Probing the phytopathogenic stem rot fungus with phytoalexins and analogues: unprecedented glucosylation of camalexin and 6-methoxycamalexin

Article abstract of DOI:10.1016/S0968-0896(02)00208-0

The remarkable metabolism of the cruciferous hytoalexins camalexin and 6-methoxycamalexin by the stem rot phytopathogen Sclerotinia sclerotiorum is reported. The biotransformations yielded camalexins glucosylated at N-1 or C-6 of the indole ring, with substantially lower antifungal activity than camalexins. A camalexin analogue with the positions N-1 and C-6 blocked was metabolized but at a much slower rate than the natural hytoalexins. The chemistry involved in the metabolism of natural camalexins and two new analogues, as well as their novel metabolites and respective antifungal activities is described. Copyright

Full text of DOI:10.1016/S0968-0896(02)00208-0

Bioorganic & Medicinal Chemistry 10 (2002) 3307–3312
Probing the Phytopathogenic Stem Rot Fungus with Phytoalexins
and Analogues: Unprecedented Glucosylation of Camalexin and
6-Methoxycamalexin
M. Soledade C. Pedras* and Pearson W. K. Ahiahonu
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK, Canada S7N 5C9
Received 19 February 2002; accepted 16 April 2002
Abstract—The remarkable metabolism of the cruciferous phytoalexins camalexin and 6-methoxycamalexin by the stem rot phyto-
pathogen Sclerotinia sclerotiorum is reported. The biotransformations yielded camalexins glucosylated at N-1 or C-6 of the indole
ring, with substantially lower antifungal activity than camalexins. A camalexin analogue with the positions N-1 and C-6 blocked
was metabolized but at a much slower rate than the natural phytoalexins. The chemistry involved in the metabolism of natural
camalexins and two new analogues, as well as their novel metabolites and respective antifungal activities is described.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
An evaluation of the biological activity of diverse phy-
toalexins against S. sclerotiorum indicated that cama-
lexin (1) could inhibit completely the growth of the
pathogen.
Camalexins are unique phytoalexins biosynthesized by a
few wild crucifers.1 Phytoalexins, induced plant chemi-
cal defenses, can selectively inhibit plant pathogens and
plant pathogens can react differently to phytoalexins. In
instances where phytopathogens can circumvent phy-
toalexins through metabolic detoxification, an envir-
onmentally attractive strategy to control such plant
pathogens could use selective inhibitors of the fungal
detoxifying enzymes.2 Towards this end, we have been
investigating the metabolism of cruciferous phytoalexins
by economically important phytopathogenic fungi.1 Our
previous work showed that camalexin (1) could be
detoxified by the fungus Rhizoctonia solani Kuhn
(Scheme 1) to 5-hydroxycamalexin (2), which was fur-
ther transformed into metabolites 3 and 4, substantially
less toxic to the pathogen than camalexin (1).3,4 How-
ever, we have also shown that not all plant pathogenic
fungi or bacteria can transform camalexin (1).5
Subsequently, we established that metabolism and
detoxification of camalexins 1 and 1a occurs via glyco-
sylation, an unusual detoxification reaction in plant
pathogenic fungi.7 To probe the selectivity of these
enzymatic reactions we designed camalexin analogues
based on the structure of the detoxification products.
The results of these studies are now reported for the first
time.8
Results and Discussion
Camalexins 1–1c were synthesized from the respective
indoles as described in the Experimental and previously
reported.9 Initial experiments established the minimum
inhibitory concentration (5 ꢀ 10ꢁ4 M) of camalexin (1)
and 6-methoxycamalexin (1a) to S. sclerotiorum. Subse-
quently, camalexins 1 and 1a were incubated separately
with S. sclerotiorum, culture samples were extracted and
analyzed. HPLC analysis of the extracts of fungal cul-
tures incubated with camalexins 1 and 1a indicated that
both were completely metabolized in ca. 48 h. After 24 h
incubation of S. sclerotiorum with camalexin (1), pro-
ducts 5 and 5a were isolated, whereas 5, 5a and 5b were
isolated after incubation with 6-methoxycamalexin (1a)
In continuation of those studies, we examined the
metabolism of naturally occurring camalexins 1 and 1a
by the stem rot fungus Sclerotinia sclerotiorum (Lib.) de
Bary. S. sclerotiorum causes stem rot disease in a vast
range of plants and is considered a worldwide problem.6
*Corresponding author. Tel.: +1-306-966-4772; fax: +1-306-966-
4730; e-mail: soledade.pedras@usask.ca
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00208-0

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M. S. C. Pedras, P. W. K. Ahiahonu / Bioorg. Med. Chem. 10 (2002) 3307–3312
(Table 2) displayed 11 carbon signals, one of which was
characteristic of a deshielded sp2 C atom (154.1 ppm).
