Tenualexin, other phytoalexins and indole glucosinolates from wild cruciferous species

Article abstract of DOI:10.1002/cbdv.201300260

In general, the chemodiversity of phytoalexins, elicited metabolites involved in plant defense mechanisms against microbial pathogens, correlates with the biodiversity of their sources. In this work, the phytoalexins produced by four wild cruciferous species (Brassica tournefortii, Crambe abyssinica (crambe), Diplotaxis tenuifolia (sand rocket), and Diplotaxis tenuisiliqua (wall rocket)) were identified and quantified by HPLC with photodioarray and electrospray mass detectors. In addition, the production of indole glucosinolates, biosynthetic precursors of cruciferous phytoalexins, was evaluated. Tenualexin, (=2-(1,4-dimethoxy-1H-indol-3-yl)acetonitrile), the first cruciferous phytoalexin containing two MeO substituents in the indole ring, was isolated from D. tenuisiliqua, synthesized, and evaluated for antifungal activity. The phytoalexins cyclobrassinin and spirobrassinin were detected in B. tournefortii and C. abyssinica, whereas rutalexin and 4-methoxybrassinin were only found in B. tournefortii. D. tenuifolia, and D. tenuisiliqua produced 2-(1H-indol-3-yl)acetonitriles as phytoalexins. Because tenualexin appears to be one of the broad-range antifungals occurring in crucifers, it is suggested that D. tenuisiliqua may have disease resistance traits important to be incorporated in commercial breeding programs. Copyright

Full text of DOI:10.1002/cbdv.201300260

9
10
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Tenualexin, Other Phytoalexins and Indole Glucosinolates from Wild
Cruciferous Species
by M. Soledade C. Pedras* and Estifanos E. Yaya
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, S7N5C9,
Canada
In general, the chemodiversity of phytoalexins, elicited metabolites involved in plant defense
mechanisms against microbial pathogens, correlates with the biodiversity of their sources. In this work,
the phytoalexins produced by four wild cruciferous species (Brassica tournefortii, Crambe abyssinica
(
crambe), Diplotaxis tenuifolia (sand rocket), and Diplotaxis tenuisiliqua (wall rocket)) were identified
and quantified by HPLC with photodioarray and electrospray mass detectors. In addition, the production
of indole glucosinolates, biosynthetic precursors of cruciferous phytoalexins, was evaluated. Tenualexin,
(
¼2-(1,4-dimethoxy-1H-indol-3-yl)acetonitrile), the first cruciferous phytoalexin containing two MeO
substituents in the indole ring, was isolated from D. tenuisiliqua, synthesized, and evaluated for antifungal
activity. The phytoalexins cyclobrassinin and spirobrassinin were detected in B. tournefortii and C.
abyssinica, whereas rutalexin and 4-methoxybrassinin were only found in B. tournefortii. D. tenuifolia,
and D. tenuisiliqua produced 2-(1H-indol-3-yl)acetonitriles as phytoalexins. Because tenualexin appears
to be one of the broad-range antifungals occurring in crucifers, it is suggested that D. tenuisiliqua may
have disease resistance traits important to be incorporated in commercial breeding programs.
Introduction. – The plant family Brassicaceae, common name crucifer, contains
more than 3,700 species of enormous diversity, including economically important
cruciferous oilseeds, vegetables, condiments, as well as numerous wild species. Brassica
species are among the most economically valuable cruciferous species, representing
sources of edible and industrial oils, food, and fodder, while wild crucifers are of great
interest as sources of agronomic traits potentially transferable to cultivated Brassica
species [1]. Among these agronomic traits, resistance to pests and diseases are much
sought after, as they facilitate production of disease-resistant species through
intercrossing or other breeding methods. Due to the susceptibility of cultivated
Brassica species to fungal diseases, it is of particular interest to uncover the chemical
defenses produced by wild cruciferous species and to evaluate their effects in the most
prevalent fungal pathogens of brassica crops.
