Phytoalexins from Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria maculans: Structures, syntheses and antifungal activity

Article abstract of DOI:10.1016/S0031-9422(03)00441-2

Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine s

Full text of DOI:10.1016/S0031-9422(03)00441-2

Phytochemistry 64 (2003) 949–956
www.elsevier.com/locate/phytochem
Phytoalexins from Thlaspi arvense, a wild crucifer resistant to
virulent Leptosphaeria maculans: structures, syntheses and
antifungal activity
M.S.C. Pedras*, P.B. Chumala, M. Suchy
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9
Received 18April 2003; received in revised form 30 May 2003
Abstract
Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial
attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine
stinkweed or pennycress (Thlaspi arvense), a potential source of disease resistance to blackleg. We have investigated phytoalexin
production in leaves of T. arvense under abiotic (copper chloride) and biotic elicitation by Leptosphaeria maculans (Desm.) Ces. et
de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], and report here two phytoalexins, wasalexin A and arvelexin (4-meth-
oxyindolyl-3-acetonitrile), their syntheses and antifungal activity against isolates of P. lingam/L. maculans, as well as the isolation of
isovitexin, a constitutive glycosyl flavonoid of stinkweed, having antioxidant properties but devoid of antifungal activity.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Thlaspi arvense; Stinkweed; Brassicaceae; Crucifer; G Leptosphaeria maculans; 4-Methoxyindolyl-3-acetonitrile; Phoma lingam; Phyto-
alexin; Wasalexin A; Wasalexin synthesis
1. Introduction
addition, the wild crucifer Arabidopsis thaliana is an
important model plant and the first flowering plant to
have its genome sequenced (The Arabidopsis Genome
Initiative, 2000). A number of cruciferous weeds are also
known for their negative impact on cultivated systems.
Our search for phytoalexins from cruciferous plants
resistant to economically important fungal diseases
(Pedras et al., 2000), led us to examine stinkweed or
pennycress (Thlaspi arvense), a potential source of dis-
ease resistance to blackleg (Gugel et al., 1990). Blackleg
of crucifers, caused by the fungus Leptosphaeria macu-
lans (Desm.) Ces. et de Not. [asexual stage Phoma lin-
gam (Tode ex Fr.) Desm.], occurs worldwide and can be
particularly devastating for the oilseed crops rapeseed
and canola. Most importantly, stinkweed is resistant to
the virulent strain of L. maculans/P. lingam but suscep-
tible to the Thlaspi strain (Petrie et al., 1995; Gugel et
al., 1990). Interestingly the resistance range of stink-
weed is similar to that of the crucifer wasabi (Wasabia
japonica, syn. Eutrema wasabi), a perennial plant widely
cultivated in Japan (Pedras and Sorensen, 1998).
Accordingly, it is of enormous importance to determine
the chemical traits responsible for the resistance of
Phytoalexins are inducible secondary metabolites
produced de novo by plants in response to diverse forms
of stress, including microbial attack (Bailey, 1982).
Until now the majority of the cruciferous phytoalexins
have been isolated from widely cultivated Brassica spe-
cies. However, because the production of phytoalexins
can have a significant impact on plant disease resistance
(Pedras et al., 2000), it is of great interest to screen a
wider range of cruciferous species for production of these
antimicrobial metabolites. Crucifers (family Cruciferae or
Brassicaceae) comprise a number of economically impor-
tant oilseed and condiment crops such as canola (Bras-
sica napus and B. rapa), rapeseed (B. napus Sinapis
alba), many vegetable species including turnip (B. rapa),
broccoli (Brassica oleracea var. botrytis), cauliflower (B.
oleracea var. italica), kale (B. oleracea var. acephala),
radish (Raphanus sativus), and cabbage (B. oleracea). In
* Corresponding author. Tel.: +1-306-966-4772; fax: +1-306-966-
4730.
E-mail address: soledade.pedras@usask.ca (M.S.C. Pedras).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00441-2

950
M.S.C. Pedras et al. / Phytochemistry 64 (2003) 949–956
stinkweed to blackleg isolates, as such agronomically
important traits are transferable to canola. We have
investigated phytoalexin production in leaves of stink-
weed under elicitation by CuCl2 and P. lingam/L.
maculans and report here two phytoalexins, wasalexin A
(8) (Scheme 1) and arvelexin (10), their syntheses and
antifungal activity against isolates of P. lingam/L.
maculans, as well as the isolation of isovitexin (11), a
constitutive glycosyl flavonoid of stinkweed, having
antioxidant properties but devoid of antifungal activity.
HPLC chromatograms (tR=13.9 and 24.4 min) of
extracts of infected leaves. Thus, in order to isolate these
phytoalexins, larger scale experiments were carried out
with plants treated with CuCl2 solution. Elicited leaves
were extracted and the residue was separated by flash
column chromatography (CH2Cl2–MeOH) as described
in the experimental. Fractions F2–F4 were combined
and further subjected to multiple chromatography to
yield the elicited compound with retention time
tR=13.9 min, whereas fractions F9–F12 were combined
and further subjected to multiple chromatography to
yield a bright yellow compound with tR=24.4 min. This
yellow compound was identified as the phytoalexin
wasalexin A (8) by comparison of its NMR, HRMS,
FTIR, UV, and HPLC data, with that of an authentic
sample available in our laboratory. Interestingly, wasa-
lexin A (8) was previously isolated from wasabi leaves
(E. wasabi) after elicitation with CuCl2 or P. lingam/L.
