added, the cooling
bath was removed and the stirring continued at rt for 1 h. The
reaction mixture was cooled to 0 ◦C, diluted with 1 M HCl
(30 ml) and extracted with Et2O. The combined organic extract
was dried, the solvent evaporated and the residue was subjected
to FCC (hexane–acetone, 5 : 1) to afford 1-methoxyindole-2-
carboxaldehyde (568 mg, 81%) as a slightly yellow oil. A solution
of NH2OH·HCl (360 mg, 5.18 mmol) and Na2CO3 (247 mg,
2.33 mmol) in water (3 ml) was added to 1-methoxyindole-2-
carboxaldehyde (568 mg, 3.24 mmol) in EtOH (12 ml). The
mixture was refluxed for 20 min, before being concentrated, diluted
with brine (30 ml) and extracted (EtOAc). The combined organic
extract was dried and the solvent was evaporated to yield crude
1-methoxyindole-2-carboxaldehyde oxime (37, 567 mg, 92%) of
sufficient purity to be used in the next step. AcONH4 (424 mg,
5.5 mmol) and NaBH3CN (314 mg, 5 mmol) were added to a
cooled solution of 1-methoxyindole-2-carboxaldehyde oxime (37,
95 mg, 0.5 mmol) in MeOH (3 ml) at 0 ◦C followed by the addition
of a solution of TiCl3 [prepared by mixing commercially available
30% TiCl3 in 2 M HCl (1.55 ml), with 1 M NaOH solution (1.1 ml)]
in one portion, and the mixture was stirred for 20 min at 0 ◦C. The
reaction mixture was then diluted with aqueous NH4OH (prepared
by mixing 1.3 ml 30% NH4OH with 33 ml water) and extracted
with EtOAc. The combined organic extract was dried, the solvent
9-Methoxy-3-methylsulfanyl-1,3-thiazino[5,6-b]indol-1-one (40).
To a solution of 39 (9 mg, 0.034 mmol) in CH2Cl2 (0.5 ml) was
added NBS (7 mg, 0.041 mmol) or NCS (6 mg, 0.041 mmol). The
mixtures were stirred at rt as follows: NBS (30 min), NCS (60 min).
The solvent was evaporated and the residues were subjected to
preparative TLC (hexane–acetone, 5 : 1) to afford 40 in the
following amounts: NBS (2 mg, 21%), NCS (3 mg, 32%), brownish
solid, mp 143–146 ◦C. HPLC: tR = 22.0 min. dH (500 MHz,
CDCl3): 7.65 (m, 2H), 7.57 (dd, J = 8, 8 Hz, 1H), 7.29 (dd, J = 8,
8 Hz, 1H), 4.31 (s, 3H), 2.80 (s, 3H); dC (125 MHz, CDCl3): 173.4
(s), 158.9 (s), 135.3 (s), 128.7 (d), 122.2 (d), 120.5 (d), 119.2 (s),
118.1 (s), 113.9 (s), 110.4 (d) 67.1 (q), 15.4 (q). HRMS (EI): calc. for
C12H12N2O2S2 (M+) m/z 278.0184, found 278.0187; MS (EI) m/z
(% relative int.): 278 [M+] (46), 205 (24), 174 (100), 120 (10), 102
(15). mmax(KBr)/cm−1: 2942, 1662, 1615, 1519, 1340, 1216, 941, 744.
Synthesis of isobrassinin (43) and 1-Boc-isobrassinin (44). To
a solution of indole-2-carboxaldehyde (41)27 or 1-Boc-indole-2-
carboxaldehyde (42, 3.4 mmol) in EtOH (10 ml), a solution
of NH2OH·HCl (720 mg, 10.4 mmol) and Na2CO3 (660 mg,
6.2 mmol) in water (5 ml) was added. The mixtures were
refluxed with stirring for 30 min, before benig concentrated and
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extracted (EtOAc). The combined organic extracts were dried and
concentrated to afford corresponding oximes in 99% yield. Oximes
(3.3 mmol) and NiCl2·6H2O (778 mg, 3.3 mmol) were dissolved
in MeOH (50 ml). NaBH4 (804 mg, 21.3 mmol) was added and
the mixtures were stirred for 5 min at rt. The black precipitates
were filtered off, the solutions were concentrated (ca. 25%) and
then poured into water (100 ml) containing 30% NH4OH (4 ml).