Phytoalexins are among the ecologically relevant natural products produced by
crucifers to defend themselves against microbial pathogens [2]. These secondary
metabolites are elicited and produced de novo in plants exposed to stress, whether
biotic or abiotic. Importantly, in general, the chemodiversity of phytoalexins correlates
with the biodiversity of their sources. For example, within crucifers, cultivated Brassica
species produce the phytoalexins brassinin (1), cyclobrassinin (2), brassilexin (3),
spirobrassinin (4), and rutalexin (5), whereas wild species such as Arabidopsis thaliana
L. and Camelina sativa L. produce camalexin (6), Erucastrum gallicum (Willd.)
ꢀ
2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
911
O.E.Shulz produces erucalexin (7), and Thlaspi arvensis L. and Thellungiella
salsuginea (Pa lla s ) O.E.Shulz produce wasalexins (8). This chemodiversity suggests
that different wild crucifers produce different phytoalexins, although the number of
species investigated to date is still rather small as to allow reasonable projections.
Toward this end, we have investigated four wild species native of various habitats:
Brassica tournefortii Gouan, an invasive species known as African mustard, Saharan
mustard, or Asian mustard, Crambe abyssinica R.E.Fr. (crambe), cultivated for its high
content of erucic acid, a non-edible but important industrial oil, and Diplotaxis
tenuifolia (L.) DC. (sand rocket) and Diplotaxis tenuisiliqua Delile (wall rocket) both
used in salads in several Mediterranean countries [3]. Herein, we report the
phytoalexins produced by these species and the discovery of tenualexin (13), the first
phytoalexin containing two MeO substituents in the indole ring. In addition, the
production of indole glucosinolates, the biosynthetic precursors of cruciferous
phytoalexins, was also evaluated over a five-day period.
Results and Discussion. – Elicitation of plant secondary metabolites can be
achieved by biotic (e.g., pathogens) or abiotic (e.g., UV radiation, metal salts) factors.
The structures and amounts of elicited metabolites depend on the plant species and
their tissues, the elicitor type, and the environmental conditions. Within the same plant
species and tissue, metal salts, and UV irradiation cause leaf damage, and subsequent
elicitation depends on salt concentrations and intensity/exposure to UV light,
respectively.
Elicitation and HPLC Analysis of Plant Metabolites. To establish conditions that
induce the biosynthesis of plant metabolites, initial experiments were carried out using
various concentrations of CuCl (2–10 mm) sprayed on whole potted plants. While B.
2
tournefortii, C. abyssinica, and D. tenuifolia showed severe foliar damage when sprayed
with 5 mm or higher concentrations of CuCl , concentrations lower than 10 mm did not
2
cause obvious damage on leaves of D. tenuisiliqua. Subsequent experiments to quantify
elicited metabolites produced by B. tournefortii, C. abyssinica, and D. tenuifolia were
carried out spraying with 2 mm solution of CuCl , while D. tenuisiliqua was sprayed
2

9
12
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
with 10 mm solution of CuCl . Leaves of each plant species were excised after different
2
incubation periods and extracted as described in the Exper. Part to give nonpolar (NP)
and polar extracts (P). Control plants (sprayed with H O) were treated similarly to
2
yield NP control and P control extracts. The NP and P extracts were analyzed by
HPLC-DAD/ESI-MS (DAD, diode array detector), and chromatograms were
compared with those of NP and P extracts of control leaves, respectively. All the NP
extracts showed the presence of elicited metabolites (i.e., either not detected in controls
or detected in trace amounts). All metabolites present in the NP extracts of D.
tenuisiliqua, except for a peak with tR 19.7 min, were available in our metabolite
libraries and were identified by comparison of their HPLC retention times (t values),
R
UV, and MS data with those of authentic samples. Quantification of each metabolite
was carried out using calibration curves built for HPLC-DAD analysis with authentic
synthetic compounds (Tables 1 and 2). Similarly, the metabolites present in P extracts
were analyzed by HPLC-DAD/ESI-MS, and the chromatograms were compared with
those of controls, but no elicited metabolites could be detected under the conditions
used.
