Biotransformation of the phytoalexin camalexin by the phytopathogen Rhizoctonia solani

Article abstract of DOI:10.1016/S0031-9422(99)00479-3

The unusual metabolism of the cruciferous phytoalexin camalexin by virulent and weakly virulent isolates of the root rot fungus Rhizoctonia solani Kuhn is reported. This biotransformation proceeded via 5- hydroxycamalexin, which was further biotransformed into more polar metabolites. Importantly, the metabolites resulting from transformation of camalexin were significantly less toxic to the pathogen than camalexin. Thus, it was concluded that R. solani can detoxify camalexin through oxidation of the indole ring. The chemistry involved in the structure determination of the intermediates of this pathway, their synthesis as well as antifungal activity is described.

Full text of DOI:10.1016/S0031-9422(99)00479-3

Phytochemistry 53 (2000) 59±69
www.elsevier.com/locate/phytochem
Biotransformation of the phytoalexin camalexin by the
phytopathogen Rhizoctonia solani
M. Soledade C. Pedras*, Abdul Q. Khan
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK, Canada S7N 5C9
Received 30 April 1999; received in revised form 13 July 1999
Abstract
The unusual metabolism of the cruciferous phytoalexin camalexin by virulent and weakly virulent isolates of the root rot
fungus Rhizoctonia solani Kuhn is reported. This biotransformation proceeded via 5-hydroxycamalexin, which was further
biotransformed into more polar metabolites. Importantly, the metabolites resulting from transformation of camalexin were
signi®cantly less toxic to the pathogen than camalexin. Thus, it was concluded that R. solani can detoxify camalexin through
oxidation of the indole ring. The chemistry involved in the structure determination of the intermediates of this pathway, their
synthesis as well as antifungal activity is described. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Rhizoctonia solani; Camalexin; Phytoalexin metabolism; Antifungal; Detoxi®cation
1. Introduction
(Desm.) Ces. et de Not.), one of the most damaging
cruciferous pathogens, brassicanal A (3) dioxibrassinin
(4), spirobrassinin (5) (Pedras, 1998), and camalexin
(6) (Pedras, Khan & Taylor, 1998) caused signi®cantly
lower inhibition at similar concentrations.
Phytoalexins from crucifers have an indole or related
ring, and at least one sulfur atom as a common struc-
tural feature (Pedras, Khan, & Taylor, 1997). Despite
a close biogenetic relationship, these phytoalexins pos-
sess di€erent ring systems and, not surprisingly, di€er-
ent biological activity. For example, brassinin (1) and
cyclobrassinin (2), the ®rst reported cruciferous phy-
toalexins produced by a variety of Brassica species
(Takasugi, Katsui & Shirata, 1986), brassicanal A (3)
(Pedras & Khan, 1996), dioxibrassinin (4) (Pedras &
Okanga, 1998), spirobrassinin (5) (Pedras, 1998), and
camalexin (6), also a cruciferous phytoalexin produced
by Arabidopsis thaliana (Tsuji, Jackson, Gage,
Hammerschmidt & Somerville, 1992) and Camelina
sativa (Browne, Conn, Ayer & Tewari, 1991) have sig-
ni®cantly di€erent antifungal activities. While brassinin
(1) and cyclobrassinin (2) completely inhibited spore
germination of Phoma lingam (Tode ex Fr.) (Pedras &
Taylor, 1993) (perfect stage Leptosphaeria maculans
* Corresponding author. Tel.: +1-306-966-4772; fax: +1-306-966-
4730.
E-mail address: pedras@sask.usask.ca (M.S.C. Pedras).
To better understand the role of phytoalexins in the
disease resistance of economically valuable brassica
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00479-3

60
M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
Scheme 1. Transformation of camalexin (6) by Rhizoctonia solani: virulent isolate AG 2-1 and weakly virulent isolate AG 4.
crops (Cruciferae family, syn. Brassicaceae ), e.g.
canola (Brassica napus, B. rapa ), rapeseed (B. napus,
B. rapa ), and mustard (B. juncea ), we have been inves-
tigating the metabolism of diverse cruciferous phytoa-
lexins by agriculturally important pathogens. Although
to date only a few examples demonstrate that crucifer-
ous pathogens can detoxify phytoalexins eciently
(Pedras, 1998), multiple examples exist for the phytoa-
lexins of other plant species (Daniel & Purkayastha,
1995; Van Etten, Sandrock, Wasmann, Soby,
McCluskey & Wang, 1995; Schafer, 1994; Van Etten,
Mathews & Mathews, 1989). We have established that
P. lingam can rapidly metabolize and detoxify brassi-
nin (1) (Pedras & Taylor, 1993), cyclobrassinin (2)
(Pedras & Okanga, 1998), brassicanal A (3) (Pedras &
by both virulent and weakly virulent isolates of R.
solani, as well as the antifungal activity of camalexin
(6) and its biotransformation products. In addition, we
report for the ®rst time the synthesis of the intermedi-
ates of camalexin biotransformation pathways, as well
as the chemistry involved in camalexin (6) oxidation.