The mixtures were extracted with EtOAc, the combined extracts
were dried and then concentrated. Obtained unstable indolyl-2-
methyl amines (orange oils) were dissolved in pyridine (2 ml) and
the solutions were cooled to 0 ◦C. Et3N (1 ml, 7.13 mmol) and
CS2 (0.97 ml, 16.12 mmol) were added and the mixtures were set
aside for 1 h at 3 ◦C, followed by addition of CH3I (0.69 ml,
(3 × q), 15.7 (q). HRMS (EI): calc. for C16H18N2O3S2 (M+) m/z
350.0759, found 350.0756; MS (EI) m/z (% relative int.): 350 [M+]
(16), 294 (14), 250 (14), 177 (41), 57 (100). mmax(KBr)/cm−1: 2927,
1719, 1600, 1464, 1352, 1303, 1253, 1156, 1089, 947, 755.
( )-Demethoxyerucalexin (46). 1-Boc-Erucalexin (45, 17 mg,
0.049 mmol) was dissolved in CH2Cl2 (0.5 ml) and TFA (110 ll,
1.46 mmol) was added. The mixture was stirred for 2 h at rt, before
being concentrated and subjected to FCC (hexane–acetone, 5 : 1).
Evaporation of the eluate afforded ( )-demethoxyerucalexin (46,
12 mg, 91%) as an orange solid, mp 128–130 ◦C. HPLC: tR
=
14.6 min. dH (500 MHz, CD3CN): 7.56 (m, 2H), 6.90 (m, 2H), 6.60
(br s, D2O exchange, 1H), 4.42 (d, J = 16 Hz, 1H), 4.33 (d, J =
16 Hz, 1H), 2.60 (s, 3H); dC (125 MHz, CD3CN): 198.7 (s), 163.2
(s), 160.6 (s), 139.7 (d), 125.9 (d), 121.0 (d), 120.0 (s), 113.6 (d),
86.6 (s), 75.3 (t), 16.2 (q). HRMS (EI): calc. for C11H10N2OS2 (M+)
m/z 250.0235, found 250.0236; MS (EI) m/z (% relative int.): 250
[M+] (84), 235 (13), 203 (38), 177 (100), 145 (32), 117 (17), 87 (44).
mmax(KBr)/cm−1: 3310, 2917, 1699, 1614, 1484, 1319, 1078, 938,
888, 748.
◦
11.16 mmol). After 16 h at 3 C the mixtures were diluted with
1.5 M H2SO4 (30 ml) and then extracted with Et2O. The combined
organic extracts were dried and then concentrated. The residues
were subjected to FCC as follows: 43 (hexane–EtOAc, 2 : 1); 44
(hexane–Et2O, 5 : 1). Evaporation of the eluates afforded residues
that were crystallized from CH2Cl2–hexane solution. Isobrassinin
(43, 234 mg, 30%, based on the aldehyde 41) was obtained as
slightly orange crystals, mp 82–84 ◦C. 1-Boc-Isobrassinin (44,
248 mg, 24%, based on the aldehyde 42) was obtained as slightly
orange crystals, mp 88–90 ◦C.
Isobrassinin (43), HPLC: tR = 21.3 min. dH (500 MHz, CDCl3):
8.96 (br s, D2O exchange, 1H), 7.58 (d, J = 8 Hz, 1H), 7.37 (m,
D2O exchange, 2H), 7.21 (dd, J = 8, 8 Hz, 1H), 7.12 (dd, J = 8,
8 Hz, 1H), 6.43 (s, 1H), 5.10 (d, J = 5.5 Hz, 1H), 2.69 (s, 3H);
dC (125 MHz, CDCl3): 201.4 (s), 136.5 (d), 134.6 (s), 127.7 (s),
122.7 (d), 120.7 (d), 120.2 (d), 111.4 (d), 102.3 (s), 44.0 (t), 18.7
(q). HRMS (EI): calc. for C11H12N2S2 (M+) m/z 236.0442, found
236.0445; MS (EI) m/z (% relative int.): 236 [M+] (20), 188 (21),
163 (13), 130 (100). mmax(KBr)/cm−1: 3386, 3310, 2917, 1498, 1294,
1068, 919, 749, 634.
1-Boc-Isobrassinin (44), HPLC: tR = 35.4 min. dH (500 MHz,
CDCl3): 8.11 (br s, D2O exchange, 1H), 8.00 (d, J = 8.5 Hz, 1H),
7.53 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 8.5, 8.5 Hz, 1H), 7.24
(dd, J = 8.5, 8.5 Hz, 1H), 6.78 (s, 1H), 5.28 (d, J = 6 Hz, 1H),
2.62 (s, 3H), 1.75 (s, 9H); dC (125 MHz, CDCl3): 198.6 (s), 151.5
(s), 136.1 (s), 134.8 (s), 129.0 (s), 124.8 (d), 123.4 (d), 121.3 (d),
115.8 (d), 112.2 (d), 85.4 (s), 44.6 (t), 28.5 (3 × q), 18.2 (q). HRMS
(EI): calc. for C16H20N2O2S2 (M+) m/z 336.0966, found 336.0975;
MS (EI) m/z (% relative int.): 336 [M+] (20), 280 (16), 189 (12),
163 (23), 130 (100). mmax(KBr)/cm−1: 3369, 2974, 1725, 1481, 1452,
1371, 1331, 1156, 1119, 1078, 923, 747.