maculans (Pedras et al., 1999). The synthesis of wasa-
lexin A (8) and desmethyl analog 7 was carried out as
outlined on Scheme 1 and described in the Experi-
mental, in seven steps and 27% overall yield. The key
intermediate 1-methoxy-2-oxoindole (3) was prepared
from phenylacetyl chloride, and methoxyl amine
hydrochloride, followed by chlorination using t-butyl
hypochlorite, to yield the corresponding N-chloro-N-
methoxyamide 2 (Kawase et al., 1989). Ag2CO3 pro-
moted electrophilic cyclization afforded 1-methoxy-2-
oxoindole (3) in 79% yield (Kikugawa and Kawase,
1984). Following formylation of 3 with ethyl formate
and NaH (Pedras and Okanga, 1999), the resulting enol
was converted to the chloromethylene intermediate 4
using thionyl chloride (Behringer and Weissauer, 1952),
followed by amination to afford 1-methoxy-2-oxo-3-
aminomethylene indole (6) in 83% yield. Treatment of
enamine 6 with carbon disulfide under basic conditions
provided the dithiocarbamate salt, which was then
methylated to afford a mixture of wasalexins A (8) and
B and/or methyl dithiocarbamate 7, depending on the
reaction conditions. Thus, when one equivalent of each
NaH and MeI was used, dithiocarbamate 7 was the
main product, two equivalents of each NaH and MeI
led to the exclusive formation of wasalexins A (8) and B
in 68% yield from 1-methoxy-2-oxoindole (3). As pre-
viously reported (Pedras et al., 1999), the ratio of wasa-
lexins A (8) and B was determined to be 2:1 by 1H
NMR spectroscopy and HPLC analysis. The naturally
occurring wasalexin A (8) can be separated from the
isomeric mixture by slow crystallization using CH2Cl2–
hexane. The Z configuration of the double bond of
methyl dithiocarbamate 7 was established on the basis
of NOE experiments. Thus, irradiation of the aromatic
proton H-4 resulted in the enhancement of the inten-
sity (9%) of the peak corresponding to the side chain
proton. The Z stereochemistry can be explained by the
presence of intramolecular hydrogen bonding between
2. Results and discussion
In preliminary experiments, a time-course response of
stinkweed plants to abiotic elicitation with CuCl2 was
investigated. After spraying and incubating plants,
leaves were excised at 24 h intervals, crushed in liquid
N2 and extracted as described in the Experimental.
Control (i.e. non-elicited) leaves were collected and
treated in a similar manner. Analysis of these extracts
by high performance liquid chromatography (HPLC)
under the conditions described in the Experimental
indicated the presence of two peaks at tR=13.9 and 24.4
min in the extracts of elicited leaves, but not detectable
on the chromatograms of extracts of control tissues.
The results of this time-course experiment indicated that
the production of these two elicited metabolites
increased as shown in Table 1. Furthermore, biotic eli-
citation of stinkweed with fungal isolates Mayfair 2
(weakly virulent on canola) or Thlaspi 9 (virulent on
stinkweed) indicated the presence of similar peaks in the
Scheme 1. Synthesis of wasalexin A (8); reagents and conditions:
.
(i) CH3ONH2 HCl, Na2CO3, C6H6–H2O (1:1), r.t., 88%; (ii) t-BuOCl,
CH2Cl2, r.t., 99%; (iii) Ag2CO3, TFA, 0 ꢀC, 79%; (iv) EtOCHO, NaH,
r.t., 99%; (v) SOCl2, r.t., 99%; (vi) NH4OH, MeOH, 83%; (vii) NaH
(1.1 eq), CS2, THF, r.t.; CH3I (1.1 eq), yields are reported in the
experimental; (viii) NaH (2.1 eq), CS2, THF, r.t.; CH3I (2.1 eq), 68%.

M.S.C. Pedras et al. / Phytochemistry 64 (2003) 949–956
951
Table 1
Production of phytoalexinsa by Thlaspi arvense upon elicitation with CuCl2
Phytoalexin
24 h
(mmol/100 g
fresh weight)
48h
(mmol/100 g
fresh weight)
72 h
(mmol/100 g
fresh weight)
96 h
(mmol/100 g
fresh weight)
Arvelexin (10)
Wasalexin A (8)
0.65ꢂ0.05
3.7ꢂ0.5
1.5ꢂ0.3
7.1ꢂ0.3
1.8ꢂ0.2
2.6ꢂ0.2
ꢂ0.6
10.3ꢂ0.89.1
a
Results are presented as meansꢂstandard deviation.
the oxygen of the carbonyl group (C-2) and the hydro-
gen on the nitrogen of the side chain (N–H).
