The cruciferous phytoalexins rapalexin A, brussalexin A and erucalexin: Chemistry and metabolism in Leptosphaeria maculans

Article abstract of DOI:10.1016/j.bmc.2012.05.020

The interactions of the cruciferous phytoalexins rapalexin A (1), brussalexin A (2) and erucalexin (3) with the fungal plant pathogen Leptosphaeria maculans were analyzed and their inhibitory activities against this pathogen were determined. The reaction of L. maculans to N-methyl S-(indolyl-3-methyl)carbamodithioate, an analogue of brussalexin A, was also investigated. Rapalexin A was resistant to metabolism and was the most inhibitory of all compounds tested, suggesting that increasing concentrations of rapalexin A in Brassica species would improve their disease resistance to L. maculans. By contrast, erucalexin was quickly detoxified by reduction to yield 3-dihydroerucalexins. The relative configurations of the diastereomeric mixture of dihydroerucalexins were established by 1D ¹H nuclear Overhauser enhancement spectroscopy (NOE). Brussalexin A was chemically unstable decomposing mainly to indolyl-3-methanol, a product with anti-cancer properties. For this reason, brussalexin A might be of interest to use as a prodrug.

Full text of DOI:10.1016/j.bmc.2012.05.020

Bioorganic & Medicinal Chemistry 20 (2012) 3991–3996
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The cruciferous phytoalexins rapalexin A, brussalexin A and erucalexin:
Chemistry and metabolism in Leptosphaeria maculans
⇑
M. Soledade C. Pedras , Vijay K. Sarma-Mamillapalle
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9
a r t i c l e i n f o
a b s t r a c t
Article history:
The interactions of the cruciferous phytoalexins rapalexin A (1), brussalexin A (2) and erucalexin (3) with
the fungal plant pathogen Leptosphaeria maculans were analyzed and their inhibitory activities against
this pathogen were determined. The reaction of L. maculans to N-methyl S-(indolyl-3-methyl)carbamod-
ithioate, an analogue of brussalexin A, was also investigated. Rapalexin A was resistant to metabolism
and was the most inhibitory of all compounds tested, suggesting that increasing concentrations of rapa-
lexin A in Brassica species would improve their disease resistance to L. maculans. By contrast, erucalexin
was quickly detoxified by reduction to yield 3-dihydroerucalexins. The relative configurations of the dia-
stereomeric mixture of dihydroerucalexins were established by 1D 1H nuclear Overhauser enhancement
spectroscopy (NOE). Brussalexin A was chemically unstable decomposing mainly to indolyl-3-methanol,
a product with anti-cancer properties. For this reason, brussalexin A might be of interest to use as a
prodrug.
Received 6 April 2012
Revised 30 April 2012
Accepted 9 May 2012
Available online 17 May 2012
Keywords:
Antifungal
Brassicaceae
Biotransformation
Brussalexin A
Crucifer
Detoxification
Dihydroerucalexin
Erucalexin
Ó 2012 Elsevier Ltd. All rights reserved.
Leptosphaeria maculans
Phoma lingam
Phytoalexin
Rapalexin A
1
. Introduction
paldoxins (phytoalexin detoxification inhibitors) have been de-
signed and synthesized as an alternative to commercial
fungicides.
1
,8
Rapalexin A (1), brussalexin A (2) and erucalexin (3) are crucif-
erous phytoalexins, that is, plant defense metabolites produced de
novo in response to biotic or abiotic stress.1 These plant metabo-
lites have uncommon chemical structures: rapalexin A (1) is the
Among the many cruciferous phytoalexins reported to date, it is
not certain which ones are resistant to fungal transformation,
although several reports regarding metabolism of phytoalexins
by plant pathogens have been published over the last few years.1
Such work is of tremendous interest as it provides targets useful
to stop pathogenic fungi from invading plants and identifies phyto-
alexins of interest to engineer plants with higher disease resistance
levels. Considering the unusual structures and antifungal activities
of rapalexin A (1), brussalexin A (2) and erucalexin (3), it was of
great importance to establish their resistance to degradation by
the blackleg fungus, one of the major pathogens of Brassica species
[Leptosphaeria maculans (Desm.) Ces. et de Not. asexual stage Pho-
,2
3
first example of an indole isothiocyanate, brussalexin A (2) con-
tains the only known N-allyl thiolcarbamate,4 and erucalexin (3)
5
has a carbon substituent at C-2 of 3-oxoindole, instead of the com-
mon C-3 substitution found in all cruciferous phytoalexins.