Given the biosynthetic relationship of indolic phytoalexins and indole glucosino-
lates, the indole glucosinolate production over the same time period and quantity were
Table 1. HPLC-DAD Analysis of Phytoalexins and Indole Glucosinolates of Brassica tournefortii (21-
day-old) and Crambe abyssinica (17-day-old)
Metabolites
HPLC method, tR min)
Incubation
period [h]
Total amount of metabolites
(nmol/g of fresh tissueꢀS.D.) )
a
(
B. tournefortii
C. abyssinica
Cyclobrassinin (2)
Method A; 25.2)
24
72
79ꢀ9
16ꢀ1
40ꢀ8
18ꢀ2
(
b
120
24
72
120
24
72
120
24
72
120
24
72
120
24
72
120
24
72
120
11ꢀ0
ND )
Spirobrassinin (4)
Method A; 12.0)
139ꢀ24
114ꢀ8
115ꢀ6
25ꢀ2
10ꢀ3
(
84ꢀ11
72ꢀ23
b
Rutalexin (5)
ND )
b
(
Method A; 14.5)
24ꢀ2
ND )
b
26ꢀ4
ND )
b
4
(
-Methoxybrassinin (9)
Method A; 19.9)
26ꢀ3
ND )
b
2.8ꢀ0.3
ND )
b
b
ND )
ND )
Arvelexin (12)
Method A; 13.8)
13ꢀ3
15ꢀ1
(
30ꢀ13
183ꢀ73
237ꢀ118
365ꢀ164
612ꢀ296
248ꢀ41
18ꢀ8
Glucobrassicin (14)
1847ꢀ442
897ꢀ540
575ꢀ102
386ꢀ108
204ꢀ119
101ꢀ30
(
Method B; 19.6)
b
4
(
-Methoxyglucobrassicin (16)
Method A; 26.2)
ND )
b
ND )
b
ND )
a) Amounts of each metabolite were determined by HPLC-DAD using calibration curves. Values are
b
averages (triplicate)ꢀstandard deviation (S.D.). ) ND, Not detected.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
913
Table 2. HPLC-DAD Analysis of Phytoalexins and Indole Glucosinolates of Diplotaxis tenuifolia (24-
day-old) and D. tenuisiliqua (28-day-old)
Metabolites
HPLC method, tR min)
Incubation
period [h]
Total amount of metabolites
(nmol/g of fresh tissueꢀS.D.) )
a
(
D. tenuifolia
D. tenuisiliqua
Arvelexin (12)
24
72
40ꢀ7
8ꢀ1
33ꢀ4
31ꢀ16
4ꢀ1
(
Method A; 13.6)
204ꢀ51
350ꢀ25
1
20
b
2
-(1,4-Dimethoxy-1H-indol-3-yl)acetonitrile
24
ND )
(
tenualexin, 13)
b
(
Method A; 19.7)
72
20
24
72
20
24
72
20
ND )
5ꢀ1
2ꢀ1
b
1
1
1
ND )
b
Glucobrassicin (14)
716ꢀ48
263ꢀ86
137ꢀ46
54ꢀ7
ND )
b
(
Method B; 19.6)
ND )
b
ND )
b
4
(
-Methoxyglucobrassicin (16)
Method B; 26.2)
ND )
b
59ꢀ9
ND )
b
34ꢀ6
ND )
a) Amounts of 12, 13, 14, and 16 determined by HPLC-DAD using calibration curves. Values are
b
averages (triplicate)ꢀstandard deviation (S.D.). ) ND, Not detected.
determined as well. HPLC-DAD/ESI-MS Data indicated that glucobrassicin (14) and
4-methoxyglucobrassicin (16) were produced by B. tournefortii and D. tenuifolia, while
only glucobrassicin (14) was detected in extracts of C. abyssinica; no indole
glucosinolates were detected in P extracts of D. tenuisiliqua. 1-Methoxyglucobrassicin
(15) was not detected in any of the four species (Tables 1 and 2).
Prior to this work, C. abyssinica was investigated and reported to produce the
elicited metabolites arvelexin (12) and rapalexin B (19) [4]; however, rapalexins A (18)
and B (19) were not detected in the current work. To the best of our knowledge, no
elicited metabolites or phytoalexins have been reported from B. tournefortii, D.
tenuifolia, and D. tenuisiliqua. It was particularly puzzling to find that no indole
glucosinolates were detected in the extracts of D. tenuisiliqua, because 2-(1H-indol-3-
yl)acetonitriles are known to be derived from glucobrassicins [5]. Previously,
glucobrassinin (14), together with its 1-methoxy and 4-methoxy derivatives, 15 and
16, respectively, were identified in several accessions of D. tenuifolia, while only
aliphatic glucosinolates were detected in D. tenuisiliqua [6].