present in control tissues, was isolated from more polar
fractions. The HRMS-FAB spectrum of this compound
showed a molecular ion at m/z 433.1134 [M++1] which
together with the NMR spectroscopic data indicated a
The molecular formula of the elicited compound with
tR=13.9 min was determined to be C11H10ON2 by
HRMS-EI, which suggested eight degrees of unsatura-
1
molecular formula of C21H20O10. The H NMR spec-
1
tion. The H NMR spectrum of this metabolite showed
trum of this compound (DMSO-d6) displayed seven pro-
tons at sp3 carbons and six aromatic protons. Four of
these aromatic proton signals at ꢀ 6.91 (2H, d, J=8Hz,
five aromatic protons, as well as two methylene and
three methoxy protons. The spin systems of the aro-
matic protons showed that the proton at ꢀ 7.14 (H-6, dd,
J=7.5, 7.5 Hz) was coupled to two protons at d 7.04
(H-7, d, J=7.5 Hz) and ꢀ 6.57 (H-5, d, J=7.5 Hz), the
coupling constants further suggesting that the orienta-
tion of H-6–H-7 and to H-5 is ortho. An exchangeable
broad proton at ꢀ 8.26 and a broad singlet at ꢀ 7.14 (H-
2) suggested the aromatic protons to be part of an
indole ring substituted at either position 4 or 6. Analysis
of the HMBC spectrum showed correlations of the most
downfield carbon at ꢀ 154.6 (C-4) with methoxy pro-
tons, and the methylene protons at ꢀ 4.07 with carbons
at ꢀ 120.2 (-CN), ꢀ 112.6 (C-2), ꢀ 105.0 (C-3), and ꢀ
117.7 (C-4a). FTIR data showed a weak absorption at
2251 cmꢁ1 which was attributed to a nitrile group, also
consistent with the 13C NMR chemical shift at ꢀ 120.2.
Therefore, structure 10 was assigned to this elicited
compound with tR=13.9 min. This structural assign-
ment was confirmed by synthesis through coupling of 4-
methoxyindole (9) with bromoacetonitrile under
Grignard conditions, in 52% overall yield (Scheme 2), as
described in the Experimental. Although our synthetic
preparation of 10 is very simple, a more economic two-step
conversion of indole-3-carboxaldehyde to 4-methoxy-
indolyl-3-acetonitrile (3) was recently reported (Yamada et
al., 1998). Compound 10 was first isolated from roots of
Chinese cabbage (B. pekinensis) infected with Plasmodio-
phora brassicae (Nomoto and Tamura, 1970); however, no
antifungal activity has been reported to date.
0
H-30 and H-50) and at ꢀ 7.90 (2H, d, J=8Hz, H-2 and
H-60) showed an ortho relationship. These data as well
as 2D NMR spectra suggested the presence of a para-
hydroxyphenyl moiety. The long range correlation of the
aromatic proton at ꢀ 6.46 (H-3, s) with a carbon at ꢀ
121.0 (C-1) as well as protons H-20 and H-60 with a car-
bon at ꢀ 164.7 (C-2) indicated the connectivity of the
para-hydroxyphenyl moiety to a flavonoid type ring.
1
Furthermore, the H NMR and 13C NMR spectra also
revealed the existence of a glucose moiety. The anomeric
proton at ꢀ 4.59 (d, J=9 Hz, H-1) and the remaining
2JHꢁH suggested that the glycoside moiety was a b-glu-
copyranoside. Since the anomeric proton (ꢀ 4.59) showed
long range correlations with carbons at ꢀ 160.7 (C-5), ꢀ
163.2 (C-7), and ꢀ 109.0 (C-6) its connectivity with an
aromatic system was evident. The unusually high field
anomeric carbon signal (ꢀ 73.1) of this compound, as
well as its resistance to acid hydrolysis was suggestive of
a C–C linked b-glucopyranoside moiety. Final analysis of
the HMBC data of this compound led to its identification
1
as isovitexin (11). Comparison of the H and 13C NMR
(Ramarathnam et al., 1989, NMR data was reported in
MeOH-d4), UV, and [a]D (17.2, c 0.37, EtOH, lit. 16.21,
Briggs and Cambie, 1958) spectroscopic data of com-
pound 11 with those published showed good agreement.
Isovitexin (11, apigenin 6-C-b-glucopyranoside) was first
isolated from Vitex lucens (Briggs and Cambie, 1958;
Horowitz and Gentili, 1964), and later on established to
show potent anti-oxidant activity (Ramarathnam et al.,
1989). To the best of our knowledge this is the first report
of isovitexin (11) production by T. arvense.
Together with the elicited compounds 8 and 10,
another bright yellow compound with tR=3.5 min, also
Scheme 2. Synthesis of arvelexin; reagents and conditions: (i) CH3I/
Mg, Et2O, r.t.; (ii) BrCH2CN, r.t., 52%.

952
M.S.C. Pedras et al. / Phytochemistry 64 (2003) 949–956
Antifungal activity assays were carried out with
anticipated that a better insight into defense mechanisms
of crucifers will be obtained with the investigation of
phytoalexin profiles of unrelated species. Eventually, as
phytoalexins from unrelated cruciferous species are dis-
covered and biological activities established, the ‘‘design’’
of species producing a phytoalexin blend active against a
range of pathogens may become a feasible strategy.
metabolites 7, 8, 10, 11 and indolyl-3-acetonitrile
(introduced for comparison with 10), against three
standard isolates of L. maculans/P. lingam: BJ 125
(virulent on canola), Mayfair 2 (weakly virulent on
canola), and Thlaspi 9 (virulent on stinkweed). Results
of these assays indicate that compound 11 displays no
antifungal activity whereas 7, 8, 10 and indolyl-3-aceto-
nitrile are inhibitory to the three isolates (5ꢃ10ꢁ4 M,
Table 2), though indolyl-3-acetonitrile appears to more
inhibitory to virulent isolate BJ 125 than any of the
other three compounds.