Although erucalexin (3) has been isolated only from the wild cru-
cifer dog mustard (Erucastrum gallicum L.), its biosynthetic precur-
sor is 1-methoxybrassinin (4a), a phytoalexin commonly produced
6
in Brassica species. Despite the variety of phytoalexins produced
in crucifers for protection against microbial pathogens, fungi are
able to counter-attack by producing enzymes that metabolize
9
ma lingam (Tode ex Fr.) Desm.]. Among more than 40 known cru-
7
and detoxify these plant defenses. Recently, a few fungal enzymes
ciferous phytoalexins, L. maculans is known to metabolize and
detoxify the phytoalexins brassinin (4), cyclobrassinin (5) and
that mediate these transformations were isolated from phytopath-
2
1,2
ogenic fungi and found to be specific targets that could stop plant
brassilexin (6), but not spirobrassinin (7) or camalexin (8). Since
fungal pathogens from invading crucifers. Toward this objective,
such transformations appear to involve enzymes that are different
in each fungal species, the structures of the detoxification products
are not predictable. For example, in a case where bioinformatic
comparison of two phylogenetically related fungal species sug-
gested that the transformation of cruciferous phytoalexins would
⇑
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966 4730.
E-mail addresses: s.pedras@usask.ca, soledade.pedras@usask.ca (M. Soledade C.
Pedras).
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.020

3
992
M. Soledade C. Pedras, V. K. Sarma-Mamillapalle / Bioorg. Med. Chem. 20 (2012) 3991–3996
involve glucosyl transferases,10 detoxification in one species oc-
curred via oxidative degradation or hydrolysis, but not through
glucosylation. Hence, for now, even with the availability of many
genome sequences of plant pathogens and bioinformatic analysis,
it is not feasible to predict metabolic reactions of fungal pathogens
to cruciferous phytoalexins.
Scheme 1. Synthesis of brussalexin (2) and N-methyl indolyl-3-methylcarbamod-
ithioate (10). Reagents and conditions: (i) NH NH O, THF, 80 °C, 84%; (ii) Et N,
ꢀH
allyl isocyanate, CH Cl , 86%; (iii) Et N, methyl isothiocyanate, CH Cl , 76%.
2
2
2
3
2
2
3
2
2
3
-indolylmethylthioacetate (9) was used to prepare N-methyl
S-(indolyl-3-methyl)carbamodithioate (10), a brassinin analogue
havingindolyl-3-methyl attached to sulfur instead of nitrogen
(Scheme 1).
The antifungal activity of each compound against L. maculans
was determined using a mycelial growth assay reported in the
experimental (Table 1). The activity of rapalexin A (1) was the
highest, completely inhibiting mycelial growth at 0.50 mM,
whereas brussalexin A (2) and erucalexin (3) displayed similar
activities causing ca. 40% inhibition at 0.50 mM and N-methyl S-
(indolyl-3-methyl)carbamodithioate (10) was slightly more inhib-
itory at similar concentration (ca. 55%).
In continuation of previous work, we analyzed for the first time
the chemistry involved in the interactions of rapalexin A (1), brus-
salexin A (2) and erucalexin (3) with L. maculans and determined
their antifungal activities. In addition, the reaction of L. maculans
to N-methyl S-(indolyl-3-methyl)carbamodithioate (10), an ana-
logue of brassinin (4) and brussalexin A (2), was also investigated.
Here we report that rapalexin A (1) was resistant to metabolism
and was the most inhibitory of all compounds tested, suggesting
that increasing concentrations of rapalexin A in Brassica species
would improve disease resistance to L. maculans. By contrast, eru-
calexin (2) was detoxified by reduction, and brussalexin A (2) was
chemically unstable decomposing to indolyl-3-methanol (11),
which was slowly oxidized by L. maculans to the corresponding
aldehyde.