These spectroscopic features suggested that metabolite 5
contained an OH groupattached to either C-5 or C-6.
That the OH groupwas attached to C-6 rather than C-5
was finally deduced from comparison of HMBC and
HMQC spectral data with that of camalexin (1) and
5-hydroxycamalexin.3 In addition, methylation of 5
with diazomethane yielded 1a, thus confirming the
structure of 5 unambiguously.
The
molecular
formulas
of
metabolites
5a
(C17H19N2O6S) and 5b (C18H21N2O6S) obtained by
HRMS-FAB indicated the presence of a hexose, which
was corroborated by NMR data (Tables 1 and 2). The
1H NMR spectra of 5a and 5b (Table 1) displayed
resonances indicative of a b-d-glucopyranosyl sub-
stituent on the indole moiety; analysis of the HMQC
and HMBC spectral data confirmed that this sub-
stituent was located at C-6 in 5a (C–H long-range
correlation between anomeric H and C-6) and at N-1 in
5b (C–H long-range correlation between anomeric H
and C-2 and C-7a).
Scheme 1. Detoxification pathway of camalexin (1) in the phyto-
pathogen Rhizoctonia solani.
(Scheme 2).10 The chemical structure of each metabolite
was deduced from comparison of their spectroscopic
data with those of camalexins 1 and 1a, as follows.
Relative to camalexin (C11H8N2S), metabolite 5 con-
tained an additional oxygen atom (C11H8N2OS), as
determined by HRMS-EI. Comparison of the 1H NMR
spectrum (Table 1) of 5 with that of camalexin (1) indi-
cated the presence of an indolic substituent, since only a
three-proton spin system was displayed in addition to
H-2 and two thiazole protons. The 13C NMR spectrum
To establish the sequence of biotransformation steps of
camalexin (1) and 6-methoxycamalexin (1a), compound
5 was administered to cultures of S. sclerotiorum. The
cultures were incubated and samples extracted and
analyzed by TLC and HPLC. As expected, 5a was
detected in the cultures incubated with 5 after 12 h;
complete metabolism of 5 to 5a occurred in ca. 24 h.
These results indicated that 1 and 1a were transformed
to 6-O-b-d-glucopyranosylcamalexin (5a) via 6-hydro-
xycamalexin (5). In addition, in 6-methoxycamalexin
(1a) was partly converted to 5b, a minor metabolite;
that is, S. sclerotiorum converted 6-methoxycamalexin
(1a) via two pathways, with the major product 5a
resulting from demethylation of the methoxy groupat
C-6, followed by glucosylation; the minor product 5b
resulted from direct N-glucosylation. The results of
these experiments are summarized in Table 3.11
Subsequently, it was anticipated that, if the fungal
detoxifying enzyme(s) was specific, blocking the C-6 of
camalexin would direct glucosylation to N-1, and
blocking both N-1 and C-6 sites would slow down if not
stopthis metabolism. Thus, camalexins 1b and 1c were
Scheme 2. Detoxification pathway of camalexins 1–1c in the phyto-
pathogen Sclerotinia sclerotiorum.
Table 1. 1H NMR chemical shifts (ppm) and multiplicities (J in Hz)a for compounds 1b–6 in CD3CN
H #
1b
1c
5
5a
5b
5c
5d
6
H-1
H-2
H-4
H-5
9.74 broad s
7.89 s
8.25 dd (9, 5.5)
7.03 ddd
(10, 9, 2.5)
—
7.68 s
8.17 dd (9, 5.5)
6.98 ddd
(10, 9, 2.5)
9.44 broad s 9.67 broad s
—
7.94 s
8.14 d (9)
6.92 dd
(9, 2)
7.12 d (2)
7.78 d (3.5)
7.35 d (3.5)
5.50 d (9)
3.87 s, OMe
3.90–3.25,
m, H-200-H-600
—
8.04 s
8.30 dd (9, 5.5
7.10 ddd
(10, 9, 2.5)
7.27 dd (10, 2.5)
7.81 d (3.5)
7.39 d (3.5)
5.44 d (9)
—
7.75 s
—
7.83 s
7.73 d (2)
8.04 d (8.5)
6.77 dd
7.82 d (3)
8.13 d (9)
6.89 dd
8.01 dd (9, 4.5) 7.66 dd (9, 5.5)
7.06 dd (12, 9)
7.09 ddd
(10, 9, 2.5)
7.30 dd (10, 2.5)
—
(8.5, 2)
(9, 2)
H-7
7.23 dd 10, 2.5) 7.09 dd (10, 2.5)
6.89 d (2)
7.75 d (3.5)
7.31 d (3.5)
—
7.22 d (2)
7.76 d (3.5)
7.33 d (3.5)
4.94 d (7)
3.90–3.25, m,
H-200-H-600
—
H-40
H-50
H-100
Other
7.78 d (3.5)
7.34 d (3.5)
—
7.74 d (3.5)
7.26 d (3.5)
—
7.76 d (3.5)
7.33 d (3.5)
5.10 d (7.5)
4.07 s, N-Me
3.70–3.15,
—
—
3.68 s, N-Me
3.90–3.25, m,
H-200-H-600
3.80 s, N-Me
m, H-200-H-600
aCoupling constants (J) are reported to the nearest 0.5 Hz.