2. Results and discussion
The metabolism of the phytoalexin camalexin (6) by
virulent and weakly virulent isolates of R. solani was
investigated. Initial experiments were carried out to
determine the minimum inhibitory concentration
4
(5 Â 10 M) of camalexin to R. solani. Subsequently,
Khan, 1996), and dioxibrassinin (4) (Pedras
&
camalexin (6) was incubated with R. solani virulent iso-
late AG 2-1 under normal laboratory light conditions
(i.e. 12 h light/12 h dark). Culture samples were with-
drawn at di€erent intervals, extracted ®rst with Et2O,
and then acidi®ed and reextracted with Et2O. Separate
cultures containing only the fungus or medium con-
taining only camalexin were incubated under similar
conditions and extracted. HPLC analyses of extracts
of fungal cultures incubated with camalexin indicated
that it was completely metabolized in ca. 72 h. In ad-
dition, the HPLC analyses indicated the optimum in-
cubation time for isolation of the putative
intermediates/products of camalexin metabolism.
Three products 7±9 were isolated after incubation of
R. solani isolate AG 2-1 with camalexin (see Scheme
1). The chemical structure of each metabolite was
deduced from comparison of their spectroscopic data
with those of camalexin, and con®rmed by chemical
syntheses.
Okanga, 1998). Conversely, spirobrassinin (5) and
camalexin (6) did not appear to be metabolized or
have a signi®cant e€ect on P. lingam mycelial growth
(Pedras, 1998). Nonetheless, because the resistance of
C. sativa to the root rot fungus Rhizoctonia solani
Kuhn appeared to correlate with the presence of cama-
lexin (6) in root tissues (Conn, Browne, Tewari, &
Ayer, 1994), we examined the metabolism of camalexin
by R. solani. Preliminary experiments established that
a virulent isolate of R. solani (more virulent to B.
napus than to C. sativa, Conn et al., 1994) metabolized
camalexin to 5-hydroxycamalexin, which was further
transformed into more polar metabolites (Pedras &
Khan, 1997). Next, we examined the metabolism of
camalexin by a weakly virulent isolate of R. solani
(weakly virulent to B. napus and no infection on C.
sativa, Conn et al., 1994), as well as the biological ac-
tivity of the metabolic products. Here we wish to
report new aspects of the metabolism of camalexin (6)
Relative to camalexin (C11H8N2S), metabolite 7 con-

M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
61
Table 1
1H-NMR chemical shifts (ppm) and multiplicities (J in Hz) for compounds 6±12 (solvent)
H no.
6 (CDCl3)
7 (CD3OD)
8 (CD3OD)
9 (CD3OD)
10 (CD3OD)
11 (CD3OD)
12 (CD3OD)
2
8.09
d (2.8)
8.13
m
7.90
br s
7.49
d (2.0)
±
8.19
s
7.81
s
7.83
s
7.88
s
7.92
s
8.09
4
7.75
d (2.8)
±
6.97
d (1.4)
±
8.03
m
8.06
m
dd (1.5, 6.5)
7.17
5
7.29
m
7.10
m
7.16
m
m
7.17
6
7.29
m
6.77
dd (2.0, 8.7)
7.30
7.02
dd (2.8, 8.8)
7.80
6.82
dd (1.4, 8.7)
7.30
7.10
m
7.16
m
m
7.41
7
7.45
m
7.42
m
7.43
m
d (8.7)
7.73
d (8.8)
8.04
d (8.7)
±
dd (1.5, 6.5)
±
4'
5'
7.81
d (3.2)
7.23
d (3.2)
4.44
t (9)
4.00
t (9)
3.73
t (6)
3.52
t (6)
d (3.4)
7.42
d (3.4)
d (3.0)
8.10
d (3.0)
±
±
tained an additional oxygen atom (C11H8N2OS), as
determined by HR±EIMS. Comparison of the 1H-
NMR spectrum (see Table 1) of 7 with that of cama-
lexin (6) indicated the presence of an indolic substitu-
(7) utilizing a route similar to that followed for cama-
lexin, as shown in Scheme 2 (Furstner & Ernst, 1995).