2.2. Metabolism, synthesis and antifungal activity of
metabolites
Rapalexin A (1) dissolved in acetonitrile was added to cultures
of L. maculans and also to non-inoculated media (control) to obtain
a final concentration of 0.10 mM. HPLC analysis of neutral, basic
and acidic extracts after different incubation times, up to 96 h, re-
vealed that rapalexin A (1) was not metabolized (stable both in
fungal cultures and in media, giving comparable recoveries). Un-
like rapalexin A (1), brussalexin A (2) was not stable in either med-
ium only (no fungal cells) or in water; HPLC analyses of solutions of
brussalexin (2) in water established that upon standing it decom-
posed spontaneously to indolyl-3-methanol (11) over a period of
72 h (t1/2 = 24 h) (Scheme 2). Although brussalexin (2) was not sta-
ble in culture medium or in water, some stabilizing factors appear
to exist in plant tissues, since it has been consistently detected and
2
. Results and discussion
2
1
.1. Chemical synthesis and antifungal activity of phytoalexins
–3 and compound 10
3
5
4
The syntheses of rapalexin A (1), and erucalexin (3) were car-
isolated from Brussels sprouts. In this connection, it is of interest
ried out as previously published, whereas the synthesis of brussa-
lexin A (2) was improved by omitting the t-Boc-protecting and
to note that the major decomposition product of 2, indolyl-3-
methanol (11) has also been isolated from diverse crucifers, includ-
ing Brassica species. For this reason, compound 11was tested for
stability in fungal cultures, water, and minimal medium. In med-
ium, indolyl-3-methanol (11) decomposed completely in 6 h to
4
1
2
deprotecting steps. In brief, 3-indolylmethylthioacetate (9) was
hydrolyzed using hydrazine hydrate11 followed immediately
by
condensation
with
allyl
isocyanate.
Similarly,
Table 1
Antifungal activity of phytoalexins 1–3 and N-methyl S-(indolyl-3-methyl)carbamodithioate (10), against Leptosphaeria maculans
Compound
Inhibition ± SD (%)a
0
.50 mM
0.20 mM
53 ± 6b
0.10 mM
Rapalexin A (1)
Brussalexin A (2)
Erucalexin (3)
N-methyl S-(indolyl-3-methyl)carbamodithioate (10)
c.i.b
21 ± 6b
c
c
d
7 ± 4c
43 ± 3
40 ± 4
25 ± 2
e
c
17 ± 4
6 ± 2
61 ± 5d
35 ± 3
e
17 ± 3b
a
The percentage of inhibition was calculated using the formula:% inhibition = 100 ꢁ [(growth on amended/growth in control) ꢂ 100]; values are averages of three
independent experiments conducted in triplicate; c.i. = complete inhibition; for statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with
adjusted set at 0.05; n = 3; different letters in the same column (b–d) indicate significant differences (P <0.05).
a

M. Soledade C. Pedras, V. K. Sarma-Mamillapalle / Bioorg. Med. Chem. 20 (2012) 3991–3996
3993
by HPLC. These chromatograms showed a major peak with
= 9.2 min and minor peak with t = 11.9 min. HPLC-ESI-MS anal-
ysis indicated that the compound with t = 9.2 min corresponded
t
R
R
R
to a compound two mass units higher than erucalexin (3). At-
tempts to purify this unknown metabolite by FCC on silica gel or
alumina were unsuccessful, resulting in conversion of the com-
pound with t
ally, the compound with t
reversed phase silica gel. HRMS-EI analysis of the purified material
indicated a molecular formula of C12 , two mass units
R
= 9.2 min into demethoxyerucalexin (14). Eventu-
R
= 9.2 min was purified by FCC using
14 2 2 2
H N O S
1
higher than erucalexin (3). The H NMR spectrum of this com-
pound showed SMe and OMe protons, four aromatic protons and
two sets of methylene protons at d
d, J = 16 Hz, 1.2H), 4.49 (d, J = 16.5 Hz, 0.6H), 4.45 (d, J = 16.5 Hz,
.6H), plus signals at d 5.24 (s, 0.3 H), 4.94 (s 0.6H), and 4.09(s,
ca. 1H, D O exchangeable), indicating the presence of two diaste-
H
4.65 (d, J = 16 Hz, 1.2H), 4.26
(
0
H
2
reomers in a 2:1 ratio. These signals suggested that the likely struc-
ture was a diastereomeric mixture of dihydroerucalexin (13), the
structures of which were further confirmed by synthesis, as fol-
Scheme 2. Spontaneous transformation of brussalexin (2) and N-methyl S-(3-
indolylmethyl) carbamodithioate (10) into indolyl-3-methanol (11) in medium and
water solutions.
4
lows. Racemic erucalexin (3) was reduced with NaBH to yield a
mixture of diastereomers in a 3:1 ratio. The major isomer could
be purified while the minor isomer was obtained as 1:1 mixture
of diastereomers. The ratio of diastereomers of dihydroerucalexin
(13), formed by fungal transformation of erucalexin (3), was deter-
0
13
yield mainly 3,3 -diindolylmethane (12) (ca. 45%) and undeter-
1
mined minor products, while in water decomposed very slowly
mined by H NMR spectrum of freeze-dried fungal cultures incu-
1
4
1
(t
1/2 >72 h). Indolyl-3-methanol (11) was incubated with mycelia
bated with erucalexin (3). The H NMR spectral data indicated
of L. maculans in water and extracts of cultures were analyzed by
HPLC over a period of 48 h, only traces (<2%) of indole-3-carboxal-
dehyde were detected, indicating a relatively slow transformation.
