The phytoalexins from cauliflower, caulilexins A, B and C: Isolation, structure determination, syntheses and antifungal activity

Article abstract of DOI:10.1016/j.phytochem.2006.05.020

Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(-)-spirobrassinin, 1-methoxybrassitin, brassicanal C) and three new (caulilexins A-C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A-C against the economically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of brassicanal C are reported.

Full text of DOI:10.1016/j.phytochem.2006.05.020

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 1503–1509
www.elsevier.com/locate/phytochem
The phytoalexins from cauliflower, caulilexins A, B and C:
Isolation, structure determination, syntheses and antifungal activity
*
M. Soledade C. Pedras , Mohammed G. Sarwar, Mojmir Suchy, Adewale M. Adio
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9
Received 8 March 2006; received in revised form 6 May 2006
Available online 27 June 2006
Abstract
Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica
oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(ꢀ)-spirobrassinin, 1-methoxybrassitin, brassicanal
C) and three new (caulilexins A–C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A–C against the eco-
nomically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of
brassicanal C are reported.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Brassica oleracea; Cruciferae; Brassicanal C synthesis; Brassitin; Cauliflower; Crucifer; Leptosphaeria maculans; 1-Methoxyindolyl-3-aceton-
itrile; Phoma lingam; Phytoalexin; Spirobrassinin
1
. Introduction
syntheses, and antifungal activity of caulilexins A (1), B (2),
and C (3), three new phytoalexins produced by cauliflower,
together with four known phytoalexins, isalexin (4) (Pedras
et al., 2004b), S-(ꢀ)-spirobrassinin (5) (Takasugi et al.,
1987; Suchy et al., 2001), 1-methoxybrassitin (6) (Takasugi
et al., 1988) and brassicanal C (7) (Monde et al., 1991). In
addition, the synthesis of brassicanal C (7) and its anti-
fungal activity against three important crucifer pathogens
is reported here for the first time.
Cauliflower (Brassica oleracea var. botrytis) is a crucifer
vegetable cultivated and consumed worldwide. Despite the
number of studies dealing with secondary metabolites of
cauliflower (Llorach et al., 2003), including volatiles (Val-
ette et al., 2006), glucosinolates (Tian et al., 2005; Menard
et al., 1999) and oviposition stimulants (Hurter et al.,
1
999), no phytoalexins have been reported to date. Phytoal-
exins are secondary metabolites produced de novo by plants
in response to various forms of stress, including microbial
attack (Bailey and Mansfield, 1982). Most, if not all, of
the phytoalexins produced by crucifers are derived from
tryptophan and therefore contain an indole or oxindole
moiety attached to a variety of functional groups (Pedras
et al., 2000, 2003b). Due to the significance of phytoalexins
in plant fitness, we have been investigating the phytoalexins
of both cultivated and wild crucifers (Pedras et al., 2000,
2. Results and discussion
2.1. Isolation and structure elucidation
Preliminary experiments were carried out to establish the
period for maximum production of induced metabolites in
cauliflower florets. Analyses of extracts of cauliflower flo-
rets exposed to UV light (abiotic elicitation) using high per-
formance liquid chromatography (HPLC) with photodiode
array detection indicated the presence of seven elicited com-
2
003b) and their interaction with economically important
pathogens. Here we wish to report the isolation, structures,
*
pounds (t = 3.8, 8.8, 9.5, 12.2, 16.6, 16.8 and 17.8 min) not
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966 4730.
R
E-mail address: soledade.pedras@usask.ca (M.S.C. Pedras).
detected in control tissues (no UV irradiation, Table 1). The
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.05.020

1504
M.S.C. Pedras et al. / Phytochemistry 67 (2006) 1503–1509
O
omethylindole-3-carboxaldehyde. The structure of com-
pound 1 was confirmed by synthesis, as described below.
The molecular formula of the compound with t = 8.8 min
H
CHO
SCH3
CN
NH
R
1
N
(2) was deduced to be C H N O , by HREIMS. The H
11 12 2 2
S
N
H
N
OCH3
NMR spectrum showed the expected 12 protons (five aro-
1
2
3
OCH3
matic, a singlet at dH 8.14 indicating the presence of a
OCH3
13
N
formamide group, MeO, and methylene) and the
C
SCH3
O
NMR spectroscopic data corroborated the structural
S
1
assignments based on H NMR analysis. Thus the struc-
O
N
H
4
O
N
ture of compound 2 was assigned as 1-methoxyindol-3-
ylmethylformamide and ultimately confirmed by synthesis.