ent, since only
a three-proton spin system was
displayed in addition to the H-2 and two thiazole pro-
tons. The 13C-NMR spectrum (Table 2) displayed 11
carbon signals, one of which was characteristic of a
deshielded sp2 C atom (153.3 ppm). These spectro-
scopic features suggested that metabolite 7 contained
an OH group attached to C-5 or C-6. That the OH
group was attached to C-5 was deduced from compari-
son of HMBC and HMQC spectral data with that of
camalexin. In addition, methylation of 7 with CH2N2
Unlike 5-hydroxycamalexin (7), metabolites 8 and 9
did not appear to accumulate to any signi®cant extent
in cultures of isolate AG 2-1 incubated with camalexin
(6), and 5-hydroxycamalexin (7) was the main com-
ponent of the culture extracts, even after six days of
incubation. The molecular formula of 8 (C11H8N2O3S)
obtained by HR±EIMS indicated the presence of two
additional oxygen atoms relative to 5-hydroxycama-
1
and comparison of the H- NMR spectral data of the
product with those of 5-methoxycamalexin and 6-
methoxycamalexin (Ayer, Peter, Ma & Miao, 1992)
indicated that the methyl derivative of 7 was 5-meth-
oxycamalexin. Furthermore, the structural assignment
was corroborated by synthesis of 5-hydroxycamalexin
1
lexin (7). While the H- NMR spectrum (Table 1) of 8
indicated spin systems similar to those of 7, its 13C-
Table 2
13C- NMR chemical shifts (ppm) of compounds 6±12 (solvent)
C no.
6a (CDCl3)
7 (CD3OD)
8 (CD3OD)
9 (CD3OD)
10 (CD3OD)
11 (CD3OD)
12 (CD3OD)
2
126.3
112.2
124.5
120.1
123.4b
121.9b
111.8
136.6
163.8
140.1
115.7
127.1
111.4
126.6
105.1
153.3
113.6
113.7
133.1
166.2
142.6
116.7
161.9
187.1
130.2
119.8
155.2
121.4
125.8
124.1
168.8
145.9
128.4
134.5
85.4
131.6
103.7
154.3
115.1
114.3
129.8
±
130.3
104.9
138.1
121.9
123.5b
121.8b
112.8
126.7
165.1
67.9
129.3
111.9
138.2
121.7
123.6b
122.1b
112.9
127.1
168.9
62.2
130.2
111.3
138.3
121.9
123.6b
122.2b
112.9
127.3
±
3
3a
4
5
6
7
7a
2'
4'
5'
Other
±
±
±
117.8-CN
54.6
43.0
±
172.0 CONH2
a Chemical shifts of camalexin (6) change with concentration; reported values are for c ˆ 10 mg/ml.
b Signals may be interchanged.

62
M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
Scheme 2. Synthesis of 5-hydroxycamalexin (7) and 5-hydroxy-2-formamidophenyl-2'-thiazolylketone (8).
NMR spectrum (Table 2) displayed two resonances in-
dicative of C1O groups (or equivalents, at 161.9 and
187.1 ppm). Furthermore, while HMQC spectral data
suggested that the thiazole ring of metabolite 8 was
intact, a carbon signal at (161.9 ppm) attached to a
singlet proton (8.19 ppm) indicated that the indole
ring had been oxidized. The structure of 8 was ulti-
mately con®rmed by synthesis, as shown in Scheme 2
and described in Section 3. The structure of nitrile 9
was readily deduced from analysis of its NMR spectra
(Tables 1 and 2) and HR±MS data and was con®rmed
by synthesis.
To establish the sequence of biotransformation
steps, compounds 7, 8, and 9 were separately adminis-
tered to cultures of R. solani isolate AG 2-1. The cul-
tures were incubated and samples withdrawn at
di€erent intervals, extracted, and analyzed by TLC
and HPLC. As expected, 8 and 9 were detected in the
cultures incubated with 7 (ca. 24 h; complete metab-
olism of 7 occurred in nine days). Furthermore, while
8 did not appear to be metabolized, cultures of R.
solani metabolized 9 to undertermined products (®fteen
days). The results of these experiments are summarized
in Table 3, and the proposed sequence of reactions
that camalexin (6) undergoes when incubated with R.
solani isolate AG 2-1 is shown in Scheme 1.
Next, camalexin (6) was incubated with R. solani
weakly virulent isolate AG 4 under conditions similar
to those used for isolate AG 2-1 (12 h light/12 h dark).