The antifungal activity of 11 against L. maculans was similar to that
of 10 (52 ± 8 at 0.50 mM, 27 ± 5 at 0.20 mM, and19 ± 3 at
the presence of two sets of proton signals that corresponded to
the major and minor diastereomers in a 2:1 ratio. That is, the major
product was formed in both chemical and enzymatic reductions of
racemic erucalexin (3), although the selectivity in chemical reduc-
tion was higher (3:1 vs 2:1). It is somewhat puzzling to find that
the chemical reduction of erucalexin (3) with NaBH4 affords a
higher diastereomeric ratio (3:1) than the enzymatic process
(2:1), however the reason for the difference is not understood at
this point. Curves for the transformation of racemic erucalexin
(3) by L. maculans and product formation are shown in Fig. 1.
Next, the relative stereochemistry at C-2 and C-3 of each diaste-
0
.10 mM). Previously, indolyl-3-methanol (11) was found to be
metabolized much faster (ca. 12 h) by the plant fungal pathogens
Rhizoctonia solani and Sclerotinia sclerotiorum.14
To probe the structural component(s) contributing to the lower
stability of brussalexin A (2) in aqueous solutions, N-methyl S-(3-
indolylmethyl)carbamodithioate (10) was similarly incubated in
water, samples were withdrawn at different times, extracted and
the extracts analyzed by HPLC. As in the case of brussalexin A
1
reomer 13a and 13b was established based on the results of 1D H
nuclear Overhauser enhancement spectroscopy (NOE). NOE differ-
ence experiments were carried out using a synthetic mixture of
diastereomers 13a and 13b (3:1) (Fig. 2A). When the methine pro-
ton (H-3) of the major diastereomer at dH 4.94 ppm was irradiated,
the intensity (0.3%) of the methylene proton signal at dH 4.26 in-
creased (Fig. 2C); similarly, when the methylene proton of the ma-
jor diastereomer at 4.26 ppm was irradiated, the intensity (0.1%) of
the methine proton signal at 4.95 ppm increased (Fig. 2D). On the
other hand, when the methine proton at dH 5.24 ppm of the minor
isomer was irradiated, no NOE difference was observed for any of
the methylene protons (Fig. 2B). Consequently, the relative
stereochemistry in the major diastereomer is assigned as (2S,3S)-
3-dihydroerucalexin (13a) (and its enantiomer(2R,3R)-3-dihydr-
oerucalexin), on which the OH and SCH2 substituents have a cis
(2), compound 10 was not stable in water, decomposing to indo-
lyl-3-methanol (t1/2 = 20 h) (11, Scheme 2). Hence, considering that
both compounds 2and 10give the same decomposition product in
aqueous solutions and that brassinin (4), an S-methyl N-(3-indo-
lylmethyl)carbamodithioate, is stable in both water and medium
solutions, the spontaneous transformation is caused mainly by
the S-(3-indolylmethyl)carbamothioate group and not by the allyl
substituent of brussalexin A (2).
Erucalexin (3) (0.10 mM) was incubated with fungal cultures of
L. maculans and medium only (control), samples were collected at
different times, extracted and the extracts were analyzed by HPLC.
After 24 h erucalexin (3, t
the cultures, but two new peaks were detected at t
minor) and t = 10.0 min (major). Chromatograms of control sam-
R
= 14.6 min) was no longer detected in
R
= 9.2 min
(
R
ples showed only one peak corresponding to erucalexin (3), indi-
cating it to be stable in the culture medium. Attempts to recover
Erucalexin (control)
Erucalexin
Dihydroerucalexin
0
0
.12
.1
the compound with t
bated in medium resulted in its transformation to the compound
with t = 10.0 min. The chemical structure of this compound was
R
= 9.2 min by extraction from cultures incu-
0.08
0.06
0.04
0.02
0
R
determined to be demethoxyerucalexin (14) by comparison of its
5
spectroscopic data with an authentic sample available in our syn-
thetic library. Subsequent biotransformation experiments to ob-
tain the minor compound (t = 9.2 min) were carried out in
R
water. Thus, mycelial mats of L. maculans grown in culture medium
0
12
24
Time (h)
36
48
60
were transferred to sterilized water and incubated with erucalexin
Figure 1. Progress curves of the transformation of erucalexin (3, 0.10 mM) and
formation of dihydroerucalexin (13) in mycelial cultures of Leptosphaeria maculans
in water.