3. Experimental
3.1. General
Compound 10 displays antifungal activity, and its for-
mation is induced by stinkweed under biotic (L. macu-
lans/P. lingam) or abiotic (CuCl2) elicitation but it is
not produced in control plants. Thus metabolite 10
satisfies the conditions to be a phytoalexin for which
we propose the name arvelexin. Arvelexin accumulates
in the leaves of stinkweed to levels comparable with
those of other phytoalexins (Pedras et al., 2002). Inter-
estingly, we found that the structurally related indolyl-
3-acetonitrile is also a phytoalexin in B. juncea (resistant
to BJ 125) but not in B. rapa or B. napus (Pedras et al.,
2002). It is noteworthy that two unrelated plant species,
wasabi and stinkweed, apparently resistant to the same
fungal isolates of L. maculans/P. lingam produce the
phytoalexin wasalexin A (8). Wasalexin production
could suggest it has a role in resistance/defense of these
two species against virulent blackleg isolates. It is
All chemicals were purchased from Sigma-Aldrich
Canada Ltd., Oakville, ON. Analytical HPLC analysis
was carried out with a high performance liquid chro-
matograph equipped with quaternary pump, automatic
injector, and photodiode array detector (wavelength range
190–600 nm), degasser, and a Hypersil ODS column (5
mm particle size silica, 4.6 i.d. ꢃ 200 mm), equipped with
an in-line filter. Mobile phase: H2O-CH3CN, 75:25 to
100% CH3CN, for 35 min, linear gradient, and 1.0 ml/
min flow rate. Samples were dissolved either in CH3CN
or in MeOH. Other conditions as previously reported
(Pedras and Okanga, 1999).
3.1.1. Plant material
Seeds of Thlaspi arvense (stinkweed or pennycress)
were obtained from Plant Gene Resources, Agriculture
Table 2
Antifungal activity of compounds 7, 8, 10, and 11 against Leptosphaeria maculans (Phoma lingam) isolates BJ 125 (virulent on canola), Mayfair 2
(virulent on brown mustard), and Thlaspi 9 (virulent on stinkweed)
Compound concentration
(M)
BJ 125
% inhibition
(mycelium diameter)a
Mayfair 2
% inhibition
(mycelium diameter)a
Thlaspi 9
% inhibition
(mycelium diameter)a
Desmethylwasalexin (7)
1 ꢃ 10ꢁ4
No inhibition
15 (17 ꢂ1)
26 (15 ꢂ1)
No inhibition
No inhibition
20 (13 ꢂ1)
No inhibition
No inhibition
14 (13 ꢂ1)
3 ꢃ 10ꢁ4
5 ꢃ 10ꢁ4
Wasalexin A (8)
1 ꢃ 10ꢁ4
No inhibition
17 (15 ꢂ1)
42 (12 ꢂ1)
No inhibition
20 (11 ꢂ1)
28(10 ꢂ1)
No inhibition
No inhibition
30 (11 ꢂ1)
3 ꢃ 10ꢁ4
5 ꢃ 10ꢁ4
Arvelexin (10)
1 ꢃ 10ꢁ4
No inhibition
11 (18 ꢂ1)
73 (8 ꢂ1)
No inhibition
33 (15 ꢂ1)
53 (12 ꢂ1)
No inhibition
No inhibition
59 (11 ꢂ1)
3 ꢃ 10ꢁ4
5 ꢃ 10ꢁ4
Isovitexin (11)
5 ꢃ 10ꢁ4
No inhibition
No inhibition
No inhibition
Indolyl-3-acetonitrile
1 ꢃ 10ꢁ4
No inhibition
77 (8 ꢂ1)
No inhibition
13 (18 ꢂ1)
25 (16 ꢂ1)
No inhibition
11 (18 ꢂ1)
57 (11 ꢂ1)
3 ꢃ 10ꢁ4
5 ꢃ 10ꢁ4
complete inhibition
a
The percentage of inhibition was calculated using the formula: % inhibition=100ꢁ [(growth on treated/growth in control) ꢃ 100]; results are
the mean of at least three independent experiments (mmꢂstandard deviation); control plates contained potato dextrose agar and 1% DMSO.

M.S.C. Pedras et al. / Phytochemistry 64 (2003) 949–956
953
and Agri Food Canada, Saskatoon Research Center,
SK. The seeds were sown in a commercial potting soil
mixture, and plants were grown in a growth chamber,
with 16 h light (fluorescent and incandescent)/8h dark,
EtOAc (12 h at r.t.). The EtOAc extract was dried
(Na2SO4), filtered, and concentrated to dryness under
reduced pressure; the crude extract residue (0.74 g) was
separated by FCC (i.d. 2 cm, silica gel, 100 ml fractions,
CH2Cl2 400 ml; CH2Cl2ꢁMeOH 97:3, 400 ml,
CH2Cl2ꢁMeOH 94:6, 400 ml).
ꢀ
at 24ꢂ2 C.
3.2. Time-course study for the production of phyto-
alexins and biotic elicitation of phytoalexins
Fractions F2–F4 from FCC on EtOAc extracts were
combined (39 mg) and subjected to FCC (i.d. 1 cm,
50 ml silica gel, CH2Cl2–hexane 30:70, 200 ml, 50:50,
200 ml, CH2Cl2 200 ml). Twelve fractions collected were
concentrated to dryness under reduced pressure. Frac-
tions F10–F12 were combined (18mg) and further sub-
jected to prep. TLC (CH2Cl2–MeOH, 98:2) to yield
arvelexin (10, 1 mg, HPLC tR=13.9 min). Fractions F7
and F8from FCC of EtOAc extracts were combined
(60.5 mg) and separated by FCC (i.d. 2 cm, CH2Cl2–
MeOH 99:1, 200 ml, 97:3, 200 ml). Fractions F4 and F5
were combined and further subjected to RP-FCC
(CH3CN–H2O, 30:70, 70 ml) to yield wasalexin A (8, 1.5
mg, HPLC tR=24.4 min).