HPLC analyses of extracts of fungal cultures incubated
with camalexin (6) indicated that it was completely
metabolized in ca. 96 h. Interestingly, while the major
product resulting from biotransformation of camalexin
(6) by weakly virulent isolate AG 4 was also 5-hydro-
Table 3
Products of metabolism of camalexin (6) and compounds 7±9 by Rhizoctonia solani virulent isolate AG 2-1 (12 h light/12 h dark)
Compound added to fungal culturesa Concentration
Products
(M) in incubation medium (%)b of metabolism
(incubation period)
4
Camalexin (6)
5 Â 10
No metabolism, complete growth inhibition
4
4
4
4
1.2 Â 10
1.2 Â 10
1.2 Â 10
1.2 Â 10
Complete biotransformation to 7 (90%) and traces of 8 and 9 in three daysc
Complete biotransformation to 8 and 9 (nine days)
No metabolism or growth inhibition
7
8
9
Slow biotransformation to undetermined products (15 days)
a Compounds were dissolved in DMSO and added to 48-h-old cultures and incubated at 24228C.
b Percentage of products detected by HPLC analysis.
c Under constant light, similar amounts of metabolites are formed in ca. 24 h.

M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
63
Table 4
Products of metabolism of camalexin (6) and compounds 7 and 9±13 by Rhizoctonia solani weakly virulent isolate AG 4 (12 h light/12 h dark)
Compound added to fungal
culturesa
Concentration (M) in incubation
medium
Products (%)b of metabolism (incubation period)
4
6
1.2 Â 10
75% biotransformation to metabolites 7 (ca. 50%), 10 (ca. 20%),
9 + 11±13 (ca. 25%), (four days)c
4
7
1.2 Â 10
Slow biotransformation to undetermined products (nine days)
Slow biotransformation to undetermined products (15 days)
Slow biotransformation to 11 (seven days)
4
9
1.2 Â 10
4
10
11
12
13
1.2 Â 10
4
1.2 Â 10
No metabolism or growth inhibition
No metabolism or growth inhibition
Slow biotransformation to 9 (nine days)
4
1.2 Â 10
4
1.2 Â 10
a Compounds were dissolved in DMSO and added to 48-h-old cultures and incubated at 24228C.
b Percentage of products detected by HPLC analysis.
c Under constant light, similar amounts of metabolites are formed in four days.
xycamalexin (7), new metabolites 10±13 were isolated.
Compound 8 was never detected in cultures of isolate
AG 4 incubated with camalexin. The structural assign-
ments of 10±13 followed from the analysis of NMR
spectroscopic data (Tables 1 and 2) as well as from
HR±MS. For example, oxazoline 10 contained oxygen
and no sulfur (C11H10N2O), as determined by HR±
EIMS. Comparison of the 1H-NMR spectrum (Table
1) of 10 with that of camalexin (6) indicated the pre-
sence of an intact indole ring and the absence of thia-
zole protons; two new triplets due to four protons
(4.44 and 4.00 ppm) indicated that the thiazole moiety
of camalexin (6) had been reduced. This hypothesis
was also supported by the 13C-NMR spectrum (Table
2) of 10, which displayed two signals indicative of sp3
C atoms (67.9 and 54.6 ppm), in addition to nine car-
bon signals due to sp2 C atoms. These spectroscopic
features suggested that metabolite 10 contained an
oxazoline instead of a thiazole moiety. The structures
of compounds 10±12 were ultimately con®rmed by
synthesis as described in Section 3, while structure 13
was con®rmed by comparison with an authentic
sample (obtained from Sigma Aldrich). The structure
of oxazoline 10 is of particular interest as the presence
of oxygen instead of sulfur suggests that this isolate
possesses an enzymatic system not present in isolate
AG 2-1.
To establish a possible pathway for the metabolism
of camalexin by weakly virulent isolate AG 4, com-
pounds 7, and 9±13 were separately administered to
liquid cultures. The cultures were incubated and
samples withdrawn at di€erent intervals, extracted,
and analyzed by TLC and HPLC. Surprisingly, the
results of these analyses indicated that isolate AG 4,
unlike isolate AG 2-1, metabolized 7 to products not
extractable with organic solvents (complete transform-
ation of 7 occurred in nine days). In addition, isolate
AG 4 metabolized 10 to 11 (seven days); however,
neither 11 or 12 appeared to be metabolized. The
results of these experiments are summarized in Table
4, and the proposed sequence of reactions that cama-
lexin (6) undergoes when incubated with R. solani iso-
late AG 4 are shown in Scheme 1. It appears that the
major pathway for metabolism of camalexin involves
the formation of 5-hydroxycamalexin (7); a minor
pathway involved transformation to oxazoline 10,
which was further metabolized to 11. Although it was
anticipated that metabolite 11 might be a precursor of
12 and 12 a precursor of 13, we were unable to con-
®rm this correlation. Our results indicated that 12 and
13 resulted from camalexin degradation by isolate AG
4, but their close precursor(s) could not be established.