(3) (0.10 mM); samples were collected at different times, freeze-
dried and the residues were dissolved in acetonitrile and analyzed

3
994
M. Soledade C. Pedras, V. K. Sarma-Mamillapalle / Bioorg. Med. Chem. 20 (2012) 3991–3996
D
C
B
HR
HS
HR + HS
H
H
OH
A
5
.3
5.2
5.1
5.0
4.9
4.8
4.7
4.6
4.5
4.4
4.3
4.2
4.1
4.0
3.9 ppm
Figure 2. Assignment of the relative stereochemistry of dihydroerucalexin diastereomers based on 1-
(
D
NOE difference experiments:(2S,3S)-3-hydroxyerucalexin (13a) and
2S,3R)-3-dihydroerucalexin (13b). (A) section of H NMR spectrum of mixture of 13a and 13b (3:1); (B) irradiation of H-3 of 13b; (C) irradiation of H-3 of 13a; (D) irradiation
of H of 13a.
1
S
relationship. In the minor isomer the relative stereochemistry is
assigned as (2S,3R)-3-dihydroerucalexin (13b) (and its enantio-
Similarly, the concentrations obtained from EtOAc extracts of solu-
tions W remained constant over 48 h; however, the concentrations
of dihydroerucalexin (13a/13 b, 3:1) obtained from EtOAc extracts
of solutions M were substantially lower, while the concentrations
of demethoxyerucalexin (14) increased. These results indicated
that decomposition of dihydroerucalexin (13) to demethoxyeruca-
lexin (14) increased during extraction/concentration of samples,
mer(2R,3S)-3-dihydroerucalexin), with the OH and SCH
2
substitu-
ents having a trans relationship.
The faster decomposition of dihydroerucalexin (13) to deme-
thoxyerucalexin (14) in culture medium than in water was inves-
tigated further using HPLC analysis, as follows. A diastereomeric
mixture of dihydroerucalexin (13a/13b, 3:1) was dissolved in
water (W) and in medium (M) (both containing 2% acetonitrile)
at identical concentration (0.20 mM); solutions W and M were
immediately injected in the HPLC and then on up to 48 h (triplicate
samples). Similarly, solutions W and M (1.0 mL, 0.2 mM) were ex-
tracted with EtOAC, the solvent evaporated under reduced pres-
sure (630 °C), the residue was dissolved in acetonitrile
2
+
3+
2+
2+
likely catalyzed by metal ions (Fe , Fe , Zn , Cu ) that were ex-
tracted as salts from the medium.
The antifungal activity of dihydroerucalexin was substantially
lower than that of erucalexin (23 ± 4% vs 40 ± 4%, at 0.50 mM;
13 ± 2% at 0.20 mM and 7 ± 2 at 0.10 mM), suggesting that detoxi-
fication of erucalexin (3) in L. maculans is mediated by a dehydro-
genase(s). The overall results demonstrate that dihydroerucalexin
(13) is a detoxification product of erucalexin (3), and that deme-
thoxyerucalexin (14) is a product of chemical degradation of
dihydroerucalexin (13), but not a product of fungal metabolism
(Scheme 3).
(
0.20 mM) and injected in the HPLC. The concentration of each
sample was determined by HPLC analysis using calibration curves
of dihydroerucalexin (13) and the conc. was plotted versus time, as
shown in Figure 3. The concentration of dihydroerucalexin
(13a/13b, 3:1) in non-extracted solutions W and M (direct
injection) remained constant over a period of 48 h (Fig. 3).
Water-direct inj
Medium direct inj
0
.3
.25
.2
.15
.1
Water extract inj
0
0
0
Medium extract inj
0
0
.05
0
0
12
24
36
48
60
Time (h)
Figure 3. Progress curves of recovery of dihydroerucalexin (13, 0.20 mM) in water
and medium solutions over a period of 48 h, obtained by HPLC analysis of solutions:
4
direct injection of solution of compound incubated in water (0.20 mM); } direct
injection of solution of compound incubated in medium (0.20 mM); N injection of
Scheme 3. Biotransformation of erucalexin (3) to dihydroerucalexin (13) by
Leptosphaeria maculans and chemical transformation of 13 to demethoxyerucalexin
(14).
extracted compound incubated in water (0.20 mM);
compound incubated in medium (0.20 mM).