Elicitation with CuCl2: 4-week-old plants were
sprayed with a CuCl2 (2 mmol) solution at 0, 24, and 48
h, and incubated for 2 additional days. Leaves were
excised at the base, were weighed, frozen in liquid
nitrogen, crushed, and then soaked in EtOAc for 24 h.
The EtOAc was decanted, dried (anhyd. Na2SO4), and
concentrated under reduced pressure. The residues from
both elicited and control leaves were analyzed by
HPLC.
Elicitation with fungal isolates: leaves of 4-week-old
plants were cut, the petioles covered with moist cotton
wool, leaves were damaged slightly by scraping with
pipette tip and dipped into a spore suspension (106 spores/
ml, isolates Mayfair 2 or Thlaspi 9). Leaves were placed
into a Petri dish, the plate was sealed with parafilm and
3.5. Wasalexin A (8)
3.5.1. 1-Methoxy-2-oxoindole (3)
ꢀ
incubated (16 h photoperiod, 20ꢂ0.5 C) for 7 days.
A solution of crude N-chloro-N-methoxy-phenyl-
acetamide (2, 1.1 g, 5.5 mmol, Kawase et al., 1989)
in TFA (5 ml) was added to a stirred suspension of
Ag2CO3 (3.04 g, 11 mmol) in TFA (10 ml) at 0 ꢀC. After
stirring for 15 min at 0 ꢀC, the pH of the reaction
mixture was adjusted to 7 using 20% solution of
Na2CO3. The mixture was extracted with CH2Cl2, the
combined organic extracts dried (over Na2SO4) and
concentrated under reduced pressure. The residue was
separated by FCC (CH2Cl2) to yield a slightly yellow oil
which was crystallized from CH2Cl2–hexane to afford
compound 3 as colorless needles (0.712 g, 79%), m.p.
84–86 ꢀC (lit. 84.5–86 ꢀC, Et2O, Somei et al., 2001). The
spectral data are in agreement with those previously
reported (Somei et al., 2001).
After incubation, the leaves were frozen, and treated as
described above. The EtOAc extracts of treated and
control leaves were analyzed by HPLC.
3.3. Antifungal bioassays
3.3.1. Mycelial radial growth
Bioassays to determine antifungal activity were car-
ried out with P. lingam/L. maculans isolates BJ 125,
Mayfair 2, and Thlaspi 9. Each isolate was grown on V8
agar under continuous light; after 15 days the fungal
ꢀ
spores were collected and stored at ꢁ20 C. A DMSO
solution of the compound to be tested (final concen-
tration of each compound 5.0 ꢃ10ꢁ4, 2.5 ꢃ10ꢁ4 and
10ꢁ4 M; final DMSO concentration 1%) was added to
ꢀ
agar medium at ca. 50 C, mixed quickly and poured
3.5.2. 1-Methoxy-3-formyl-2-oxoindole (4)
onto 12-well plates (1 ml). An agar plug (4 mm diameter)
cut from edges of 7-day-old solid cultures was placed
upside down on the center of each plate, the plꢀates were
sealed with parafilm, and incubated at 23ꢂ2 C under
constant light for 96 h. The diameter of the mycelia (in
mm) was then measured and compared with control
plates containing only DMSO. Each assay was con-
ducted in triplicate and repeated at least three times.
NaH (60% suspension in mineral oil, washed with
hexane, 185 mg, 5.5 mmol) was added to a stirred solu-
tion of 1-methoxy-2-oxoindole (3, 82 mg, 0.5 mmol) in
ethyl formate (1.5 ml). After stirring for further 2 min
the reaction mixture became a paste which was cooled
to 0 ꢀC, diluted with water (10 ml) and acidified to pH 5
with 1 M HCl. The product was extracted with Et2O,
the extracts were combined, dried (over Na2SO4), and
concentrated under reduced pressure to afford a brown-
ish solid (95 mg, 99%), of sufficient purity to use in the
next step. An analytical sample was obtained by crys-
tallization from diethyl ether–hexane solution, m.p.
3.4. Isolation of phytoalexins
Four-week-old plants (T. arvense) were sprayed with
CuCl2 solution (2 ꢃ10ꢁ3 M) every 24 h for 3 consecutive
days. All leaves were collected (182 g) 48 h after the last
spray, frozen in liquid N2, crushed, and extracted with
ꢀ
183–185 C, (lit. 190 ꢀC, EtOH, Sakan et al., 1964).