It might be that the enzyme catalyzing the degradation
of 11 requires induction by camalexin (6) or that the
solubility of 11 prevents its transport into the appro-
priate cellular site. Metabolites 10±13 were not
detected in cultures of isolate AG 2-1 incubated with
camalexin (6).
Additional experiments carried out to determine the
e€ect of light on camalexin (6) metabolism, demon-
strated that continuous light a€ected the rate of its
metabolism by virulent isolate AG 2-1 and weakly
virulent isolate AG 4 di€erently. Camalexin (6) was
metabolized by isolate AG 2-1 (liquid culture) in ca.
24 h under continuous light, while in darkness its
metabolism occurred over a much longer period (ca.
ten days). Unexpectedly, light did not appear to a€ect
to a detectable level the rate of metabolism of cama-
lexin by isolate AG 4 in liquid culture; however, cama-
lexin was more inhibitory in the absence of light to
both isolates AG 2-1 and AG 4 grown on solid med-
ium, as shown in Table 5. Furthermore, measurements
of the diameter of mycelia showed a similar growth
pattern for both fungal isolates in the absence of
camalexin (6) (Table 5, control plates).
Since on several instances we found that m-CPBA
oxidation of indolic phytoalexins yielded products
identical to those resulting from fungal oxidation

64
M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
Table 5
Mycelium radial growth of Rhizctonia solani virulent isolate AG2-1 and weakly virulnt isolate AG 4 incubated with camalexin (6) in constant
light and constant darkness
Concentration of camalexin
(M)
Isolate AG 2-1 % of inhibitiona
(mycelium diameter mm)
Isolte AG-4 % of inhibitiona
(mycelium diameter mm)b
Constant light
Constant dark
Constant light
Constant dark
4
5.0 Â 10
100 (5)
100 (5)
100 (5)
100(5)
4
2.5 Â 10
57 (3321)
42 (4522)
0 (7821)
66 (2621)
53 (3722)
0 (7821)
71 (2121)
28 (5622)
0 (7821)
81 (1521)
45 (4322)
0 (7821)
4
1.2 Â 10
Controlc
a The percentage of inhibition was calculated using the formula: % inhibition = 100
are the mean of at least three separate experiments (SD20.7).
[(growth on treated/growth in control) Â 100]; results
b The size of mycelium plug to initiate cultures was ca. 5 mm (described in Section 3).
c Control plates contained potato dextrose agar and 1% dimethylsulfoxide and no camalexin.
(Pedras et al., 1997), camalexin (6) was oxidized with
m-CPBA. Contrary to other brassica phytoalexins, the
oxidation of camalexin (6) proceeded rather slowly
yielding two isomeric products 14 and 15 in a 1:1
ratio. The HR±MS analysis of each product
(C11H8N2O2S) indicated the presence of two additional
oxygen atoms relative 6. The 13C-NMR and HMQC
spectroscopic data of 14, displayed signals that re-
sembled those of metabolite 8, namely, two C1O
groups at 187.4 and 162.2 ppm, and a direct corre-
lation between the latter signal and a singlet proton at
8.32 ppm. In this regard, it is interesting to note that
14 was recently reported as an intermediate in the syn-
thesis of camalexin (6) (Furstner & Ernst, 1995).
Alkaline hydrolysis of 14 yielded quantitatively the
expected product 16 (C10H8N2OS). The structure of 15
was determined from analysis of its spectroscopic data.
Although thiazoles are known to yield N-oxides on
treatment with m-CPBA (Begtrup & Hanson, 1992)
oxidation of camalexin (6) occured at the indole moi-
ety to yeild the isomeric products 14 and 15 (see
Scheme 3).
R. solani can detoxify camalexin (6) e€ectively under
normal light conditions, but in darkness this process
was signi®cantly slower. Considering that R. solani is a
root pathogen, it is likely that plants able to biosynthe-
size camalexin (6) in their root tissues will show higher
resistance to root diseases. Therefore, camalexin (6)
might be a useful metabolite to control root disease
caused by R. solani. In this context it is worthy to note
that a higher resistance of C. sativa to R. solani was
attributed to the presence of camalexin (6) in root,
stem, and leaf tissues (Conn et al., 1994).
The antifungal activity of camalexin (6) to R. solani
was determined utilizing the radial mycelial growth in-
hibition assay described in Section 3 (see Table 6). The
bioactivity of metabolites 7±13 was compared with
that of camalexin (6) ultilizing identical bioassays.