j injection of extracted

M. Soledade C. Pedras, V. K. Sarma-Mamillapalle / Bioorg. Med. Chem. 20 (2012) 3991–3996
3995
3
. Conclusion
triethylamine (17
l
L, 0.62 mmol) in CH
2
Cl
2
(2 mL) and stirred at
rt for 20 min. After complete conversion of the starting material,
the reaction mixture was concentrated and separated by chroma-
tography (silica gel, EtOAc–hexane, 20:80, v/v) to afford brussalex-
in (2, 65 mg, 0.26 mmol) in 86% yield. All spectroscopic data were
This work established that rapalexin A (1) is a strongly anti-
fungal phytoalexin that was resistant to metabolism in cultures
of L. maculans, whereas erucalexin (3) was quickly metabolized
by reduction at C-3 (ca. 24 h, t1/2<6 h) to give a diastereomeric mix-
ture (13a and 13b). This mixture of dihydroerucalexins was less
inhibitory to L. maculans than erucalexin (3), indicating that the
enzymatic transformation was a detoxification reaction likely ben-
eficial to the pathogen. Somewhat surprising, the chemical reduc-
tion of erucalexin (3) afforded a higher diastereomeric ratio of
products (3:1) than the enzymatic process (2:1). The relative con-
figurations of these products were established by 1D 1H NOE dif-
ferential spectroscopy. Brussalexin A (2) was not stable upon
standing in medium or in water, converting spontaneously to indo-
lyl-3-methanol (11). For this reason, it was not possible to establish
if L. maculans was able to metabolize brussalexin A (2).
4
identical to previously reported data.
4.2.2. N-Methyl S-(3-methylindolyl)carbamodithioate (10)
Methyl isothiocyanate (49 mg, 0.67 mmol) was added to a solu-
tion of indolyl-3-methanethiol (100 mg, 0.613 mmol) and triethyl-
2 2
amine (200 lL, 1.47 mmol) in CH Cl (3 mL) and stirred at rt for
20 min. After complete conversion of the starting material, the
reaction mixture was concentrated, and separated by column chro-
matography (silica gel, EtOAc–hexane, 20:80, v/v) to afford the
product 10 (110 mg, 0.46 mmol) in 76% yield.
1
HPLC t
R
= 11.2 min. H NMR (500 MHz, CDCl
3
): d 9.20 (br s, NH),
8.01 (br s, NH), 7.63 (d, J = 8 Hz, 1H), 7.41 (d, J = 8 Hz, 1H), 7.29 (d,
J = 2 Hz, 1H), 7.17 (dd, J = 8, 8 Hz, 1H), 7.08 (dd, J = 7.5, 7.5 Hz, 1H),
4.69 (s, 2H), 3.12 (s, 3H), minor rotamer peaks were found at d 4.78,
The overall results of this work suggest that rapalexin A (1) is a
phytoalexin of great significance to improve the disease resistance
of crucifers to L. maculans, one of the most damaging pathogens of
1
3
and 2.93 (ca. 1.7: 0.3). C NMR (125 MHz, CDCl
3
): d 199.4, 137.8,
9
oilseed crops. However, considering that no genes in the biosyn-
128.0, 125.7, 123.3, 120.6, 120.0, 112.9, 110.8, 34.3, 32.1. FTIR
ꢁ1
thetic pathway of rapalexin A (1) have been cloned, increasing its
production in stressed plants is currently a difficult proposition.
In addition, considering the spontaneous transformations of brus-
salexin A (2) and N-methyl S-(3-methylindolyl)carbamodithioate
(KBr)
744. UV (HPLC, CH
measured 236.0435 ([M] , calcd 236.0442 for C11
m
max cm : 3403, 3323, 2928, 1509, 1455, 1339, 1031, 940,
3
OH–H O) kmax (nm): 219, 271. HRMS-EI m/z:
2
+
12 2 2
H N S ). MS
+
(EI) m/z (% relative int.): 236 [M ] (1), 163 (19), 130 (100).
(
10) to the anticarcinogenic compound indolyl-3-methanol (11),
1
5,16
their potential use as prodrugs might be of interest in medic-
inal chemistry.