M. S. C. Pedras, P. W. K. Ahiahonu / Bioorg. Med. Chem. 10 (2002) 3307–3312
3309
Table 2. 13C NMR chemical shifts (ppm) of compounds 1b–1c (CD3CN), 5a (DMSO-d6), 5, 5b–5d (CD3CN), 6(CD3OH)a
C #
1b
1c
5
5a
5b
5c
5d
6
C-2
C-3
C-3a 122.7
C-4
C-5
C-6
C-7
127.3
112.4
133.1
113.4
124.6
124.6 126.5 125.2
112.0 113.1 112.4
118.9 120.5 119.8
127.1
112.5
122.5
132.5
110.4
124.3
137.8
84.7
124.4
120.2, d jC-F=10
2
3
3
3
3
3
122.3, d jC-F=10 124.8, d jC-F=10 121.6 121.4 121.9
2
122.6, d jC-F=10 117.1, d jC-F=9
2
2
2
110.4, d jC-F=25 112.1, d jC-F=25 111.4 111.4 111.9
161.7, d jC-F=238 162.9, d jC-F=237 154.1 155.1 157.6
110.3, d jC-F=25 111.0, d jC-F=22 110.8, d jC-F=26
160.5, d jC-F=238 151.7, d jC-F=237 161.0, d, jC-F=240
2
2
2
2
2
99.2, d jC-F=26
3
99.4, d jC-F=27
3
97.4 100.0
94.9
98.2, d jC-F=28 130.0, d jC-F=28
3
97.4, d jC-F=27
3
3
C-7a 138.5, d jC-F=12 140.4 jC-F=12
137.6 137.9 138.6
165.6 163.8 163.0
142.9 143.4 143.1
116.2 116.9 116.7
138.6, d jC-F=13 130.9, d jC-F=13 137.0, d jC-F=12
C-20
C-400
C-500
C-100
C-2
C-300
C-400
C-500
C-600
Other
165.6
143.2
117.5
165.6
145.5
118.8
162.8
143.2
117.1
85.7
72.7b
77.6b
70.3b
79.5b
61.9b
162.9
143.1
—
—
—
—
—
—
—
—
—
116.5
104.4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
102.6
74.3b
77.5b
70.6b
77.9b
61.6b
—
85.2
72.7b
74.7b
77.8b
76.7b
70.4b
79.3b
61.9b
55.7, OCH3
70.4b
77.1b
61.5b
36.9, N-CH3
35.6, N-CH3
—
32.8, N-CH3 115.5, CN
aChemical shifts of camalexins 1b and 1c change with concentration; reported values are for c=10 mg/mL.
bSignals in the same column may be interchanged.
Table 3. Products of metabolism of camalexins 1a–1c and compound 5 at 1 ꢀ 10ꢁ4 M by Sclerotinia sclerotiorum
Compound added to fungal culturesa
Products (%)b of metabolism after incubation for 24 and 48 h
Camalexin (1)
6-Methoxycamalexin (1a)
6-Fluorocamalexin (1b)
6-Fluoro-1-methylcamalexin (1c)
6-Hydroxycamalexin (5)
Camalexin (1)+6-fluorocamalexin (1b)
Complete biotransformation to 5 (28%) and 5a (22%) with no camalexin recovered after 24 h
Complete biotransformation to 5 (18%), 5a (20%) and 5b (12%) with no camalexin recovered after 24 h
Complete biotransformation to 5c (52%) with no camalexin recovered after 48 h
Biotransformation to 5d (17%) and 6 (17%) with 35% camalexin recovered after 48 h
Complete biotransformation to 5a in 24 h
Biotransformation to 5a (80%) and no 5c in 24 h, 5a (91%) and 5c (5%) in 48 hc
aCompounds were dissolved in DMSO, added to 6-day-old cultures and incubated at 24ꢂ2 ꢃC.