1
HPLC tR=8.8 min; H NMR (500 MHz, DMSO-d6):
ꢀ 12.63 (br s, 1H, D2O exchangeable), 7.87 (s, 1H), 7.58

954
M.S.C. Pedras et al. / Phytochemistry 64 (2003) 949–956
(d, J=8Hz, 1H), 7.20 ( dd, J=8, 8 Hz, 1H), 7.03 (dd,
J=8, 8 Hz, 1H), 6.98 (d, J=8Hz, 1H), 3.90 ( s, 3H); 13
3.5.5. Methyl 1-methoxy-3-aminomethylene-2-oxoindole
dithiocarbamate (7)
C
NMR (125 MHz, DMSO-d6): ꢀ 164.7 (s), 156.6 (d),
136.7 (s), 127.1 (d), 122.9 (2ꢃ d), 119.1 (s), 107.1 (d),
103.6 (s), 64.3 (q); HREIMS m/z (% relative abundance)
measured: 191.0582 (191.0582 calc. for C10H9NO3);
EIMS m/z (% relative abundance): 191 [M+] (10), 162
(100), 148(11), 132 (7), 104 (7), 119 (31), 77 (8); FTIR
vmax 3051, 2630, 1684, 1614, 1461, 1421, 1394, 1206,
NaH, (60% suspension in mineral oil, washed with
hexane, 15 mg, 0.43 mmol) and CS2 (30 ml, 0.52 mmol)
were added to a solution of crude 1-methoxy-3-amino-
methylene-2-oxoindole (83 mg, 0.43 mmol) in dry THF
ꢀ
(1ꢀml) at 0 C. After stirring the mixture for 5 min at
0 C, methyl iodide (30 mL, 0.48mmol) was added and
the stirring was continued for further 1 h at 0 C. The
ꢀ
1104 cmꢁ1
.
solvent was evaporated and the residue was chromato-
graphed on silica gel (hexane–acetone, 5:1). Concen-
tration of the less polar fraction afforded 7 as a yellow
residue (50 mg, 36%, m.p. 140–141 ꢀC, acetone–hex-
ane); the more polar fraction contained an oily yellow
material containing wasalexins A and B (13 mg, 9%).
3.5.3. 1-Methoxy-3-chloromethylene-2- oxoindole (5)
Crude 1-methoxy-3-formyl-2-oxoindole (4, 95 mg, 0.5
mmol) was dissolved in thionyl chloride (2.5 ml, 34.3
mmol) and allowed to stand for 1 h at r.t. The thionyl
chloride was evaporated to yield a yellow residue (104
mg, 99%), of sufficient purity to use in the next step. An
analytical sample was obtained by crystallization from
1
HPLC tR=29.4 min; H NMR (500 MHz, CDCl3): ꢀ
12.17 (d, J=10 Hz, 1H, D2O exchangeable), 8.68 (d,
J=10 Hz, 1H), 7.50 (d, J=8Hz, 1H), 7.30 ( dd, J=8, 8
Hz, 1H), 7.12 (dd, J=8, 8 Hz, 1H), 7.05 (d, J=8Hz,
1H), 4.10 (s, 3H), 2.79 (s, 3H); 13C NMR (125 MHz,
CDCl3): ꢀ 200.4 (s), 164.1 (s), 137.2 (d), 133.3 (s), 128.2
(d), 123.3 (d), 119.5 (d), 118.7 (s), 108.0 (d), 106.3 (s),
64.5 (q), 19.2 (q); HREIMS m/z (% relative abundance)
measured: 280.0340 (280.0340 calc. for C12H12N2O2S2);
EIMS m/z (% relative abundance): 280 [M+] (61), 249
(78), 232 (75), 201 (19), 173 (100), 159 (24), 144 (30), 129
(12), 115 (25), 90 (56); FTIR vmax: 3059, 2946, 1683,
ꢀ
CH2Cl2–hexane solution, m.p. 98–100 C.
1
HPLC tR=18.1 min; H NMR (500 MHz, CDCl3): d
7.97 (d, J=8Hz, 1H), 7.50 ( s, 1H), 7.37 (dd, J=8, 8 Hz,
1H), 7.10 (dd J=8, 8 Hz, 1H), 6.98 (d, J=8Hz, 1H),
3.90 (s, 3H); 13C NMR (125 MHz, CDCl3): ꢀ 161.5 (s),
139.8( s), 130.8( d), 130.3 (d), 127.2 (s), 125.0 (d), 123.4
(d), 117.4 (s), 107.6 (d), 64.2 (q); HREIMS m/z (% rela-
tive abundance) measured: 209.0240 (209.0244 calc. for
C10H8ClNO2); EIMS m/z (% relative abundance): 209
[M+] (100), 194 (14), 174 (49), 159 (30), 146 (24), 129
(6), 115 (16), 102 (10), 89 (7), 59 (6); FTIR vmax: 3059,
2943, 1721, 1616, 1461, 1421, 1320, 1220, 1073, 769,
1645, 1455, 1321, 1230, 1101, 1058, 998, 740 cmꢁ1
.
3.5.6. Wasalexin A (8)
741, 690 cmꢁ1
.
NaH, (60% suspension in mineral oil, washed with
hexane, 37 mg, 1.1 mmol) and CS2 (30 ml, 0.52 mmol)
were added to a solution of crude 1-methoxy-3-amino-
methylene oxoindole (83 mg, 0.43 mmol) in dry THF (1
ml) at 0 ꢀC. After stirring the mixture for 5 min at 0 ꢀC,
methyl iodide (70 ml, 1.1 mmol) was added and the stir-
3.5.4. 1-Methoxy-3-aminomethylene-2-oxoindole (6)
Ammonium hydroxide (28% solution, 20 ml, 340
mmol) was added to a solution of crude 1-methoxy-3-
chloromethylene-2-oxoindole (104 mg, 0.5 mmol) in
MeOH (2.5 ml). After stirring for 90 min at r.t., the
reaction mixture was diluted with brine (20 ml), extrac-
ted with CH2Cl2, the extracts were combined, dried over
Na2SO4, and concentrated under reduced pressure to
yield a yellow solid (83 mg, 83%), of sufficient purity to
use in the next step. An analytical sample was obtained
by crystallization from CH2Cl2–hexane solution, m.p.