After 96 h of incubation, the mycelium of control
plates incubated with R. solani covered 100% of the
agar surface area, while agar plates containing cama-
4
lexin (6) at 5 Â 10 M showed no mycelial growth,
i.e. camalexin (6) inhibited completely the growth of
isolates AG 2-1 and AG 4. Among metabolites 7±13,
only nitrile 13 showed some inhibitory e€ect towards
both isolates. These results demonstrated that the
metabolism of camalexin (6) leads to products having
no signi®cant antifungal activity, thus suggesting this
metabolism is a detoxi®cation process. It is clear that
Scheme 3. Oxidation of camalexin (6) with m-chloroperbenzoic acid
(m-CPBA).

M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
65
Table 6
mm. Flash column chromatography (FCC): silica gel
Merck, grade 60, mesh size 230±400, 60 A. HPLC
analysis was carried out with a high performance
Hewlett Packard liquid chromatograph equipped with
a quaternary pump, automatic injector, and diode
array detector (wavelength range 190±600 nm), degas-
ser, and a Hypersil ODS column (5 mm particle size
silica, 4.6 i.d. Â 200 mm), equipped with an in-line ®l-
ter. Mobile phase: 75% H2O-25% CH3CN to 100%
CH3CN, for 35 min, linear gradient, and a ¯ow rate
1.0 ml/min. The samples to be analyzed by HPLC
were dissolved in CH3CN and ®ltered through a tight
cotton wool plug. NMR spectra were recorded on a
Bruker AMX 300 or on a Bruker AMX 500 spec-
trometer; for 1H-NMR (300 or 500 MHz), d values
were referenced to CD3OD (CD2HOD 3.31 ppm),
CDCl3 (CHCl3 7.27 ppm), or CD3CN (CD2HCN 1.94
ppm) and for 13C-NMR (75.5 or 125.8 MHz) chemical
shifts were referenced to CD3OD (49.15 ppm), CDCl3
(77.23 ppm), or CD3CN (1.39, 118.69 ppm). Fourier
transform infrared (FTIR) spectra were obtained on a
Bio-Rad FTS-40 spectrometer using a di€use re¯ec-
tance cell. Mass spectra (MS) were obtained on a VG
70 SE mass spectrometer [high resolution (HR), elec-
tron impact (EI) or chemical ionization (CI) with
ammonia], employing a solids probe.
Percentage of inhibition of Rhizoctonia solani virulent isolate AG 2-1
and weakly virulent isolate AG 4 incubated with camalexin (6) and
compounds 7±13 (after 96h, constant light)
Compound
added
Concentration Rhizoctonia solani Rhizoctonia solani
(M) isolate AG 4 isolate AG 2-1
(% of inhibition)a (% of inhibition)a
to fungal
cultures
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Camalexin (6) 5.0 Â 10
2.5 Â 10
100
71
28
10
0
100
57
42
25
8
1.2 Â 10
7
5.0 Â 10
2.5 Â 10
1.2 Â 10
5.0 Â 10
2.5 Â 10
1.2 Â 10
5.0 Â 10
2.5 Â 10
1.2 Â 10
5.0 Â 10
2.5 Â 10
1.2 Â 10
5.0 Â 10
2.5 Â 10
1.2 Â 10
5.0 Â 10
2.5 Â 10
1.2 Â 10
5.0 Â 10
2.5 Â 10
1.2 Â 10
0
4
8
0
0
0
0
0
0
9
0
5
0
5
0
5
10
11
12
13
0
23
5
0
0
0
0
0
0
0
0
0
12
0
0
0
0
0
52
23
10
57
36
16
3.2. Fungal cultures
a The percentage of inhibition was calculated using the formula: %
inhibition = 100
results are the mean of at least three separate experiments
[(growth on treated/growth in control) Â 100];
Standard isolates of Rhizoctonia solani, virulent iso-
late AG 2-1 and weakly virulent isolate AG 4 utilized
in this study were obtained from the AAFC collection
(Saskatoon, SK). The fungal isolates were grown on
potato dextrose agar (PDA) plates at 24228C, under
constant light for ®ve days.
(SD20.7).
3. Experimental
3.1. General
3.3. Fungal growth assays
All chemicals were purchased from Sigma-Aldrich
Canada, Oakville, ON. All solvents were HPLC grade
and used as such, except for CH2Cl2 and CHCl3 which
were redistilled. Solvents utilized in syntheses were
dried over the following drying agents prior to use:
benzene, THF, and EtO2±Na/benzophenone; CH2Cl2±
CaH2. Organic extracts were dried over anhydrous
Na2SO4 and solvents removed under reduced pressure
in a rotary evaporator. Yields reported are based on
recovered starting material.