bPercentage yields of products represents isolated amounts; due to losses during extraction and separation, yields of glucosyl derivatives 5a–5d
determined by HPLC analysis are 25–30% higher than isolated yields.
cPercentage yields of products represents HPLC determined yields.
synthesized and separately administered to cultures of
S. sclerotiorum. After incubation and isolation of the
resulting products, metabolites 5c and 5d were obtained;
while 1b was completely metabolized to 5c, 1c was
transformed to 5d (17%) and 6 (17%) (Table 3). The
chemical structures of 5c and 5d were deduced from
analysis of NMR spectroscopic data (Tables 1 and 2),
and HMQC and HMBC spectral data (substituent
located at N-1 in 5c displayed C–H long-range corre-
lation between anomeric H and C-2 and C-7a; sub-
stituent located C-7 in 5d displayed C–H long-range
correlation between anomeric H and C-7). The chemical
structure of 6 was deduced from spectroscopic data and
confirmed by synthesis as described in the Experimental.
Results of the metabolism of camalexin (1) suggest that
the first stepfor camalexin detoxification involves oxi-
dation of C-6 to the 6-hydroxy derivative 5, whereas in
the case of 1a, the first stepinvolves oxidative deme-
thylation to 5. The second metabolic stepinvolves glu-
cosylation of 5 to 5a; however, a minor pathway for
detoxification of 1a leads to glucosylation of N-1. Next,
to probe the specificity of the oxygenase(s) involved in
the first transformation stepof 1 and 1a, in additional
experiments, camalexin (1) and 6-fluorocamalexin (1b)
were administered together to fungal cultures, and the
cultures incubated and analyzed as previously descri-
bed. Camalexin (1) was metabolized to 5 and 5a almost
completely (ca. 95%) in 48 h, whereas analogue 1b was
metabolized only after camalexin. On the other hand,
although analogue 1c was metabolized much slower
than camalexin (ca. 24 h versus 7 days) it did not affect
the rate of camalexin transformation when co-incu-
bated. These results indicate that there is competition
between 1 and 1b but no competition between substrates
1 and 1c. Nonetheless, a better understanding of the
mechanisms involved in these biotransformations will
require kinetic studies with the cell-free cultures and
purified enzymes, which are of pivotal importance in the
design of detoxification inhibitors of camalexins.
The antifungal activity of camalexins 1–1c and metabo-
lites 5a–5d to S. sclerotiorum was compared (Table 4).
After 7 days of incubation, the mycelium of control
plates incubated with S. sclerotiorum covered 100% of
the plates, while plates containing 1 or 1c (5 ꢀ 10ꢁ4 M)
showed no mycelial growth, and plates containing 1a or
1b (5 ꢀ 10ꢁ4 M) showed slower growth than controls.
By contrast, plates containing 5a–5d were similar to
control plates. These results demonstrated for the first
time that metabolism of camalexins 1–1c in S. scler-
otiorum leads to products having no detectable anti-
fungal activity, thus this metabolism is a detoxification
process.
Although glucosylation is a very common detoxifica-
tion mechanism among plants, it is less usual in

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M. S. C. Pedras, P. W. K. Ahiahonu / Bioorg. Med. Chem. 10 (2002) 3307–3312
Table 4. Percentage of inhibitiona of Sclerotinia sclerotiorum incubated with camalexins 1–1c (after 7 days, constant light)
Compound assayed against fungal cultures
Concentration (M)
% of InhibitionꢂSD (average diameter of mycelium growth, mm)
Control
Camalexin (1)
—
0ꢂ0.5 (35)
100ꢂ0.5 (8)b
0ꢂ0.5 (35)
41ꢂ0.5 (24)
0ꢂ0.5 (35)
70ꢂ0.5 (16)
0ꢂ0.5 (35)
100ꢂ0.5 (8)b
0ꢂ0.5 (35)
5 ꢀ 10ꢁ4
5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ4
5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ4
5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ4
5 ꢀ 10ꢁ5
6-Methoxycamalexin (1a)
6-Fluorocamalexin (1b)
6-Fluoro-1-methylcamalexin (1c)
aThe percentage of inhibition was calculated using the formula: % inhibition=100ꢁ[(growth on treated/growth in control) ꢀ 100]; results are the
mean of at least three independent experiments conducted in triplicate.
bThe size of mycelium plug used to initiate cultures was 8 mm.