4.2.3. Dihydroerucalexin (13)
4
NaBH (1 mg, 0.03 mmol) was added to a solution of erucalexin
(3, 15 mg, 0.053 mmol) in methanol (1 mL) at 0 °C and stirred at
4
4
. Experimental
the same temperature. After 5 min, the reaction mixture was di-
luted with water (1 mL) and directly loaded onto a WP C18 silica
gel column (H O–CH CN, 60:40, v:v) to afford the mixture of dia-
.1. Materials and general procedures
Chemicals were purchased from Sigma–Aldrich Canada Ltd,
2
3
stereomeric products 13a and 13b as a white solid in 3:1 ratio
(9 mg, 0.03 mmol, 60% yield). Further chromatography (WP C18 sil-
ica gel column, H O–CH CN, 60:40, v:v) of the mixture yielded the
Oakville, ON; solvents were HPLC grade and used as such. Flash
column chromatography (FCC) was carried out using silica gel
grade 60, mesh size 230–400 Å or WP C18 prepscale bulk packing
2
3
major diastereomer 13a containing <5% of the minor component.
HPLC t = 9.2 min (major, 13a) and 11.9 min (minor, 13b). Major
R
1
2
75 Å (J.T. Baker, NJ, USA)
isomer13a (containing <5% of the minor): H NMR (500 MHz,
Nuclear magnetic resonance (NMR) spectra were recorded on
Bruker 500 MHz Avance spectrometers, for H, 500.3 MHz and for
C, 125.8 MHz; chemical shifts (d) are reported in parts per million
ppm) relative to TMS; spectra were calibrated using the solvent
peaks; spin coupling constants (J) are reported to the nearest
.5 Hz. Fourier transform infrared (FT-IR) data were recorded on
CD CN): d 7.31–7.23 (m, 2H), 7.00 (ddd, J = 8, 8, 1 Hz, 1H), 6.93
3
1
(d, J = 8 Hz, 1H), 4.94 (s, 1H), 4.65 (d, J = 16 Hz, 1H), 4.26 (d,
1
3
13
J = 16 Hz, 1H), 4.09 (br s, OH), 3.86 (s, 3H), 2.51 (s, 3H). C NMR
(
(125 MHz, CDCl ) (containing <5% of the minor): d 164.9, 150.7,
3
130.4, 124.5, 123.9, 112.5, 74.3, 70.2, 66.8, 65.3, 15.5. Minor isomer
1
0
13b (1:1 mixture): H NMR (500 MHz, CD CN): d 7.31–7.23 (m,
3
a spectrometer and spectra were measured by the diffuse reflec-
tance method on samples dispersed in KBr. MS [high resolution
2H), 7.00 (ddd, J = 8, 8, 1 Hz, 1H), 6.93 (d, J = 8 Hz, 1H), 5.24 (s,
1H), 4.49 (d, J = 16.5 Hz, 1H), 4.45 (d, J = 16.5 Hz, 1H), 4.09 (br s,
1
3
(
HR), electron impact (EI)] were obtained on a VG 70 SE mass spec-
OH), 3.88 (s, 3H), 2.51 (s, 3H). C NMR (125 MHz, CDCl ) (1:1 mix-
3
trometer employing a solids probe.
ture): d 163.8, 152.0, 131.1, 130.5, 126.3, 124.1, 113.0, 103.9, 76.3,
66.7, 15.5. FTIR (KBr) mmax cm (1:1 mixture): 3317, 2929, 1686,
ꢁ1
HPLC analysis was carried out with Agilent high performance li-
quid chromatographs equipped with quaternary pump, automatic
injector, and diode array detector (DAD, wavelength range 190–
1614, 1566, 1463, 1193, 994, 954, 758. UV (HPLC, CH CN–H O)
3
2
kmax (nm): 208, 240, 285. HRMS-EI m/z: measured 282.0494
+
6
00 nm), degasser, and a column Eclipse XDB-C18 (5
size silica, 4.6 id ꢂ 150 mm), having an in-line filter, using mobile
phase 50% H O–50% CH OH to 100% CH OH, for 25.0 min, linear
gradient, and at a flow rate of 0.75 mL/min.
lm particle
([M] , calcd 282.0497 for C12H14N O S ). MS (EI) m/z (% relative
2
2 2
+
int.): 282 [M ] (12), 250 (51), 191 (100), 177 (68), 149 (22), 132
(57), 117 (34).