H
O
5
The compound with t = 16.8 min (3) had a molecular for-
R
SCH3
1
mula of C H N O (HREIMS) and displayed in the H
NH
CHO
11 10
2
NMR spectrum the expected 10 protons (five aromatic,
1
3
OCH3
three MeO, and methylene). Both the C NMR (d 119.6)
S
c
N
N
H
ꢀ1
and FTIR (mmax 2249 cm ) spectra indicated the presence
of a nitrile group. Therefore, the chemical structure of 3
was provisionally deduced to be 1-methoxyindol-3-ylaceto-
nitrile. The proposed structure was confirmed by synthesis
O
OCH3
6
7
as described below. The compound with t = 9.5 min (7)
R
1
incubation period required for maximum production of
these elicited compounds was about four days. Of the elic-
ited metabolites, three of them (4–6) appeared to be known
compounds (UV spectra identical to those available in our
HPLC-UV library) whose chemical structures were con-
firmed upon comparison of their spectroscopic data with
those of authentic samples available in our laboratory.
displayed in the H NMR spectrum nine protons (four
aromatic, one aldehydic, OMe, and (N)H) consistent with
1
3
its molecular formula of C H NO (HREIMS) and
C
1
0
9
3
NMR spectroscopic data. Comparison of the spectroscopic
data with literature data indicated that the compound with
tR = 9.5 min was brassicanal C (7) (Monde et al., 1991),
whose optical rotation could not be measured accurately
due to the very small amount isolated. Eventually, this indi-
cation was confirmed by synthesis of brassicanal C (7), as
described below.
Thus, compound 4 (t = 3.8 min) was found to be isalexin,
R
previously isolated from rutabaga tubers (Pedras et al.,
2
004b), compound 5 (t = 12.2 min) was identified as
R
24
S-(ꢀ)-spirobrassinin (½aꢁ ꢀ 109, CH Cl , c 0.35) having
D
2
2
1
absolute configuration S as confirmed by H NMR chiral
2.2. Syntheses of 1–3 and 7
solvation (Pedras et al., 2004a), and compound 6 (t =
R
1
7.8 min) was found to be 1-methoxybrassitin (Takasugi
Synthesis of metabolites 1–3 and brassicanal C (7) was
carried out in order to confirm their structures and to
obtain sufficient amounts for testing for antifungal activity.
Caulilexin A (1) was prepared from 2-sulfanylindole-3-car-
boxaldehyde (8) (Pedras and Okanga, 1999) by treatment
first with iodine in pyridine followed by sodium thiometh-
oxide (Scheme 1). This one-pot process afforded caulilexin
A in 33% yield from aldehyde 8. Although brassicanal C
(7) has been isolated previously, its synthesis has not been
reported to date. Thus, a very simple preparation of 7,
similar to that of 1 but replacing sodium thiomethoxide
et al., 1988).
Of the unknown elicited compounds, the compound
1
with t = 16.6 min (1) showed in the H NMR spectrum
nine protons (four aromatic, one aldehydic, Me, and one
R
(
(
N)H) consistent with its molecular formula of C H NOS
10 9 2
HREIMS). The C NMR spectrum confirmed the pres-
1
3
ence of 10 carbons and suggested that the Me group was
attached to a sulfur atom. Further structural analysis
suggested the presence of a disulfide bridge and thus the
chemical structure of compound 1 was assigned as 2-dithi-
Table 1
a
Production of phytoalexins in cauliflower florets (Brassica oleraceae var. botrytis) upon elicitation with UV light (250 nm)
b
b
b
b
Phytoalexin
48 h lmol/100 g
72 h lmol/100 g
96 h lmol/100 g
120 h lmol/100 g
Caulilexin A (1)
Caulilexin B (2)
Caulilexin C (3)
Isalexin (4)
0.059 ± 0.004
0.084 ± 0.018
0.012 ± 0.001
0.062 ± 0.014
0.051 ± 0.007
0.17 ± 0.00
0.078 ± 0.019
0.18 ± 0.02
0.055 ± 0.013
0.23 ± 0.03
2.44 ± 0.65
0.075 ± 0.010
0.49 ± 0.09
0.17 ± 0.01
0.23 ± 0.05
0.039 ± 0.008
0.49 ± 0.05
3.97 ± 0.18
0.054 ± 0.006
0.43 ± 0.09
0.13 ± 0.08
0.064 ± 0.009
0.020 ± 0.001
0.46 ± 0.06
1.11 ± 0.25
0.030 ± 0.00
0.30 ± 0.09
S-(ꢀ)-spirobrassinin (5)
1-Methoxybrassitin (6)
Brassicanal C (7)
0.22 ± 0.05
a
Results are presented as means ± standard deviation.
Fresh floret tissue.
b

M.S.C. Pedras et al. / Phytochemistry 67 (2006) 1503–1509
CHO
SCH3
1505
ate 11. Subsequently, H O –Na WO -mediated oxidation
CHO
SH
2
2
2
4
i; ii
of 11 (Somei and Kawasaki, 1989), followed by treatment
with dimethyl sulfate afforded caulilexin C (3, Scheme 3)
in 10% yield, based on recovered indolyl-3-acetonitrile
(11). Although the yields of caulilexin C (3) obtained by
both methods are rather low, previous syntheses appear
to be less efficient (Acheson et al., 1984; Somei et al., 1985).