microorganisms.12 To the best of our knowledge this is
the first example of phytoalexin glucosylation by a plant
pathogen. Microorganisms able to transform mycotox-
ins to glucosyl derivatives may have potential applica-
tions in treating contaminated cereals13 or as models of
mammalian metabolites.14 Here we demonstrate that
S. sclerotiorum can detoxify natural camalexins and
analogues through unusual glycosylation of the indole
ring. In this context, because N-1 glucosylation of
indoles are often low yield and time consuming reac-
tions, the fungus S. sclerotiorum might be a potential
biotransformation agent in some of these synthetic
preparations.15
10ꢁ5 and 1 ꢀ 10ꢁ5 M); control solutions contained 1%
DMSO in minimal media.2 Sterile tissue culture plates
(6-well, 35 mm diameter) containing test solutions and
control solution (2 mL per well) were inoculated with
mycelium plugs placed upside down on the center of
each plate (8 mm cut from 3-day-old PDA plates of
S. sclerotiorum, clone # 33) and incubated under con-
stant light for 7 days. All bioassay experiments were
carried out in triplicate, at least three times.
Time-course study and metabolism of camalexins 1–1c
and 6-hydroxycamalexin (5). Erlenmeyer flasks
(250 mL) containing 100 mL of minimal media were
employed. Each flask was inoculated with sclerotia of S.
sclerotiorum clone # 33 and was incubated at 25ꢂ2 ꢃC
on a shaker at 120 rpm under constant light. After six
days a solution of each compound in DMSO was added
to fungal cultures (1 ꢀ 10ꢁ4 or 2 ꢀ 10ꢁ4 M) and to
uninoculated medium (control); DMSO (200 mL) was
added to control cultures. Samples (10 mL each) were
taken from the flasks immediately after adding the
compounds. Subsequently, 15 mL samples were taken
every 24 h for 4 days and final samples after 7 days. The
samples were either immediately extracted or frozen for
later extraction. Each sample was first extracted with
EtOAc; the resulting aqueous layer was acidified (to pH
2 with dil HCl) and extracted with EtOAc. Finally the
acidic aqueous layer was made alkaline (to pH 10 with
28% aqueous ammonia) and extracted with chloroform.
After concentration of the solvent, the extracts were
analyzed by HPLC. Metabolite 5 in DMSO (1 ꢀ
10ꢁ4 M) was subjected to further biotransformation by
S. sclerotiorum clone # 33. The biotransformation was
monitored by taking samples after 3 h, 6 h, 12 h, 24 h,
48 h, and 7 days. The samples were worked up and
analyzed by HPLC.
Experimental
General experimental procedures
All chemicals were purchased from Sigma-Aldrich
Canada Ltd., Oakville, ON, Canada. All solvents were
HPLC grade and used as such, except for CH2Cl2 and
CHCl3 which were redistilled. Remaining conditions as
previously reported.4
HPLC analysis was carried out with a high performance
liquid chromatograph equipped with quaternary pump,
automatic injector, and diode array detector (wave-
length range 190–600 nm), degasser, and a Hypersil
ODS column (5 mm particle size silica, 4.6 id ꢀ 200 mm),
equipped with an in-line filter. Mobile phase: 75% H2O-
25% CH3CN to 100% CH3CN, for 35 min, linear gra-
dient, and a flow rate 1.0 mL/min. Specific rotations,
[a]D were determined at ambient temperature on a Per-
kin–Elmer 141 polarimeter using a 1 mL, 10 cm path
length cell; the units are 10ꢁ1 deg cm2 gꢁ1 and the con-
centrations (c) are reported in g/100 mL.
Isolation of metabolites 5–5d and 6. To obtain larger
amounts of extract to isolate the products of metabo-
lism of each camalexin, experiments were carried out
with 1-L batches, as described above for time-course
studies. Only the chromatograms of the EtOAc extracts
of the neutral broth showed peaks not present in chro-
matograms of extracts of control cultures. Thus, the
EtOAc extracts were fractionated by FCC on silica gel
(gradient elution: CH2Cl2–MeOH, 100:0–85:15), and
each fraction was analyzed by HPLC. The metabolites
were isolated by preparative TLC (silica gel, CH2Cl2–
Fungal cultures and bioassays
S. sclerotiorum was obtained from C. Lefol, AAFC,
Saskatoon, Canada. The fungal isolates were grown on
potato dextrose agar (PDA) plates at 24ꢂ2 ꢃC, under
constant light for 7 days. The antifungal activity of
compounds 1–1c and 5a–5d was determined using the
following mycelial radial growth bioassay. Solutions of
each compound in DMSO (5 ꢀ 10ꢁ2 M) were used to
prepare assay solutions in minimal media (5 ꢀ 10ꢁ4, 5 ꢀ

M. S. C. Pedras, P. W. K. Ahiahonu / Bioorg. Med. Chem. 10 (2002) 3307–3312
3311
MeOH, 90:10, multiple development) and/or reverse-
phase preparative TLC (RP C-18 silica gel, H2O–
CH3CN, 60:40).