Acknowledgements
Financial support from the Natural Sciences and Engineering
Research Council of Canada (Discovery Grant to M. S. C. P.) is
gratefully acknowledged. We thank D. Kessler and R. K. Gugel,
Plant Gene Resources Canada, Saskatoon, SK, for providing
seeds of E. gallicum and isolates of R. solani, and C. Lefol,
Agriculture and Agri Food Canada, Saskatoon, SK, for sclerotia
of S. sclerotiorum.
References and notes
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Glasgow, 1982, pp. 1–334.
2 For recent reviews of crucifer phytoalexins, see: M. S. C. Pedras, F. I.
Okanga, I. L. Zaharia and A. Q. Khan, Phytochemistry, 2000, 53, 161;
M. S. C. Pedras, M. Jha and P. W. K. Ahiahonu, Curr. Org. Chem.,
2003, 7, 1635.
3 M. Pilatova, M. Sarissky, P. Kutschy, A. Mirossay, R. Mezencev, Z.
Curillova, M. Suchy, K. Monde, L. Mirossay and J. Mojzis, Leukemia
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4 ‘The Arabidopsis Genome Initiative’, Nature (London), 2000, 408, 796.
5 S. I. Warwick and D. A. Wall, Can. J. Plant Sci., 1998, 78, 155.
6 E. Lefol, G. Se´guin-Swartz and R. A. A. Morrall, Can. J. Plant Pathol.,
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7 Y. Kohli, L. J. Brunner, H. Yoell, M. G. Milgroom, J. B. Anderson,
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13 J. W. Scheeren, P. H. J. Ooms and R. J. F. Nivard, Synthesis, 1973, 149.
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CrO3-mediated oxidation of 1-Boc-isobrassinin (44). To a
stirred suspension of 1-Boc-brassinin (44, 67 mg, 0.2 mmol) in
acetic acid (1 ml), a solution of CrO3 in water (0.5 ml) was
added at rt. The mixture was stirred at rt for 18 h, before being
diluted with brine (20 ml) and then extracted with EtOAc. The
combined organic extract was dried, concentrated, and the residue
was subjected to FCC (hexane–EtOAc, 4 : 1). Evaporation of the
eluate afforded ( )-1-Boc-erucalexin (45, 18 mg, 26%) as a yellow
oil. HPLC: tR = 30.1 min. dH (500 MHz, CDCl3): 8.27 (d, J =
8 Hz, 1H), 7.77 (d, J = 8 Hz, 1H), 7.69 (ddd, J = 8, 8, 1 Hz,
1H), 7.20 (ddd, J = 8, 8, 1 Hz, 1H), 4.71 (d, J = 16 Hz, 1H),
4.62 (d, J = 16 Hz, 1H), 2.58 (s, 3H), 1.59 (s, 9H); dC (125 MHz,
CDCl3): 196.2 (s), 171.4 (s), 152.8 (s), 150.4 (s), 138.4 (d), 124.9
(d), 124.2 (d), 121.2 (s), 117.1 (d), 85.7 (s), 84.4 (s), 74.7 (t), 28.2
15 A. S. Bourlot, E. Desarbre and J. Y. Merour, Synthesis, 1994, 411.
16 1-Methoxyisatin (17) could also be prepared by oxidation of 15 with
CrO3; a previous synthesis of 17 from 2-nitrobenzoyl chloride was less
efficient, see: H. Tomioka, N. Ichikawa and K. Komatsu, J. Am. Chem.
Soc., 1993, 115, 8621.
17 L. Marchetti and A. Andreani, Ann. Chim. (Rome), 1973, 63, 681.
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23 M. Somei and T. Kawasaki, Heterocycles, 1989, 29, 1251.
24 T. Kawasaki, A. Kodama, T. Nishida, K. Shimizu and M. Somei,
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25 M. S. C. Pedras and I. L. Zaharia, Phytochemistry, 2000, 55, 213.
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27 M. D. Meyer and L. I. Kruse, J. Org. Chem., 1984, 49, 3195.
28 P. Kutschy, M. Dzurilla, M. Takasugi, M. Torok, I. Achbergerova, R.
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20 M. S. C. Pedras and S. Montaut, Chem. Commun., 2004, 452.
21 Our hypothesis is supported by biosynthetic studies showing that
the biotransformation of model compound 47 yielded a compound
resulting from C-3–C-2 bond migration, 48, and a compound resulting
from oxidative spirocyclization, 49; see: K. Monde, M. Takasugi and
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30 K. Monde, M. Takasugi and A. Shirata, Phytochemistry, 1995, 39,
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