S
N
H
N
H
1
8
CHO
i; iii
OCH3
S
N
H
O
7
2.3. Antifungal activity
Scheme 1. Synthesis of caulilexin A (1) and brassicanal C (7); reagents and
2 3
conditions: (i) I , pyridine; (ii) CH SNa, 33% from 8; (iii) MeOH, 22%
from 8.
The antifungal activity of metabolites 1–3, 7, 13 and 14
against the phytopathogenic fungi Leptosphaeria maculans
Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode
(
ex Fr.) Desm.], Sclerotinia sclerotiorum (Lib.) de Bary and
Rhizoctonia solani Kuhn, was determined using a mycelial
radial growth antifungal assay. The phytoalexins brassica-
nals A (13) and C (7) and arvelexin (14) were used in the
bioassays as standards due to their structural resemblance
to caulilexins A (1) and C (3). The results of these anti-
fungal bioassays are shown in Table 2. Caulilexin A (1)
appeared to exhibit the strongest antifungal activity among
the various metabolites, causing complete growth inhibi-
with methanol, was developed (22% yield, Scheme 1). To
prepare caulilexin B (2), 1-methoxyindole-3-carboxalde-
hyde oxime (9) was reduced to the corresponding 1-meth-
oxyindol-3-ylmethyl amine (Pedras et al., 2003b), which
was then formylated in refluxing ethyl formate (85% yield,
Scheme 2). Caulilexin C (3) was prepared by Ac O-medi-
2
ated dehydration of 1-methoxyindol-3-yl acetaldoxime
(
12), which was obtained as previously reported (Pedras
ꢀ
4
et al., 2003b) (42% yield, Scheme 3). An alternate route
tion of R. solani (at 5.0 · 10 M) and S. sclerotiorum (at
ꢀ4
to 3 started with NaBH CN-AcOH-mediated reduction
3
1.0 · 10 M), that is substantially higher activity than
brassicanal A (13) but showed lower activity against L.
maculans (slightly lower than that of brassicanal A (13)).
Caulilexin C (3) caused complete growth inhibition
(
Gribble, 1998) of commercially available indol-3-ylaceto-
nitrile (10, Scheme 3) which yielded the indoline intermedi-
ꢀ
4
(
5.0 · 10 M) of R. solani and had a smaller effect on L.
maculans (77% inhibition), appearing to be slightly more
antifungal than arvelexin (14), whereas caulilexin B (2)
exhibited the lowest antifungal activity against the tested
fungi. In the TLC bioassay against C. cucumerinum, cauli-
O
OH
H
N
NH
i; ii
lexin A (1) caused complete growth inhibition at
N
N
ꢀ8
1
.0 · 10 M, whereas all other tested compounds caused
OCH3
OCH3
2
inhibition of mycelial growth at 100 times higher concen-
9
ꢀ
6
trations (1.0 · 10 M).
Scheme 2. Synthesis of caulilexin B (2); (i) NaBH
3
CN, TiCl
3
, AcONH
4
,
MeOH/H O; (ii) HCOOEt, reflux, 85% yield from 9.
2
OCH3
CHO
SCH3
CN
N
N
H
CN
CN
H
i
13
14
N
N
H
H
10
11
ii; iii
N OH
iv
3. Conclusions
CN
Three metabolites were isolated from stressed cauliflower
N
N
florets (1–3), their structures were elucidated, the syntheses
were carried out and the antifungal activity was established.
Compounds 1–3 display antifungal activity, are elicited
metabolites produced by cauliflower under abiotic elicita-
tion (UV light) and are not detectable in control plants.
Thus, metabolites 1–3 satisfy the conditions to be new
OCH3
OCH3
12
3
Scheme 3. Synthesis of caulilexin C (3); (i) NaBH
Na WO Æ 2H O; (iii) K CO , (CH SO , MeOH/H
0; (iv) Ac O, reflux, 42% yield from 12.
3
CN, AcOH; (ii) H
2 2
O ,
2
4
2
2
3
3
)
2
4
2
O; 10% yield from
1
2

1506
M.S.C. Pedras et al. / Phytochemistry 67 (2006) 1503–1509
Table 2
a
Antifungal activity of caulilexins A-C (1–3), brassicanals A (13) and C (7), and arvelexin (14) against Leptosphaeria maculans, Sclerotinia sclerotiorum and
Rhizoctonia solani
Compound
Concentration
M)
Leptosphaeria
b
maculans
Sclerotinia
sclerotiorum
Rhizoctonia
d
solani
c
(
ꢀ4
ꢀ4
ꢀ4
ꢀ5
ꢀ5
ꢀ4
ꢀ4
ꢀ4
ꢀ4
ꢀ4
ꢀ4
ꢀ4
ꢀ4
ꢀ4
ꢀ5
ꢀ4
ꢀ4
ꢀ4
ꢀ5
ꢀ4
ꢀ4
ꢀ4
Caulilexin A (1)
5.0 · 10
55 ± 7
39 ± 3
21 ± 7
10 ± 6
n.d.
31 ± 8
17 ± 6
n.i.