9
14
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Isolation, Identification, Synthesis, and Antifungal Activity of New Metabolite 13
from Diplotaxis tenuisiliqua. To isolate the new metabolite with t of 19.7 min present
R
in NP extracts of D. tenuisiliqua, a larger-scale extraction of elicited leaves (300 g) was
carried out. The NP extract was fractionated using multiple chromatographic methods
to yield the desired metabolite with t of 19.7 min in sufficient amount (0.8 mg) for
R
spectroscopic characterization. The HR-EI-MS of this sample showed a molecular-ion
þ
peak consistent with the molecular formula C H N O (m/z 216.0897 (M ; calc.
12
12
2
2
2
16.0899)). Consistent with the degree of unsaturation of eight and the molecular
1
formula, the H-NMR spectrum exhibited signals of twelve H-atoms: a singlet at d(H)
.20 (1 H), a doublet of doublet at d(H) 7.18 (1 H), two doublets at d(H) 7.03 (1 H) and
7
d(H) 6.51 (1 H), two Me singlets at d(H) 4.06 and 3.92, and a CH singlet at d(H) 4.02.
2
The presence of four H-atom signals in the aromatic region (d(H) 7.20–6.51), their
multiplicity, and the degree of unsaturation suggested the presence of an indole ring
substituted at N(1), C(3), and either C(4) or C(7). Furthermore, by biosynthetic
analogy to other cruciferous indolic structures, the two MeO groups (d(H) 4.06 and
3.92) were assumed to be attached to the indole ring at N(1) and C(4), and the CH2
substituent located at C(3). Finally, considering that the indole ring counted for six
unsaturations, the two remaining unsaturations should be due to the functional group
that was assigned as a nitrile. Based on this reasoning, the structure of the new
metabolite with t of 19.7 min was assigned as 2-(1,4-dimethoxy-1H-indol-3-yl)aceto-
R
nitrile (13). This structure was confirmed by synthesis, as outlined in Scheme 1 and
described in the Exper. Part.
In short, commercially available 4-methoxy-1H-indole (20) was reduced to the
corresponding 4-methoxyindoline [7], followed by oxidation and methylation [8] to
yield 1,4-dimethoxyindole (21). Formylation of 21 yielded 1,4-dimethoxy-1H-indole-3-
carboxaldehyde that was condensed with excess of MeNO [9] to yield 1,4-dimethoxy-
2
3
-(2-nitroethenyl)-1H-indole (22). Compound 22 was reduced with NaBH [10] to
4
yield 23 that was then dehydrated to yield 2-(1,4-dimethoxy-1H-indol-3-yl)acetonitrile
(13; Scheme 1).
The antifungal activity of 2-(1,4-dimethoxy-1H-indol-3-yl)acetonitrile (13) was
assayed against four economically important plant pathogenic fungi (Alternaria
brassicicola, Leptosphaeria maculans, Rhizoctonia solani, and Sclerotinia sclerotiorum)
as described in the Exper. Part. At the highest concentration (0.50 mm), 13 inhibited
completely the growth of mycelia of A. brassicicola, R. solani, and S. sclerotiorum,
Scheme 1. Synthesis of 2-(1,4-Dimethoxy-1H-indol-3-yl)acetonitrile (13)
i) NaBH CN, AcOH. ii) Na WO , H O , K CO , (Me) SO ; 41%. iii) POCl , DMF. iv) MeNO ,
3
2
4
2
2
2
3
2
4
3
2
AcONH
4
, 1058. v) NaBH
4
, THF, MeOH. vi) CS
2
, Et
3
N, 408; 77%.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
915
a
Table 3. Antifungal Activity ) of Tenualexin (13) against the Plant Pathogenic Fungi Alternaria
brassicicola, Leptosphaeria maculans, Rhizoctonia solani, and Sclerotinia sclerotiorum
Tenualexin (13)b)
A. brassicicola [%]
L. maculans [%]
R. solani [%]
S. sclerotiorum [%]
0.50 mm
0.20 mm
0.10 mm
100ꢀ0
47ꢀ4
31ꢀ4
69ꢀ4
31ꢀ4
18ꢀ4
100ꢀ0
86ꢀ0
67ꢀ4
100ꢀ0
57ꢀ2
23ꢀ2
a) Percentage inhibition¼100 – [(diameter of mycelium on medium containing compound – plug) )/
c
c
(
diameter of mycelium on control medium – plug) )ꢁ100)]ꢀstandard deviation. Results are given
b
averages (triplicate)ꢀstandard deviations. ) Dissolved in DMSO and added to potato dextrose agar
c
medium. ) Plug diameter, 4 mm.