Analytical TLC: (Merck, Kieselgel 60 F254, alumi-
num sheets) 5  2 cm  0.2 mm; compounds were
visualized under UV light and by dipping the plates in
a 5% aqueous (w/v) phosphomolybdic acid solution
containing 1% (w/v) of ceric sulfate and 4% (v/v)
H2SO4, followed by heating to 2008C. Preparative
TLC: Merck, Kieselgel 60 F254, 20  20 cm  0.25
The antifungal activity of compounds 6±13 was
investigated using the following mycelial radial growth
bioassay.
A DMSO solution (®nal concentration
R 1%) of the compound to be tested (®nal concen-
4
tration of each compound 5.0 Â 10 4, 2.5 Â 10 and
4
1.2 Â 10 M) was added to agar medium at ca. 508C,
mixed quickly and poured onto Petri plates. An agar
plug (5 mm diameter) cut from edges of 5-day-old
solid cultures of R. solani was placed upside down on
the center of each plate, the plates were sealed with
para®lm, and incubated under constant light at
24228C for 96 h. The diameter (in mm) of the myce-
lial area was measured at 24-h intervals for 96 h.
Control plates containing only DMSO were prepared
and incubated similarly. Each assay was repeated at
least three times.

66
M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
3.4. Metabolism of camalexin (6)
CH2Cl2 (3 ml) at room temperature. After 24 h, Me2S
(200 ml) was added to the reaction mixture, the mixture
was concentrated in vacuo and the residue was separ-
ated by FCC (CH2Cl2±MeOH, 98:2 v/v) to yield 14,
(21.7 mg, 22%), 15 (22.7 mg, 23%), and unreacted
camalexin (6) (15 mg).
Liquid shake cultures (100 ml of media in 250 ml
Erlenmeyer ¯asks) were initiated by inoculating potato
dextrose broth (PDB) with ®ve agar plugs cut from
edges of ®ve-day-old solid cultures. Solutions of cama-
lexin (6), synthesized as previously reported (Ayer et
4
al., 1992), (®nal concentration 1.2 Â 10
M) in
3.8. 5-Hydroxycamalexin (7)
DMSO (®nal concentration R 1%) were added to 48-
h-old liquid cultures and to uninoculated media.
Cultures were incubated either in 12 h light/12 h dark,
constant light, or constant darkness, on a shaker at
130 rpm, at 24228C. Samples (5±10 ml) were with-
drawn at di€erent intervals up to two weeks, and were
either immediately frozen or were ®ltered and
extracted with Et2O, followed by n-butanol. The
extracted broth was acidi®ed to pH 2 with HCl and
extracted with Et2O, followed by neutralization with
NaOH and extraction with CHCl3 [containing 1%
NH4OH, (v/v)]. Camalexin (6) was stable in uninocu-
lated medium for at least ®fteen days.
A suspension of 5-acetoxy-2-formamidophenyl-2'-
thiazolylketone (25 mg, 0.086 mmol) (prepared from 5-
hydroxy-2-formamidophenyl-2'-thiazolylketone
(8),
acetic anhydride-pyridine), TiCl3 (423 mg, 0.15 mmol),
and Zn dust (20 mg, 0.3 mmol) in DME (3 ml) was
re¯uxed for 1 h under Ar atmosphere (Furstner &
Ernst, 1995). The reaction mixture was allowed to cool
to room temperature, diluted with EtOAc (5 ml), a sol-
ution of EDTA (558 mg) in H2O (5 ml) was added,
and the mixture was vigorously stirred at room tem-
perature for 20 min. The suspension was ®ltered, the
inorganic residue was washed with EtOAc, the organic
phases were combined, dried (Na2SO4), and concen-
trated. The residue was separated by prep. TLC
(CH2Cl2±MeOH, 97:3, v/v, multiple development) to
yield 5±acetoxycamalexin (7.5 mg, 34%). 5-
Acetoxycamalexin in 5% aqueous HCl was re¯uxed
for 30 min to yield 7 quantitatively (Rf 0.38, CH2Cl2±
3.5. Metabolism of compounds 7±13
Solutions of compounds 7±13 (®nal concentration
4
1.2 Â 10
M) in DMSO (®nal concentration of
DMSO in PDB was R 1% v/v) were added separately
to 48-h-old liquid cultures and to uninoculated media,
and samples were withdrawn and analyzed as
described for camalexin (6). All the compounds were
stable in uninoculated medium for at least ®fteen days.
1
MeOH, 95:5, v/v). H-NMR spectral data in Table 1;
13C-NMR spectral data in Table 2; FTIR nmax 3307,
2923, 1581, 1543, 1472, 1444, 1243, 1205 cm 1; EIMS,
m/z 216.0364 [M+] (100), (calculated for C11H8N2OS,
216.0357), 158 (33), 149 (25); CIMS, m/z 217 [M++1]
(100).