77 ± 2
45 ± 5
30 ± 10
70 ± 5
62 ± 7
30 ± 5
13 ± 4
70 ± 5
62 ± 7
30 ± 5
13 ± 4
59 ± 3
36 ± 3
15 ± 2
100 ± 0
100 ± 0
100 ± 0
90 ± 7
71 ± 12
31 ± 1
20 ± 2
n.i.
30 ± 8
n.i.
n.i.
33 ± 9
11 ± 4
n.i.
100 ± 0
83 ± 9
50 ± 8
13 ± 2
n.d.
18 ± 7
n.i.
n.i.
100 ± 0
80 ± 7
48 ± 11
53 ± 4
33 ± 7
n.i.
2
1
5
1
.5 · 10
.0 · 10
.0 · 10
.0 · 10
Caulilexin B (2)
Caulilexin C (3)
Brassicanal C (7)
5.0 · 10
2
1
.5 · 10
.0 · 10
5.0 · 10
2
1
.5 · 10
.0 · 10
5.0 · 10
2
1
5
.5 · 10
.0 · 10
.0 · 10
n.i.
n.i.
Brassicanal A (13)
5.0 · 10
33 ± 9
12 ± 5
8 ± 2
n.i.
37 ± 3
17 ± 3
n.i.
53 ± 4
33 ± 7
n.i.
2
1
5
.5 · 10
.0 · 10
.0 · 10
n.i.
Arvelexin (14)
5.0 · 10
70 ± 3
17 ± 7
n.i.
2
1
.5 · 10
.0 · 10
a
%
Inhibition = 100 ꢀ [(growth on treated/growth on control) · 100] ± SD; results are the means of at least three separate experiments; n.i., no
inhibition; n.d., not determined.
b
Results after 4 days of incubation.
Results after 2 days of incubation.
Results after 3 days of incubation.
c
d
phytoalexins, for which we propose the names caulilexins A
in mammalian systems (Seow et al., 2005; Hsu et al., 2005;
Eberhardt et al., 2005). Therefore, not surprisingly, some
indolyl phytoalexins (brassinin, spirobrassinin, brassilexin,
camalexin, 1-methoxyspirobrassinin) have been found to
possess significant activity against various cancer cells
(Mezencev et al., 2003). While it is likely that the effect of
phytoalexins on human health is not readily noticeable
(due to the very small amounts produced), structures like
caulilexin A (1) might be good leads for the design of com-
pounds with potential antiproliferative and chemopreven-
tive activities in mammals.
(
1), B (2) and C (3). Caulilexins 1–3 accumulate in florets of
cauliflower to levels comparable to those of other phytoal-
exins (Pedras et al., 2003a). Caulilexin A (1) is the first
example of an indolyldisulfide phytoalexin and is the most
active of all phytoalexins against S. sclerotiorum. Caulilexin
B (2) represents yet another example of a non-sulfur con-
taining indolyl phytoalexin. Caulilexin C (3) is the third
indol-3-ylacetonitrile found to be a phytoalexin; previously,
indolyl-3-acetonitrile was found to be a phytoalexin of B.
juncea and arvelexin (1-methoxyindol-3-ylacetonitrile) a
phytoalexin of stinkweed (Thlaspi arvense) (Pedras et al.,
2
003a). It is worthy to note that nitriles seem to be rather
active against L. maculans and R. solani but less active
against S. sclerotiorum. Nitrile 3 was previously isolated
from Chinese cabbage (Nomoto and Tamura, 1970) and
was also obtained from incubations of an indole glucosino-
late with myrosinase (Hanley et al., 1990; Agerbirk et al.,
4. Experimental
4.1. General
All chemicals were purchased from Sigma–Aldrich Can-
ada Ltd., Oakville, ON. Analytical HPLC analysis was car-
ried out with a high performance liquid chromatograph
equipped with quaternary pump, automatic injector, and
photodiode array detector (wavelength range 190–
600 nm), degasser, and a Hypersil ODS column (5 lm par-
ticle size silica, 4.6 i.d. ·200 mm), equipped with an in-line
filter. Mobile phase: H O–CH CN, 75:25 to 100% CH CN,
1
998). However, this is the first time that compound 3 is
reported to be an induced metabolite and to display anti-
fungal activity. In agreement with previous results (Pedras
et al., 2004b, 2003a), the current bioassay results using three
different plant pathogens suggest that crucifer phytoalexins
display selective antifungal activities.
Crucifer vegetables are known to be biologically active
against carcinogenesis due to their ability to induce phase
II conjugating enzymes, and have additional positive effects
2
3
3
in 35 min, linear gradient, and 1.0 ml/min flow rate. Sam-
ples were dissolved either in CH CN or in MeOH.