whereas inhibition of L. maculans was not complete (69%; Table 3). Therefore,
considering that 2-(1,4-dimethoxy-1H-indol-3-yl)acetonitrile is an elicited metabolite
(
not present in control plants) and displays antifungal activity, it should be considered a
phytoalexin, for which the name tenualexin (13) is proposed. Tenualexin (13) is a
phytoalexin in D. tenuisiliqua and a new plant metabolite that does not appear to have
been synthesized previously.
2
-(1H-Indol-3-yl)acetonitriles 10–12 were found as phytoalexins in some crucifers,
including caulilexin C (11) produced by B. oleraceae var. botrytis [11] and arvelexin (12)
produced by T. arvensis [12]. Compound 10 was identified in various species, but
sometimes it is also produced in controls, although in much smaller amounts; in such
cases, it is more appropriate to consider them as phytoanticipins. Interestingly, although
13 has not been reported to date, its potential biosynthetic precursor 1,4-dimethox-
yglucobrassicin (17) was isolated as the desulfo derivative from roots of Barbarea
vulgaris ssp. arcuata [13][14]. Since 17 was not detected in any of the extracts of leaves
of D. tenuisiliqua, we assume that 2-(1,4-dimethoxy-1H-indol-3-yl)acetaldehyde oxime
(24) is a more likely immediate precursor of tenualexin (13; Scheme 2). Nonetheless,
the precursor role of 17 cannot be ruled out. Due to its strong antifungal activity, the
biosynthetic pathway of 13 is of great interest and warrants further investigation.
Scheme 2. Proposed Biosynthetic Pathway of Tenualexin (13) in Diplotaxis tenuisiliqua
Conclusions. – Elicitation of B. tournefortii, C. abyssinica, D. tenuifolia, and D.
tenuisiliqua with CuCl solutions, followed by incubation and HPLC analyses of
2
extracts of their elicited and control leaves showed that all species produced
phytoalexins. B. tournefortii and C. abyssinica produced arvelexin (12) together with
phytoalexins common to Brassica species (cyclobrassinin (2) and spirobrassinin (4)),

9
16
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
whereas no Brassica phytoalexins were detected in D. tenuifolia and D. tenuisiliqua
under identical experimental conditions. It is pertinent to point out that all cruciferous
phytoalexins reported to date are derived from the same basic building block, l-
tryptophan, and tenualexin (13) is no exception.
Based on previous antifungal assays using caulilexin C (11) and arvelexin (12) [15],
tenualexin (13) appears to be the most potent antifungal agent among these three
metabolites. These results suggest that D. tenuisiliqua could have disease resistance
traits of interest in commercial breeding programs.
Financial support of this work was obtained from the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant to M. S. C. P.), the Canada Research Chairs Program, the Canada
Foundation for Innovation, the Saskatchewan Government, and the University of Saskatchewan
(
teaching assistantship to E. E. Y.).