3 mL).4 After 3 h, SeO2 (13.7 mg, 0.12 mmol) was added
to the reaction mixture and refluxing proceeded for 3 h.
The reaction mixture was allowed to cool, anhydrous
MgSO4 (2 mg) was added and stirring was continued for
10 min at room temperature. The mixture was then fil-
tered and the solvent was removed under reduced pres-
sure. The residue was separated by prep. TLC (CH2Cl2,
developed three times) to give 6 as off-white powder
(9.1 mg, 74%, Rt=15.9 min) and unreacted 6-fluoro-1-
methylindole-3-carboxaldehyde (6.8 mg).
Synthesis of camalexins 1–1c. Camalexin (1) was syn-
thesized as previously reported.9 6-Methoxycamalexin
(1a, 94 mg, 67% yield based on recovered starting
material) was synthesized similarly but replacing indole
with 6-methoxy indole (100 mg, 0.68 mmol). 6-Fluor-
ocamalexin (1b, 131 mg, 59% yield based on recovered
starting material) was synthesized similarly but repla-
cing indole with 6-fluoroindole (150 mg, 1.1 mmol).
6-Fluoro-1-methylcamalexin (1c, 112 mg, 96%) was
synthesized from 6-fluorocamalexin (1b, 110 mg,
0.51 mmol) by treatment with MeI/NaH.
Camalexin (1). Rt=16.5 min (HPLC, broad peak);
remaining data in supporting information.
6-Methoxycamalexin (1a). Rt=23.4 min (HPLC, broad
peak); remaining data in supporting information.
Synthesis of 6-fluoroindole-3-carbonitrile (6). Phospho-
rus oxychloride (50 mL, 0.55 mmol, freshly distilled) was
added dropwise with stirring to DMF (160 mg, 1.12 mL)
under argon atmosphere. 6-Fluoroindole (67.5 mg,
0.5 mmol) in DMF (40 mg, 30 mL) was then added
dropwise with continuous stirring, at room temperature.
The reaction mixture was kept at 35 ꢃC for 45 min and
then poured into crushed ice. Aqueous NaOH (96 mg in
50 mL, 2.4 mmol) was added slowly to the reaction
mixture until it became alkaline. The solution was
boiled for 1 min, cooled to room temperature and then
extracted with EtOAc (50 mL, 2ꢀ), the organic phases
were combined, washed with water, dried (Na2SO4) and
concentrated under vacuum to give 6-fluoroindole-3-
6-Fluorocamalexin (1b). Rt=19.3 min (HPLC, broad
1
peak); H NMR in Table 1; 13C NMR in Table 2;
HRMS-EI m/z: measured 218.0315 (M+, calcd
218.0314 for C11H7N2FS,); MS-EI m/z (relative inten-
sity): 218 (100); FTIR (cmꢁ1) 3162, 1624, 1555, 1451,
1134, 839; UV (lmax, CH3CN) 218 (log e 4.4), 287 (log e
4.1), 314 (log e 4.1).
6-Fluoro-1-methylcamalexin (1c). Rt=23.6 min (HPLC,
1
broad peak); H NMR in Table 1; 13C NMR in Table
2; HRMS-EI m/z: measured 232.0473 (M+, calcd
232.0471 for C12 H9 N2 FS); MS-EI m/z (relative inten-
sity): 232 (100), 174 (49), 173 (27); FTIR (cmꢁ1) 2922,
2853, 1462, 943; UV (lmax, CH3CN) 224 (log e 4.4), 290
(log e 4.1), 322 (log e 4.3).