3

M.S.C. Pedras et al. / Phytochemistry 67 (2006) 1503–1509
1507
Specific rotations, [a] were determined at ambient tem-
MeOH, 9:1: silica gel reversed phase, CH CN–H O, 9:1);
3 2
D
perature on a polarimeter using a 1 ml, 10 cm path length
S-(ꢀ)-spirobrassinin (5, 4 mg), FCC (silica gel, hexane–
ꢀ
1
2
ꢀ1
cell; the units are 10 deg cm g and the concentrations
c) are reported in g/100 ml. NMR spectra were recorded
Et O, 1:1; silica gel reversed phase, CH CN–H O, 1:1); 1-
2
3
2
(
methoxybrassitin (6, 2 mg), FCC (silica gel, hexane–Et O,
2
1
on 500 MHz spectrometers. For H NMR (500 MHz) the
3:2; silica gel reversed phase, CH CN–H O, 1:1); brassica-
3
2
chemical shifts (d) are reported in parts per million (ppm)
nal C (7, 1 mg), FCC (silica gel, CH Cl –MeOH, 9:1; silica
2 2
relative to TMS. The d values were referenced to CD CN
gel reversed phase, CH CN–H O, 4:1).
3 2
3
(
CD HCN at 1.94 ppm). Other conditions are as previ-
2
ously reported (Pedras and Okanga, 1999).
Plant material: cauliflower florets were purchased from
local markets.
4
.4. Caulilexin A (1)
A solution of 2-sulfanylindole-3-carboxaldehyde (8,
5
3 mg, 0.30 mmol) in pyridine (1 ml) was added to a solu-
4
.2. Fungal cultures and antifungal bioassays
tion of iodine (76 mg, 0.30 mmol) in pyridine (0.5 ml),
cooled to 0 ꢁC. The mixture was stirred for 10 min at
Leptosphaeria maculans isolate BJ-125, R. solani isolate
0
ꢁC, CH SNa (42 mg, 0.60 mmol) was added and the cool-
3
AG 2-1 and S. sclerotiorum clone #33 were obtained from
AAFC, Saskatoon, Canada. Cultures were handled as
described previously: L. maculans (Pedras and Okanga,
999); R. solani (Pedras and Liu, 2004); S. sclerotiorum
Pedras and Ahiahonu, 2002). Radial growth antifungal
bioassays were carried out as described previously: L. mac-
ulans (Pedras et al., 2003a); R. solani (Pedras and Liu,
004); S. sclerotiorum (Pedras and Ahiahonu, 2002). Clad-
osporium cucumerinum TLC bioassay was performed as
described previously (Pedras and Sorensen, 1998).
ing bath was removed. After stirring for 2 h at r.t., the mix-
ture was diluted with 1.5 M H SO (15 ml) and was
2
4
extracted with EtOAc. The combined organic extract was
dried, concentrated and the residue subjected to FCC hex-
ane–EtOAc (4:1) to afford caulilexin A (1) (22 mg, 33%) as
1
(
a slightly yellow solid, m.p. 114–116 ꢁC. HPLC t =
R
1
1
6.2 min; H NMR (500 MHz, CD CN): d 10.42 (br s,
3
2
D O exchangeable, 1H), 10.20 (s, 1H), 8.09 (d, J = 8 Hz,
2
1
7
H), 7.56 (d, J = 8 Hz, 1H), 7.33 (dd, J = 8, 8 Hz, 1H),
.28 (dd, J = 8, 8 Hz, 1H), 2.60 (s, 3H). C NMR
1
3
(
1
125 MHz, CD CN): d 185.5 (s), 143.8 (s), 138.1 (s),
3
4.3. Isolation and identification of phytoalexins from UV
elicited cauliflower florets
28.0 (s), 126.7 (s), 125.4 (d), 124.1 (d), 121.0 (d), 113.1
+
(
(
d), 24.1 (q). HREIMS m/z [M ] measured: 223.0121
223.0126 calc. for C H NOS ); EIMS m/z (% relative
1
0
9
2
Cauliflowers were cut vertically in 10–15 mm thick slices
or pieces and were incubated for 24 h in a closed chamber,
+
abundance): 223 [M ] (58), 176 (100), 121 (19), 77 (16).
FTIR mmax: 3213, 2920, 1632, 1578, 1433, 1373, 1242,
8
1
00% humidity. The slices were irradiated with a UV light
ꢀ
1
46, 745 cm . UV (MeOH) kmax (loge): 213 (4.4), 252
(
250 nm) for 15 min on each side. Control tissues were not
(4.2), 307 (4.0) nm.
UV elicited but otherwise were treated similarly. Control
and elicited tissues were incubated further at 20 ꢁC in dark-
ness; one of the control slices and one of the elicited slices
were collected at 24 h intervals for five days. Each slice was
ground in a blender and the resulting material was stirred
in EtOAc (150 ml) for 12 h. The macerates were filtered,
the filtrates were dried and concentrated. The residues were
4.5. Caulilexin B (2)
Crude 1-methoxyindolyl-3-methylamine (34 mg, 0.20
mmol) obtained from 1-methoxyindole-3-carboxaldehyde
oxime (9) (Pedras et al., 2003b) was dissolved in HCOOEt
(2 ml) and the mixture was refluxed for 16 h with stirring.