Experimental Part
General. Chemicals were purchased from SigmaꢂAldrich Canada Ltd., Oakville, ON, Canada, or
Alpha Aeser, Ward Hill, MA, USA. Org. extracts were dried (Na SO ), and solvents were removed
2
4
under reduced pressure in a rotary evaporator. Flash column chromatography (FC): silica gel, grade 60;
mesh size, 230–400 ꢂ. Prep. TLC: silica-gel plates, Kieselgel 60 F254, visualization under UV light. HPLC:
Agilent chromatographs equipped with quaternary pump, automatic injector, and diode array detector
(
DAD; wavelength range 190–600 nm), degasser, and a Hypersil ODS column (5-mm particle size silica,
4
.6 i.d.ꢁ200 mm), having an in-line filter; mobile phase, Method A (for NP extracts): H O/MeCN linear
2
gradient (75 :25 to 25 :75 in 35 min, to 0 :100 in 5 min), and at a flow rate 1.0 ml/min; Method B (for P
extracts): H O/MeCN (100 :0 to 25 :75 in 35 min, to 0 :100 in 5 min) and a flow rate of 1 ml/min. Samples
2
were dissolved in MeCN for Method A, and in H O/MeOH 1:1 for Method B. HPLC-DAD-ESI-MS:
2
Agilent 1100 series HPLC system equipped with an autosampler, binary pump, degasser, and a DAD
connected directly to a mass detector (Agilent G2440A MSD-Trap-XCT ion trap mass spectrometer)
with an electrospray ionization (ESI) source. Chromatographic separation was carried out at r.t. using an
Eclipse XSB C-18 column (5-mm particle size silica, 150ꢁ4.6 mm i.d.). The mobile phase consisted of a
linear gradient of 0.2% HCOOH in H O and 0.2% HCOOH in MeCN (75 :25 to 25 :75 in 35 min, to
2
0
:100 in 5 min) and a flow rate of 1 ml/min. Data acquisition was carried out in positive and negative
polarity modes in a single LC run. Data processing was carried out using Agilent Chemstation software.
1
13
H- and C-NMR spectra: Bruker Avance 500 MHz spectrometers; d in ppm rel. to Me Si as internal
4
standard, J in Hz. HR-EI-MS: VG 70 SE mass spectrometer, employing a solids probe; in m/z (rel. %).
Plant Material, Elicitation, and Extraction. Seeds of Brassica tournefortii, Crambe abyssinica,
Diplotaxis tenuisiliqua, and Diplotaxis tenuifolia were obtained from Plant Gene Resources, AAFC,
Saskatoon, Canada. The seeds were sown in a commercial potting soil mixture, and plants were grown in
a growth chamber under controlled environmental conditions (at 258/16 h, light, 168/8 h, dark) for 2–
4
weeks (B. tournefortii, 21 d; C. abyssinica, 17 d; D. tenuifolia, 24 d; and D. tenuisiliqua, 28 d).
Plants were sprayed with CuCl solns. (2–10 mm) to the point of run-off and incubated in a growth
2
chamber for different periods. Leaves from elicited and control plants were harvested at 24, 72, and 120 h,
weighed, separately frozen in liquid N , crushed, and extracted with MeOH (2ꢁ20 ml) on a shaker for
2
1
h. MeOH Extracts were filtered and concentrated under reduced pressure. The residue was rinsed with
CH Cl (6 ml) and decanted to yield the nonpolar (NP) extract and a residue (polar (P) extract). The NP
2
2
extract was concentrated, dissolved in MeCN (500 ml), and analyzed by HPLC-DAD-ESI-MS. The P
extract was dissolved in MeOH/H O 1:1 (2 ml) and analyzed by HPLC-DAD-ESI-MS.
2
Isolation of New Metabolite 13. A larger-scale experiment was conducted to isolate and characterize
the new metabolite (tR 19.7 min) from 21-day-old leaves of D. tenuisiliqua. Plants were elicited with
CuCl soln. (10 mm), leaves were harvested, weighed (300 g), and crushed in liquid N . The crushed
2
2
leaves were divided into two parts and extracted with MeOH overnight. The plant tissue was filtered, and

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
917
the filtrate was concentrated. The crude MeOH extract was washed with CH Cl and concentrated. The
2
2
NP extract was subjected to FC (hexane/AcOEt 80 :20 to 0 :100 and CH Cl /MeOH 95 :5). The fractions
2
2
were analyzed by HPLC-DAD, and the fraction containing a peak at t 19.7 min was further fractionated
R
by FC (hexane/AcOEt 90 :20 to 70 :30). Final fractionation of the fraction containing the peak at tR
1
9.7 min was carried out by prep. TLC (hexane/AcOEt 85 :15) to yield 2-(1,4-dimethoxy-1H-indol-3-
yl)acetonitrile (13) as an oil (0.8 mg).