ꢀ
ring was continued for further 30 min at 0 C and then
ꢀ
for 2 h at r.t. The mixture was then cooled to 0 C,
diluted with water (20 ml), extracted with EtOAc, dried
over Na2SO4, concentrated under reduced pressure and
the residue chromatographed on silica gel (hexane–ace-
tone, 2:1). Evaporation of the solvent afforded 100 mg
(68%) of yellow oily material, which contained wasalex-
ins A and B in the ratio 2:1. The naturally occurring
wasalexin A separated as follows: 50 mg of the mixture
of both isomers was dissolved in CH2Cl2 (2 ml) and
hexane (5 ml) and the mixture allowed to stand for 78h
at 0 ꢀC and then for 5 h at ꢁ10 ꢀC. This process afforded
ꢀ
162–164 C.
HPLC tR=3.3 min; H NMR (500 MHz, CDCl3): ꢀ
1
8.26 (br s, 1H, D2O exchangeable), 7.59 (m, 1H), 7.28( d,
J=8Hz, 1H), 7.15 ( t, J=8Hz, 1H), 7.03 ( m, 2H), 5.40
(br s, 1H, D2O exchangeable), 4.08( s, 3H); 13C NMR
(125 MHz, CDCl3): ꢀ 164.6 (s), 144.1 (d), 134.4 (s), 124.4
(d), 121.9 (d), 120.6 (s), 116.0 (d), 107.1 (d), 95.7 (s), 64.3
(q); HREIMS m/z (% relative abundance) measured:
190.0743 (190.0743 calc. for C10H10N2O2); EIMS m/z
(% relative abundance): 190 [M+] (95), 175 (11), 159
(48), 144 (8), 132 (100), 104 (17), 89 (6), 77 (15); FTIR
vmax: 3171, 2934, 1672, 1561, 1466, 1306, 1199, 1100,
ꢀ
wasalexin A as orange cubes, m.p. 139–140 C (24 mg,
48%). HPLC tR=24.4 min; spectroscopic data were con-
sistent with those previously reported (Pedras et al., 1999).
3.6. Arvelexin (10)
Magnesium turnings (20 mg, 0.82 mmol) in dry Et2O
(under argon) were allowed to react with iodomethane
1033, 953, 740, 604 cmꢁ1
.

M.S.C. Pedras et al. / Phytochemistry 64 (2003) 949–956
955
(70 ml, 1.1 mmol) at r.t. until all magnesium was con-
References
sumed (30 min). Next, after distilling off Et2O and MeI
and the residue dissolved in Et2O, a solution of
4-methoxyindole (25 mg, 0.17 mmol) in Et2O (500 ml)
was added dropwise to the reaction mixture under stir-
ring at r.t. After stirring for 15 min, bromoacetonitrile
(90 ml, 1.3 mmol) was added dropwise at r.t., and the
mixture stirred for further 60 min at r.t. (Filler et al.,
1989). The solvent was evaporated under reduced pres-
sure, the residue was suspended in distilled water (40
ml), and was neutralized with 2 M HCl (0.5 ml). The
aqueous phase was extracted with CH2Cl2, the organic
layer dried over MgSO4, filtered, and concentrated to
dryness to yield the crude product (54 mg). After FCC
(CH2Cl2–hexane, 40:60) arvelexin (8) was obtained in
52% yield (16.5 mg).
HPLC tR=13.9 min; 1H NMR (CD2Cl2): ꢀ 8.26
(bs, 1H, D2O exchangeable), 7.14 (bs, 1H), 7.11 (dd,
J=8, 8 Hz, 1H), 7.04 (d, J=8Hz, 1H), 6.57 ( d, J=8
Hz, 1H), 4.07 (s, 2H), 3.93 (s, 3H); 13C NMR
(CD2Cl2): ꢀ 154.6 (s), 138.5 (s), 123.7 (d), 120.2 (s),
117.7 (s), 112.6 (d), 105.3 (d), 105.0 (s), 100.0 (d),
55.3 (q), 15.6 (t); FTIR vmax 3396, 2955, 2850,
2251,1619, 1590, 1259, 1089 cmꢁ1; HRMS-EI: m/z
186.0788 (186.0793 calc. for C14H18O3), EI-MS: m/z
(relative intensity) 186.07 [M+] (99%), 171.05
(100%).
Bailey, J.A., Mansfield, J.W. (Eds.), 1982. Phytoalexins. Blackie and
Son, Glasgow, UK, p.334.
Behringer, H., Weissauer, H., 1952. Uber eine neue Synthese des b-
Oxindolyl-(3)-alanins (a-Oxy-tryptophans); ein Betrag zum Anwen-
dungsbereich der Sorensenschen Aminosaure-Synthese. Chemische
Berichte 85, 743–751.
Briggs, L.H., Cambie, R.C., 1958. The extractives of Vitex lucens—I.
Tetrahedron 3, 269–273.
Filler, R., Woods, S.M., White, W.L., 1989. Polyfluoroaralkyl amines.
Further studies on the reactivity of 4,5,6,7-tetrafluoroindole. Cana-
dian Journal of Chemistry 67, 1837–1841.
Gugel, R.K., Seguin-Swartz, -G., Petrie, A., 1990. Pathogenicity of
three isolates of Leptosphaeria maculans on Brassica species and
other crucifers. Canadian Journal of Plant Pathology 12, 75–82.
Horowitz, R.M., Gentili, B., 1964. Structure of vitexin and isovitexin.
Chemistry and Industry 498–499.