The mixture was concentrated and the residue was sub-
jected to FCC (CH Cl –MeOH, 95:5) to afford caulilexin
B (23 mg, 85% based on oxime 11), m.p. 71–73 ꢁC,
CH Cl –MeOH. HPLC: t = 8.9 min. H NMR (500
MHz, CD CN): d 8.14 (s, 1H), 7.65 (d, J = 8 Hz, 1H),
7.46 (d, J = 8 Hz, 1H), 7.38 (s, 1H), 7.27 (dd, J = 8, 8 Hz,
dissolved in CH CN and were analysed by HPLC.
3
To isolate phytoalexins, cauliflower florets (12 kg) were
treated as described above. After four days of incubation,
slices were ground in a blender and the resulting material
was stirred in EtOAc (18 l) for 24 h. The macerates were fil-
tered, the filtrates were dried and were concentrated. The
residue (4.05 g) was fractionated by FCC (CH Cl –hexane
2
2
1
2
2
R
3
2
2
and CH Cl –MeOH gradient elution). Fractions contain-
1H), 7.13 (dd, J = 8, 8 Hz, 1H), 6.76 (br s, D O exchange-
able, 1H), 4.52 (d, J = 6 Hz, 1H), 4.07 (s, 3H); C NMR
2
2
2
1
3
ing elicited metabolites (HPLC analysis) were combined
and were further fractionated using FCC, preparative
TLC and reversed phase chromatography. Elicited metab-
olites were obtained as follows: caulilexin A (1, 1 mg), pre-
parative TLC (CH Cl –Et O, 95:5); caulilexin B (2, 1 mg),
(125 MHz, CD CN): d 162.2 (s), 133.8 (s), 124.3 (s),
3
124.0 (d), 123.8 (d), 121.2 (d), 120.6 (d), 110.6 (s), 109.7
+
(d), 67.0 (q), 33.7 (t); HREIMS m/z [M ] measured:
204.0898 (204.0899 calc. for C H N O ); EIMS m/z (%
2
2
2
11 12
2
2
+
FCC (hexane–Et O, 1:4, reversed phase chromatogra-
relative abundance) 204 [M ] (92), 173 (64), 145 (39), 118
2
phy, CH CN–H O, 3:7); caulilexin C (3, 1 mg), FCC (silica
(100), 91 (21); FTIR mmax: 3282, 2935, 1661, 1523, 1452,
1382, 1226, 1101, 1010, 740 cm ; UV (MeOH)max (loge):
3
2
ꢀ1
gel, hexane–EtOAc, 9:1; silica gel reversed phase CH CN–
3
H O, 1:3); isalexin (4, 1 mg), FCC (silica gel, CH Cl –
221 (4.4), 290 (3.7) nm.
2
2
2

1508
M.S.C. Pedras et al. / Phytochemistry 67 (2006) 1503–1509
4
4
9
.6. Caulilexin C (3)
Acknowledgments
.6.1. Method A
A solution of 1-methoxyindolyl-3-acetaldoxime (12,
5 mg, 0.46 mmol) (Pedras et al., 2003b) in Ac O (1 ml)
Financial support from the Natural Sciences and Engi-
neering Research Council of Canada (NSERC Discovery
Grant to M.S.C.P), and the University of Saskatchewan
(graduate teaching assistantship to M.G.S.) is gratefully
acknowledged.
2
was stirred under reflux for 1 h. The reaction mixture was
concentrated and the residue was subjected to FCC
(
CH Cl –MeOH, 98:2) to afford caulilexin C (3, 36 mg,
2 2
4
2%) as a semi-solid.
References
4
.6.2. Method B
To a stirred solution of indol-3-ylacetonitrile (10, 100 mg,
Acheson, R.M., Aldridge, G.N., Choi, M.C.K., Nwankwo, J.O., Ruscoe,
M.A., Wallis, J.D., 1984. An improved preparation of 1-hydroxyindole
and the synthesis of some related 3-carboxylic acids and 1-methoxy-
indole-3-acetonitrile. Journal of Chemical Research (Miniprints),
0
.64 mmol) in AcOH (2 ml), NaBH CN (120 mg, 1.8 mmol)
3
was added and the reaction mixture was stirred at r.t. After
h, the reaction mixture was diluted with H O (40 ml) and
3
2
1
301–1319.
was extracted with CH Cl . The combined organic extract
was dried, was concentrated and the residue was subjected
2
2
Agerbirk, N., Olsen, C.E., Sorensen, H., 1998. Initial and final products,
nitriles, and ascorbigens produced in myrosinase-catalyzed hydrolysis
of indole glucosinolates. Journal of Agricultural and Food Chemistry
to FCC (CH Cl ) to afford indoline 11 (40 mg, 40% yield)
2
2
4
6, 1563–1571.
and recovered indolyl-3-acetonitrile (10, 60 mg, 60%). Indo-
line 11 (40 mg, 0.25 mmol) was dissolved in MeOH (2 ml)
Bailey, J.A., Mansfield, J.W. (Eds.), 1982. Phytoalexins. Blackie and Son,
Glasgow, UK, p. 334.
and a solution of Na WO Æ 2H O (22 mg, 0.07 mmol) in
2
4
2
Eberhardt, M.V., Kobira, K., Keck, A.S., Juvik, J.A., Jeffery, E.H., 2005.