Synthesis of Tenualexin (13). NaBH CN (382 mg, 6.06 mmol) was added during 10 min to a soln. of
3
4
-methoxy-1H-indole (20; 300 mg, 2.04 mmol) in AcOH (6 ml), the mixture was stirred at r.t. for 1 h,
diluted with H O, basified with 2m NaOH aq. soln., and extracted with Et O. The combined org. phase
2
2
was dried and concentrated to yield crude 4-methoxyindoline (303 mg, 97%). Na WO ·2 H O (126 mg,
2
4
2
0
.38 mmol) in H O (1 ml) was added to a stirred soln. of 4-methoxyindoline (333 mg, 2.25 mmol) in
2
MeOH (8 ml) and cooled to ꢂ208. Next, a soln. of H O (1.5 ml, 19.6 mmol) in MeOH (2 ml) was added
2
2
dropwise to the cooled suspension, the mixture was stirred at r.t. for 10 min, and K CO (2.5 g, 18 mmol)
2
3
and (MeO) SO (341 ml, 3.60 mmol) were added under vigorous stirring. After stirring for 10 min, the
2
2
mixture was diluted with H O and extracted with Et O (3ꢁ40 ml). The org. extract was dried,
2
2
concentrated, and subjected to FC (CH Cl 100%) to yield 21 (163 mg, 0.92 mmol) in 41% yield.
2
2
POCl (127 ml, 1.36 mmol) was added dropwise to a soln. of 21 (160 mg, 0.91 mmol) in DMF (2 ml) at
3
0
8. The mixture was stirred for 1 h at r.t., basified with NH ·H O (28%), diluted with H O, and extracted
3
2
2
with Et O (3ꢁ30 ml). The org. layer was dried, concentrated, and subjected to FC (CH Cl /MeOH
2
2
2
9
(
0
8 :2) to yield 1,4-dimethoxyindole-3-carboxaldehyde (149 mg, 0.73 mmol) in 81% yield. AcONH4
26 mg, 0.34 mmol) was added to a soln. of 1,4-dimethoxy-1H-indole-3-carboxaldehyde (139 mg,
.68 mmol) in MeNO (1.5 ml) and refluxed for 90 min. The mixture was allowed to cool, diluted with
2
H O, extracted with CH Cl , and concentrated to yield crude 22, which was used in the next step without
2
2
2
further purification. NaBH (78 mg, 2.04 mmol) was added to a soln. of crude 22 (169 mg, 0.68 mmol) in
4
THF (4 ml) and MeOH (500 ml). The mixture was stirred for 3 h at r.t. and diluted with H O. The mixture
2
was extracted by CH Cl , concentrated, and subjected to FC (CH Cl 100%) to yield 23 (65 mg,
2
2
2
2
0
.38 mmol) in 26%. Et N (500 ml, 3.60 mmol) and CS (217 ml, 3.60 mmol) were added to a soln. of 23
3 2
(
45 mg, 0.18 mmol) in MeCN (4 ml). The mixture was stirred at 408 for 20 h in an air-tight reaction vial,
the solvent was evaporated, and the residue was diluted with H O and extracted (CH Cl , 3ꢁ20 ml). The
2
2
2
org. layer was dried, concentrated, and the crude mixture was fractionated by FC (CH Cl 100%) to yield
2
2
(
13; 30 mg, 0.14 mmol, 77%). Brown oil. tR 19.7 min (Method A). UV (HPLC, H O/MeCN): 220, 265,
2
1
2
1
3
1
2
95. FT-IR (KBr): 2935, 2359, 1502, 1456, 1303, 1260, 1060, 1033. H-NMR (500 MHz, CDCl ): 7.20 (s,
3
H); 7.18 (dd, J¼8, 8, 1 H); 7.03 (d, J¼8, 1 H); 6.51 (d, J¼8, 1 H); 4.06 (s, 3 H); 4.02 (s, 2 H); 3.92 (s,
13
3
H). C-NMR (125 MHz, CDCl ): 154.6; 134.0; 124.3; 120.3; 118.9; 112.8; 101.9; 101.1; 100.2; 66.1; 55.4;
þ
þ
þ
2
6.1.l. MS-ESI: 217.0 (100, [Mþ1] ), 186.0 (20). HR-EI-MS: 216.0897 (87, M , C H N O ; calc.