1
carboxaldehyde (70.1 mg, 86%, Rt=8.5 min). H NMR
(300 MHz, CD3CN) d 10.03 (br s, 1H, D2O exchange-
able), 9.95 (s, 1H), 8.11 (dd, J=9, 5.5 Hz, 1H), 7.98 (s,
1H), 7.26 (dd, J=10, 2.5 Hz, 1H), 7.04 (ddd, J=10, 9,
2.5 Hz, 1H); 13C NMR (75 MHz, CD3CN) d 188.1,
163.3 (d, J=238 Hz), 140.9, 140.1 (d, J=13 Hz), 125.3
(d, J=10 Hz), 123.9, 121.6, 113.6 (d, J=24 Hz), 101.5
(d, J=27 Hz); HRMS-EI m/z: measured 163.0430 (M+,
calcd 163.0433 for C9 H6 NOF); FTIR (cmꢁ1) 3131,
2922, 2862, 1630, 1535, 1453, 1158, 1074, 951, 823; UV
(lmax, CH3CN) 221 (log e 4.5), 247 (log e 4.2), 295 (log e
4.2), 318 (log e 4.2). 6-Fluoroindole-3-carboxaldehyde
(40 mg, 0.25 mmol) was dissolved in anhydrous THF
under argon atmosphere. Sodium hydride (8.2 mg,
0.34 mmol) was added with constant stirring at 0 ꢃC
followed by methyl iodide (32 mL, 0.34 mmol) and the
reaction mixture was stirred at 0 ꢃC for 30 min. The
reaction mixture was poured into ice-cold water and
then extracted with EtOAc (50 mL, 2ꢀ). The extract
was dried (Na2SO4) and the solvent removed under
reduced pressure to give 6-fluoro-1-methylindole-3-car-
1
6-Hydroxycamalexin (5). Rt=11.4 min (HPLC);
H
NMR in Table 1; 13C NMR in Table 2; HRMS-EI m/z:
measured 216.0361 (M+, calcd 216.0357 for
C11H8N2OS); MS-EI m/z (relative intensity): 216 (100),
158 (35). FTIR (cmꢁ1) 3380, 2924, 2854, 1534, 1361,
1312, 1085; UV (lmax, CH3CN) 221 (log e 4.5), 251 (log
e 4.1), 295 (log e 4.2), 322 (log e 4.1).
6-(O-ꢀ-D-Glucopyranosyl)camalexin (5a). Rt=4.2 min
1
(HPLC) ; [a]D=ꢁ12.2 (c 0.36, MeOH); H NMR in
Table 1; 13C NMR in Table 2; HRMS-FAB m/z: mea-
sured 379.0967 ([M+H]+, calcd 379.0964 for
C17H19N2O6S); MS-FAB m/z (relative intensity): 379
(15), 329 (40), 176 (100); FTIR (cmꢁ1) 3312, 2924, 1549,
1248, 1077; UV (lmax, CH3CN) 222 (log e 4.0), 251 (log
e 3.7), 286 (log e 3.9), 321 (log e 3.7).
6-Methoxy
1-(ꢀ-D-glucopyranosyl)camalexin
(5b).
1
1
boxaldehyde (42.4 mg, 96%, Rt=11.4 min). H NMR
Rt=7.1 min (HPLC); [a]D=ꢁ14.4 (c 0.08, MeOH);
H
(300 MHz, CD3OD) d 9.82 (s, 1H), 8.15 (dd, J=9,
5.5 Hz, 1H), 8.06 (s, 1H), 7.27 (dd, J=10, 2.5 Hz, 1H),
7.05 (ddd, J=10, 9, 2.5 Hz, 1H), 3.87 (s, 3H); 13C NMR
(75 MHz, CD3OD) d 186.8, 162.2 (d, J=240 Hz), 143.7,
140.1 (d, J=12 Hz), 123.8 (d, J=9 Hz), 122.9, 119.2,
112.2 (d, J=24 Hz), 98.3 (d, J=24 Hz), 34.1; HRMS-EI
mass m/z: measured 177.0587 (M+, calcd 177.0590 for
C10H8NOF,). Pyridine (10 mL in 0.5 mL CHCl3) was
NMR in Table 1; 13C NMR in Table 2; HRMS-FAB m/
z: measured 393.1117 ([M+H]+, calcd 393.1120 for
C18H21N2O6S); MS-FAB m/z (relative intensity): 393
(20), 207 (40), 115 (100). FTIR (cmꢁ1) 3335, 2924, 2853,
1557, 1456, 1079; UV (lmax, CH3CN) 216 (log e 4.1),
254 (log e 3.7), 297 (log e 3.7), 318 (log e 3.7).
6-Fluoro-1-(ꢀ-D-glucopyranosyl)camalexin (5c).
Rt=7.4 min (HPLC); [a]D=ꢁ5.0 (c 0.09, MeOH); H
1
.
added to a refluxing mixture of HONH2 HCl (8.6 mg,
0.12 mmol)
aldehyde (20 mg, 0.11 mmol) in CHCl3–EtOH (7:3, v/v,
and
6-fluoro-1-methylindole-3-carbox-
NMR in Table 1; 13C NMR in Table 2; HRMS-FAB
m/z: measured 381.0921 ([M+H]+, calcd 381.0920 for

3312
M. S. C. Pedras, P. W. K. Ahiahonu / Bioorg. Med. Chem. 10 (2002) 3307–3312
C17H17N2FSO5); MS-FAB m/z (relative intensity): 381
(90), 329 (25), 218 (38), 176 (100), 149 (33); FTIR
(cmꢁ1) 3342, 2924, 2852, 1553, 1499, 1459, 1127, 1083,
1022, 960, 637. UV (lmax, CH3CN) 221 (log e 3.8), 315
(log e 3.9).