Correlation analyses of phytochemical composition, chemical, and
cellular measures of antioxidant activity of broccoli (Brassica oleracea
L. var. italica). Journal of Agricultural and Food Chemistry 53, 7421–
H O (160 ll) was added with stirring. The stirred mixture
2
was cooled to ꢀ18 ꢁC (ice + NaCl) and an aqueous solution
of H O (300 ll, 3 mmol) was added dropwise over a 15 min
2
2
7
431.
period. After stirring for further 30 min at ꢀ18 ꢁC, K CO
2
3
Gribble, G.W., 1998. Sodium borohydride in carboxylic acid media: a
phenomenal reduction system. Chemical Society Reviews 27, 395–
404.
Hanley, A.B., Parsley, K.R., Lewis, J.A., Fenwick, G.R., 1990. Chemistry
of indole glucosinolates: intermediacy of indol-3-ylmethyl isothiocya-
nates in the enzymic hydrolysis of indole glucosinolates. Journal of
Chemical Society, Perkin Transactions 1, 2273–2276.
Hsu, J.C., Zhang, J., Dev, A., Wing, A., Bjeldanes, L.F., Firestone, G.L.,
2005. Indole-3-carbinol inhibition of androgen receptor expression and
down regulation of androgen responsiveness in human prostate cancer
cells. Carcinogenesis 26, 1896–1904.
Hurter, J., Ramp, T., Patrian, B., Stadler, E., Roessingh, P., Baur, R., De
Jong, R., Nielsen, J.K., Winkler, T., Richter, W.J., Muller, D., Ernst,
B., 1999. Oviposition stimulants for the cabbage root fly: isolation
from cabbage leaves. Phytochemistry 51, 377–382.
Llorach, R., Gil-Izquierdo, A., Ferreres, F., Tomas-Barberan, F.A., 2003.
HPLC-DAD-MS/MS ESI characterization of unusual highly glycos-
ylated acylated flavonoids from cauliflower (Brassica oleracea L. var.
botrytis). Journal of Agricultural and Food Chemistry 51, 3895–
3899.
Menard, R., Larue, J.P., Silue, D., Thouvenot, D., 1999. Glucosinolates in
cauliflower as biochemical markers for resistance against downy
mildew. Phytochemistry 52, 29–35.
(
300 mg, 2.2 mmol) and (CH ) SO (80 ll, 0.85 mmol) were
3 2 4
added and the mixture was stirred for 5 h at r.t. The mixture
was diluted with H O (5 ml) and was extracted with CH Cl ,
2
2
2
with the combined organic extract dried and concentrated.
The residue was subjected to FCC (EtOAc–hexane, 3:7) to
afford 7 mg (10% yield from indolyl-3-acetonitrile (10)) of
caulilexin C (3).
1
HPLC: t = 16.8 min. H NMR (500 MHz, CD CN): d
R
3
7
7
4
.65 (d, J = 8 Hz, 1H), 7.50 (d, J = 8 Hz, 1H), 7.46 (s, 1H),
.32 (dd, J = 8, 8 Hz, 1H), 7.18 (dd, J = 8, 8 Hz, 1H),
1
3
.24 (s, 3H); 3.91 (s, 2H); C NMR (125 MHz, CDCl3):
d 133.4 (s), 124.3 (d), 123.6 (s), 123.4 (d), 121.3 (d), 119.7
d), 119.6 (s), 109.6 (d), 102.2 (s), 67.0 (q), 14.5 (t); HRE-
(
+
IMS m/z [M ] measured: 186.0795 (186.0793 calc. for
C H N O); EIMS m/z (% relative abundance) 186 [M ]
+
1
1
10
2
(
2
98), 171 (37), 155 (100), 128 (58), 101 (23); FTIR mmax:
937, 2249, 1454, 1359, 1240, 1097, 1029, 739 cm ; UV
ꢀ1
(
MeOH)max (loge): 219 (4.4), 271 (3.8) nm.
Mezencev, R., Mojzis, J., Pilatova, M., Kutschy, P., 2003. Antiprolifer-
ative and cancer chemopreventive activity of phytoalexins: focus on
indole phytoalexins from crucifers. Neoplasma 50, 239–245.
Monde, K., Sasaki, K., Shirata, A., Takasugi, M., 1991. Brassicanal C and
two dioxindoles from cabbage. Phytochemistry 30, 2915–2917.
Nomoto, M., Tamura, S., 1970. Isolation and identification of indole
derivatives in clubroots of Chinese cabbage. Agricultural and Biolog-
ical Chemistry 34, 1590–1592.
Pedras, M.S.C., Ahiahonu, P.W.K., 2002. Probing the phytopathogenic
stem rot fungus with phytoalexin analogues: unprecedented glucosy-
lation of camalexin and 6-methoxycamalexin. Bioorganic & Medicinal
Chemistry 10, 3307–3312.