2
3
3
4
.3. Antifungal bioassays and metabolism by Leptosphaeria
4
4
.2. Synthesis and characterization of new compounds
maculans
.2.1. Brussalexin A (2)
Hydrazine hydrate (109 mg, 2.19 mmol) was added to a solu-
The antifungal activity of compounds was determined using a
mycelial radial growth bioassay on potato dextrose agar (PDA)
1
7
tion of indolyl-3-methylthioacetate (9, 150 mg, 0.73 mmol) in
THF (4 mL) and refluxed for 45 min.11 The reaction mixture was
concentrated, adsorbed onto silica gel, and fractionated (5% EtOAc
in hexane) to afford indolyl-3-methanethiol (100 mg, 0.61 mmol)
in 84% yield. Allyl isocyanate (28 mg, 0.34 mmol) was added to a
solution of indolyl-3-methanethiol (50 mg, 0.31 mmol) and
medium. In brief, isolates of L. maculans (isolates BJ-125 or
UAMH-9410) were grown on V8 agar plates for 14 days at 23 °C
under constant light. Sterile tissue culture plates (6-well, 33 mm
diameter) containing test (0.50, 0.20 and 0.10 mM) and control
solutions (2 mL per well containing 1% CH CN) in PDA medium
were inoculated with mycelium plugs 4 mm, cut from 7-day-old
3

3
996
M. Soledade C. Pedras, V. K. Sarma-Mamillapalle / Bioorg. Med. Chem. 20 (2012) 3991–3996
V8 agar plates of L. maculans placed upside down on the center of
each plate and incubated under constant light for 5 days. Mycelial
growth in each treatment was measured and % inhibition values
were calculated as previously reported. All bioassay experiments
were carried out in triplicate, at least two times.
and the University of Saskatchewan (graduate assistantship to
V.K.S.M.). We acknowledge the technical assistance of K. Brown
(NMR) and K. Thoms (MS) from the Department of Chemistry.
Supplementary data
Metabolism studies of compounds in fungal cultures were car-
ried out as previously reported. Erlenmeyer flasks (250 mL) each
containing 100 mL minimal media were inoculated with spores
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.020.
6
of L. maculans (isolate BJ-125, 10 /mL) and incubated at 23 °C on
a shaker at 120 rpm under constant light. After two days, com-
References and notes
pounds (in CH
.10 mM) as well as uninoculated media (control). Samples (2 mL
or 5 mL) were withdrawn immediately and at intervals between
and 72 h after incubation and were either frozen or immediately
extracted with EtOAc, the organic extracts were concentrated and
the residue dissolved in CH CN and analyzed by HPLC. Calibration
3
CN) were added to the cultures (final conc.
0
1. Pedras, M. S. C.; Yaya, E. E. Phytochemistry 2010, 71, 1191.
2.
3.
4.
Pedras, M. S. C.; Yaya, E. E.; Glawischnig, E. Nat. Prod. Rep. 2011, 28, 1381.
Pedras, M. S. C.; Zheng, Q. A.; Gadagi, R. S. Chem. Commun. 2007, 368.
Pedras, M. S. C.; Zheng, Q. A.; Sarwar, M. G. Org. Biomol. Chem. 2007, 5, 1167.
6
5. Pedras, M. S. C.; Suchy, M.; Ahiahonu, P. W. K. Org. Biomol. Chem. 2006, 4, 691.
6
7
8
.
.
.
Pedras, M. S. C.; Okinyo, D. P. O. Chem. Commun. 1848, 2006.
Pedras, M. S. C.; Ahiahonu, P. W. K. Phytochemistry 2005, 66, 391.
Pedras, M. S. C.; Minic, Z.; Hossain, S. Bioorg. Med. Chem. 2012, 20, 225.
3
curves with synthetic samples were built for quantitative analysis
of each compound.
9. Pedras, M. S. C.; Yu, Y. Nat. Prod. Commun. 2009, 4, 1291.
1
0. Pedras, M. S. C.; Hossain, S.; Snitynsky, R. B. Phytochemistry 2011, 72, 199.
1. Tomlinson, I. D.; Grey, J. L.; Rosenthal, S. J. Molecules 2002, 7, 777.
2. Agerbirk, N.; De Vos, M.; Kim, J. H.; Jander, G. Phytochem. Rev. 2009, 8, 101.
1
1
1
Acknowledgements
0
3. The formation of 3,3 -diindolylmethane from indolyl-3-methanol is discussed
in Ref. 12
Financial support for the authors’ work was obtained from the
Natural Sciences and Engineering Research Council of Canada
1
1
4. Pedras, M. S. C.; Hossain, S. Phytochemistry 2011, 72, 2308.
5. Ahmad, A.; Sakr, W. A.; Rahman, K. M. W. Curr. Drug Targets 2010, 11, 652.
16. Safe, S.; Papineni, S.; Chintharlapalli, S. Cancer Lett. 2008, 269, 326.
7. Pedras, M. S. C.; Jha, M. Bioorg. Med. Chem. 2006, 14, 4958.
(
Discovery Grant to M.S.C.P.), the Canada Research Chairs program,
1
Canada Foundation for Innovation, the Saskatchewan Government,