Kawase, M., Kitamura, T., Kikugawa, Y., 1989. Electrophilic aro-
matic substitution with N-methoxy-N-acylnitrenium ions generated
from N-chloro-N-methoxyamides: syntheses of nitrogen heterocyclic
compounds bearing a N-methoxyamide group. Journal of Organic
Chemistry 54, 3394–3403.
Kikugawa, Y., Kawase, M., 1984. Electrophilic aromatic substitu-
tion with a nitrenium ion generated from N-chloro-N-methox-
yamides. Application to the synthesis of 1-methoxy-2-
oxoindoles. Journal of the American Chemical Society 106,
5728–5729.
Nomoto, M., Tamura, S., 1970. Isolation and identification of indole
derivatives in clubroots of Chinese cabbage. Agricultural and Bio-
logical Chemistry 34, 1590–1592.
Pedras, M.S.C., Nycholat, C.M., Montaut, S., Xu, Y., Khan, A.K.,
2002. Chemical defenses of crucifers: elicitation and metabolism of
phytoalexins and indole-3-acetonitrile in brown mustard and turnip.
Phytochemistry 59, 611–625.
3.7. Isovitexin (11)
Pedras, M.S.C., Okanga, F.I., 1999. Strategies of cruciferous patho-
genic fungi: detoxification of the phytoalexin cyclobrassinin by
mimicry. Journal of the Agricultural and Food Chemistry 47, 1196–
1202.
HPLC tR=3.5 min; [a]D=+17.2 (c 0.37, EtOH, lit.
+16.21; Briggs and Cambie, 1958); H NMR (DMSO-
1
d6): ꢀ 13.72 (s, 1H, D2O exchangeable), 7.90 (d, J=8
Hz, 2H), 6.91 (d, J=8Hz, 2H), 6.74 ( s, 1H), 6.46 (s,
1H), 4.59 (d, J=9 Hz, 1H), 4.08( dd, J=9, 9 Hz, 1H),
3.68( d, J=11 Hz, 1H), 3.41 (dd, J=11, 5 Hz, 1H),
3.22–3.13 (m, 3H); 13C NMR (DMSO-d6): ꢀ 181.7 (s),
164.7 (s), 163.3 (s), 161.4 (s), 160.7 (s), 156.4 (s), 128.4
(d), 121.0 (s), 116.0 (d), 109.0 (s), 102.8( s), 102.6 (d),
93.9 (d), 81.5 (d), 79.0 (d), 73.1 (d), 70.6 (d), 70.1 (d),
61.4 (t) (previous NMR data was reported in MeOH-
d4, Ramarathnam et al., 1989). HR-FAB m/z: 433.1134
[M+1]+.
Pedras, M.S.C., Sorensen, J.L., 1998. Phytoalexin accumulation and
production of antifungal compounds by the crucifer wasabi. Phy-
tochemistry 49, 1959–1965.
Pedras, M.S.C., Sorensen, J.L., Okanga, F.I., Zaharia, I.L., 1999.
Wasalexins A and B, new phytoalexins from wasabi: isolation,
synthesis, and antifungal activity. Bioorganic & Medicinal Chem-
istry Letters 9, 3015–3020.
Pedras, M.S.C., Okanga, F.I., Zaharia, I.L., Khan, A.K., 2000. Phy-
toalexins from crucifers: synthesis, biosynthesis, and bio-
transformation. Phytochemistry 53, 161–176.
Petrie, A., Seguin-Swartz, G., Gugel, R.K., 1995. Latent infection of
Brassicaceae in the field by Leptosphaeria maculans (blackleg).
Canadian Journal of Plant Pathology 17, 75–81.
Ramarathnam, N., Osawa, T., Namiki, M., Kawakishi, S., 1989.
Chemical studies on novel rice hull antioxidants. 2. Identification of
isovitexin, a C-glycosyl flavonoid. Journal of the Agricultural and
Food Chemistry 37, 316–319.
Acknowledgements
Sakan, T., Kusuda, K., Miwa, T., 1964. The base-catalyzed con-
densation of o-nitroacetophenone. II. The ozonolysis of methyl
ether of compound A, a condensation product, and the structure of
compound B, another product. Bulletin of the Chemical Society of
Japan 37, 1681–1684.
We thank Dallas Kessler and Richard Gugel, Plant
Gene Resources, Agriculture and Agri Food Canada,
Saskatoon, SK, for a generous gift of seeds of T. arvense.
Financial support from the Natural Sciences and Engi-
neering Research Council of Canada (NSERC Dis-
covery Grant to M.S.C.P), NATO (postdoctoral
fellowship to M.S.) and the University of Saskatchewan
(graduate teaching assistantship to P.B.C.) is gratefully
acknowledged.
Somei, M., Yamada, F., Kurauchi, T., Nagahama, Y., Hasegawa, M.,
Teranishi, S., Sato, H., Kaneko, C., 2001. The chemistry of Indoles.
CIII. Simple syntheses of serotonin, N-methylserotonin, bufotenine,
5-methoxy-N -methyltryptamine, and lespedamine based on 1-
hydroxyindole chemistry. Chemical and Pharmaceutical Bulletin 49,
87–96.

956
M.S.C. Pedras et al. / Phytochemistry 64 (2003) 949–956
The Arabidopsis Genome Initiative, 2000. Nature (London) 408, 796–
826.
Yamada, F., Hashizume, T., Somei, M., 1998. Chemistry of
indoles. 83. Simple one step syntheses of indole-3-acetoni-
triles from indole-3-carboxaldehydes. Heterocycles 47, 509–
516.