4
.7. Brassicanal C (7)
A solution of 2-sulfanylindole-3-carboxaldehyde (8,
0 mg, 0.28 mmol) in MeOH (1.5 ml) was added to a solu-
5
tion of iodine (140 mg, 0.55 mmol) in MeOH (1.5 ml) and
pyridine (200 ll) and the mixture was stirred for 4 h at
r.t. The reaction mixture was concentrated and the residue
was subjected to FCC (hexane–EtOAc, 4:1) to afford brass-
icanal C (14 mg, 22%) as a slightly yellow solid, m.p. 148–
1
49 ꢁC, hexane–EtOAc. HPLC: t = 9.5 min. Except for
R
Pedras, M.S.C., Chumala, P.B., Suchy, M., 2003a. Phytoalexins from
Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria
maculans: structures, syntheses and antifungal activity. Phytochemistry
64, 949–956.
the optical rotation, the spectroscopic data of the synthetic
sample were identical to those previously reported (Monde
et al., 1991).

M.S.C. Pedras et al. / Phytochemistry 67 (2006) 1503–1509
1509
Pedras, M.S.C., Hossain, M., Sarwar, M.G., Montaut, S., 2004a.
Determination of the enantiomeric purity of the phytoalexins spiro-
Somei, M., Kawasaki, T., 1989. A new and simple synthesis of 1-
hydroxyindole derivatives. Heterocycles 29, 1251–1254.
Somei, M., Sato, H., Komura, N., Kaneko, C., 1985. Synthetic study for
1-methoxyindoles and 1-methoxy-2-oxindoles. Heterocycles 23, 1101–
1106.
Suchy, M., Kutschy, P., Monde, K., Goto, H., Harada, N., Takasugi, M.,
Dzurilla, M., Balentova, E., 2001. Synthesis, absolute configuration
and enantiomeric enrichment of a cruciferous oxindole phytoalexin,
(S)-(ꢀ)-spirobrassinin, and its oxazoline analog. Journal of Organic
Chemistry 66, 3940–3947.
1
brassinins by H NMR using chiral solvation. Bioorganic & Medicinal
Chemistry Letters 14, 5469–5471.
Pedras, M.S.C., Jha, M., Ahiahonu, P.W.K., 2003b. The synthesis and
biosynthesis of phytoalexins produced by cruciferous plants. Current
Organic Chemistry 7, 1635–1647.
Pedras, M.S.C., Liu, J., 2004. Designer phytoalexins: probing camalexin
detoxification pathways in the phytopathogen Rhizoctonia solani.
Organic & Biomolecular Chemistry 2, 1070–1076.
Pedras, M.S.C., Montaut, S., Suchy, M., 2004b. Phytoalexins from the
crucifer rutabaga: structures, syntheses, biosyntheses and antifungal
activity. Journal of Organic Chemistry 69, 4471–4476.
Pedras, M.S.C., Okanga, F.I., 1999. Strategies of cruciferous pathogenic
fungi: detoxification of the phytoalexin cyclobrassinin by mimicry.
Journal of Agricultural and Food Chemistry 47, 1196–1202.
Pedras, M.S.C., Okanga, F.I., Zaharia, I.L., Khan, A.Q., 2000. Phyto-
alexins from crucifers: synthesis, biosynthesis, and biotransformation.
Phytochemistry 53, 161–176.
Takasugi, M., Monde, K., Katsui, N., Shirata, A., 1987. Spirobrassinin, a
novel sulfur-containing phytoalexin from the daikon Raphanus sativus
L. var. hortensis (Cruciferae). Chemistry Letters, 1631–1632.
Takasugi, M., Monde, K., Katsui, N., Shirata, A., 1988. Novel sulfur-
containing phytoalexins from the Chinese cabbage Brassica campestris
L. ssp. pekinensis (Cruciferae). Bulletin of the Chemical Society of
Japan 61, 285–289.
Tian, Q.G., Rosselot, R.A., Schwartz, S.J., 2005. Quantitative determi-
nation of intact glucosinolates in broccoli, broccoli sprouts, Brussels
sprouts, and cauliflower by high-performance liquid chromatography–
electrospray ionization–tandem mass spectrometry. Analytical Bio-
chemistry 343, 93–99.
Pedras, M.S.C., Sorensen, J.L., 1998. Phytoalexin accumulation and
production of antifungal compounds by the crucifer wasabi. Phyto-
chemistry 49, 1959–1965.
Seow, A., Vainio, H., Yu, M.C., 2005. Effect of glutathione-S-
transferase polymorphisms on the cancer preventive potential of
isothiocyanates: an epidemiological perspective. Mutation Research
Valette, L., Fernandez, X., Poulain, S., Lizzani-Cuvelier, L., Loiseau,
A.M., 2006. Chemical composition of the volatile extracts from
Brassica oleracea L. var. botrytis ‘‘Romanesco’’ cauliflower seeds.
Flavor and Fragrance Journal 21, 107–110.
592, 58–67.