2. Pedras, M. S. C.; Okanga, F. I. J. Agric. Food Chem. 1999,
47, 1196.
3. Pedras, M. S. C.; Khan, A. Q. Bioorg. Med. Chem. Lett.
1997, 7, 2255.
4. Pedras, M. S. C.; Khan, A. Q. Phytochemistry 2000, 53,
59.
5. Pedras, M. S. C.; Khan, A. Q.; Taylor, J. L. Plant Sci.
1998, 139, 1.
6. Kohn, L. M.; Brunner, L. J.; Morrall, R. A. A.; Yoell, H.;
Milgroom, M. G.; Anderson, J. B.; Kohli, Y. Mol. Ecol. 1995,
4, 69.
7. See, for example: Daniel, M., Purkayastha, R. P., Eds.
Handbook of Phytoalexin Metabolism and Action. Marcel
Dekker: New York, 1995 p. 615.
8. Pedras, M. S. C.; Ahiahonu, P. W. K. Abstracts of Papers,
37th National Organic Symposium, June, Bozeman, MT,
2001; Abstract 0197.
6-Fluoro-1 -methyl -7 -( O-ꢀD-glucopyranosyl) camalexin
(5d). Rt=7.2 min (HPLC); [a]D=12.4 (c 0.06, MeOH); 1
H NMR in Table 1; 13C NMR in Table 2; HRMS-FAB
m/z: measured 411.1028 ([M+H]+ calcd 411.1026 for
C18H20N2FSO6); MS-FAB m/z (relative intensity): 411
(10), 329 (22), 176 (100); FTIR (cmꢁ1) 3331, 2924, 1629,
1466, 1381, 1074; UV (lmax, CH3CN) 218 (log e 2.9),
326 (log e 3.3).
6-Fluoro-1-methylindole-3-carbonitrile (6). Rt=15.9 min
1
9. Ayer, W. A.; Peter, A. C.; Ma, Y.; Miao, S. Tetrahedron
1992, 48, 2919.
(HPLC); H NMR in Table 1; 13C NMR in Table 2;
HRMS-EI m/z: measured 174.0596 (M+, calcd
174.0593 for C10H7N2F); MS-EI m/z (relative intensity):
174 (100), 173 (38); FTIR (cmꢁ1) 2922, 2219, 1739,
1463, 1246, 1110; UV (lmax, CH3CN) 215 (log e 4.0),
283 (log e 3.4).
10. b-d-Glucopyranosyl camalexins 5a and 5b were further
transformed into more polar undetermined products.
11. The yields of metabolic products were determined
by HPLC analysis and compared with isolated yields.
The isolated yields of glucosides 5a–5d were ca. 25–
30% lower than yields determined by HPLC; butanol
extractions did not improve the recovery of glucosyl
camalexins.
12. See for example, Hall, J. C.; Hoagland, R. E.; Zabloto-
wicz, R. M. Pesticide Biotransformation in Plants and Micro-
organisms; ACS Symposium Series; ACS: Washington, DC, ,
2000; p. 432.
13. (a) El-Sharkawy, S.; Abul-Hajj, Y. J. Nat. Prod. 1987, 50,
520. (b) Kamimura, H. Appl. Environ. Microbiol. 1986, 52,
515.
14. Chatterjee, P.; Pezzuto, J. M.; Kouzi, S. A. J. Nat. Prod.
1999, 62, 761.
Acknowledgements
Support for the authors’ work was obtained from the
Natural Sciences and Engineering Research Council of
Canada and the University of Saskatchewan. We thank
C. Lefol, Agriculture and Agri-Food Canada Research
Station, Saskatoon SK, for kindly providing clones of
Sclerotinia sclerotiorum.
15. (a) For example, some glycosylated indolocarba-
zoles represent an important series of antitumor
drugs; for example: Winneroski, L. L.; Krumrich,
C. A. J. Org. Chem. 1999, 64, 2465. (b) Voldoire, A.;
Sancelme, M.; Prudhomme, M.; Colson, P.; Houssier, C.;
Bailly, C.; Leonce, S.; Lambel, S. Bioorg. Med. Chem. 2001, 9,
357.
References and Notes
1. For a recent review of cruciferous phytoalexins see, Pedras,
M. S. C.; Okanga, F. I.; Zaharia, I. L.; Khan, A. Q. Phy-
tochemistry 2000, 53, 161.