12
12
2
16.0897), 185.0 (100), 170.0 (34).
Antifungal Bioassays. Leptosphaeria maculans, Rhizoctonia solani AG 2–1, Sclerotinia sclerotiorum,
and Alternaria brassicicola were obtained from AAFC Research Centre, Saskatoon, Canada. Solid
cultures were initiated with spores of A. brassicicola and L. maculans, sclerotia of S. sclerotiorum, and
mycelia of R. solani. Spores of A. brassicicola and L. maculans, sclerotia of S. sclerotiorum, mycelia of R.
solani were grown on potato dextrose agar (PDA) plates at 23ꢀ28 under constant light. Solns. of 13 in
DMSO were used to prepare sterile assay plates (6 wells per plate, 36-mm diameter, 2 ml per well) in
PDA media (0.50, 0.20, and 0.10 mm; final DMSO concentration, 1%); control plates contained 1%
DMSO in PDA. Plates containing test solns. and the solvent were inoculated with mycelial plugs (4-mm
diameter) cut from the edge of actively growing cultures and placed upside down on the center of each
plate. The plates were sealed with parafilm and incubated at 23ꢀ28 under constant light for 24 h for S.
sclerotiorum, 72 h for R. solani, and 120 h for L. maculans and A. brassicicola. The diameter of the
mycelia in control media and in tenualexin-containing media was measured every 24 h. The antifungal
activity was calculated using the equation given in Table 3. The experiment was conducted in triplicate
and repeated three times.

9
18
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
REFERENCES
[
1] S. I. Warwick, ꢃGenetics and Genomics of the Brassicaceae, Plant Genetics and Genomics: Crops
and Models 9ꢄ, Eds. R. Schmidt, I. Bancroft, 2011, Vol. 9, pp. 33–65.
2] M. S. C. Pedras, E. E. Yaya, E. Glawischnig, Nat. Prod. Rep. 2011, 28, 1381.
3] A. Protap, S. K. Gupta, ꢃBiology and Breeding of Crucifersꢄ, Ed. S. K. Gupta, CRC Press, 2009,
pp. 37–67.
[
[
[
[
[
[
[
[
4] M. G. Sarwar, M.Sc. Thesis, University of Saskatchewan, Canada, 2007.
5] N. Agerbirk, M. De Vos, J. H. Kim, G. Jander, Phytochem. Rev. 2009, 8, 101.
6] L. F. DꢄAntuono, S. Elementi, R. Neri, Phytochemistry 2008, 69, 187.
7] X. Wang, Y. Gao, Y. M. Xu, L. Li, Z. Zhang, J. Liu, Synth. Commun. 2009, 39, 4030.
8] M. Somei, T. Kawasaki, Heterocycles 1989, 29, 1251.
9] L. Canoira, J. G. Rodriguez, J. B. Subirats, J.-A. Escario, I. Jimenez, A. R. Martinez-Fernandez, Eur.
J. Med. Chem. 1989, 24, 39.
[
[
[
[
10] A. B. Robertson, N. P. Botting, Tetrahedron 1999, 55, 13269.
11] M. S. C. Pedras, M. G. Sarwar, M. Suchy, A. M. Adio, Phytochemistry 2006, 67, 1503.
12] M. S. C. Pedras, P. B. Chumala, M. Suchy, Phytochemistry 2003, 64, 949.
13] N. Agerbirk, B. L. Petersen, C. E. Olsen, B. A. Halkier, J. K. Nielsen, J. Agric. Food Chem. 2001, 49,
1502.
[
[
14] N. Agerbirk, C. E. Olsen, Phytochemistry 2012, 77, 16.
15] M. S. C. Pedras, S. Hossain, Phytochemistry 2011, 72, 2308.
Received August 1, 2013