3.6. Analysis and isolation of metabolites
Analysis of the organic extracts and biotransform-
ation products was performed with the HPLC system
described above. Only the chromatograms of the Et2O
extracts of the neutral broth showed peaks not present
in chromatograms of extracts of control cultures; n-
butanol extracts, as well as Et2O extracts of acidic and
basic broths, were similar to those of control cultures.
The isolation of products resulting from the fungal
metabolism of camalexin (6) was carried out by prep.
TLC (CH2Cl2±MeOH, 95:5 v/v). The structures of the
products were determined from analyses of the spec-
troscopic data (NMR, MS, FTIR, UV) of the puri®ed
metabolites, and con®rmed by synthesis, as described
below. The structure of metabolite 13 was con®rmed
by direct comparison of its spectroscopic data with
those of commercially available indole-3-carbonitrile.
3.9. 5-Hydroxy-2-formamidophenyl-2'-thiazolylketone
(8)
A solution of 5-hydroxy-2-nitrobenzaldehyde (250
mg, 1.5 mmol) in 3 ml of CH2Cl2±Et2O (4:1 v/v) was
added with stirring to a mixture of 3,4-dihydro-2H-
pyran (630 mg, 7.5 mmol) and p-toluenesulfonic acid
monohydrate (2.5 mg, 0.015 mmol) in CH2Cl2 (4 ml)
dropwise (Boom & Herschied, 1973). The reaction
mixture was stirred at room temperature for 4 h, then
diluted with CH2Cl2 (10 ml), washed with a saturated
solution of NaHCO3, and ®nally with water. The or-
ganic phase was dried (Na2SO4), concentrated under
vacuum, and the residue separated by FCC (CH2Cl2±
MeOH, 99:1 v/v) to yield 5-(2-tetrahydropyranyloxy)-
1
2-nitrobenzaldehyde (340 mg, 90%). H-NMR spectral
data (CD3CN) d 10.32 (1H, s), 8.14 (1H, d, J = 8.9
Hz), 7.36 (2H, m ), 5.65 (1H, br s ), 3.75 (1H, m ), 3.59
(1H, m ), 1.85±1.65 (6H, m ); 13C-NMR spectral data
(CD3CN) d 190.6, 162.8, 144.1, 135.7, 128.5, 121.5,
3.7. Oxidation of camalexin (6)
m-Chloroperbenzoic acid (200 mg, 1 mmol, in
CH2Cl2, 2 ml) was added to a stirred solution of cama-
lexin (6) (Ayer et al., 1992) (100 mg, 0.5 mmol) in
117.2, 98.0, 63.4, 30.9, 26.0, 19.5; FTIR nmax, 2946,
1
1697, 1584, 1518, 1338, 1284, 1119 and 1023 cm
;

M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
67
EIMS, m/z 167.0251 [M+ 84] (47), (calculated for
C7H5NO4, 167.0218); CIMS, m/z 269 [M++18] (100).
To a stirred solution of n-buthyllithium (0.264 ml,
0.66 mmol in 2.5 ml hexane) was added 2-bromothia-
zole (153 mg, 0.93 mmol) in Et2O (1 ml), at 788C
under Ar atmosphere over a period of 1 h (Furstner &
Ernst, 1995). The resultant suspension was stirred at
788C for another 30 min and a solution of 5-(2-tetra-
hydropyranyloxy)-2-nitrobenzaldehyde (156 mg, 0.62
mmol) in THF (3 ml) was added over a period of 1 h
and the mixture was stirred for another 45 min at
788C. The cooled mixture was poured onto a sol-
ution of NH4Cl (1g/10 ml) and the aqueous layer was
extracted with EtOAc. The combined organic phases
were washed with brine, dried (Na2SO4), and the sol-
vent was removed under vaccum. The crude material
was separated by FCC (hexane±acetone, 3:2 v/v) to
yield 5-(2-tetrahydropyranyloxy)-2-nitrophenyl-2'-thia-
zolylmethanol (17, 159 mg, 84%). 1H-NMR spectral
data (CD3CN) d 8.05 (1H, d, J = 9 Hz), 7.60 (1H, d,
J = 3 Hz), 7.44 (2H, m ) 7.10 (1H, dd, J = 3.0, 9.0
Hz), 6.81 (1H, d, J = 5.7 Hz, singlet on D2O
exchange), 5.57 (1H, br s ), 4.83 (1H, m ), 3.76 (1H,
m ), 3.59 (1H, m ) and 1.81±1.65 (6H, m ); 13C-NMR
spectral data (CD3CN) d 174.2, 162.3, 143.4, 142.4,
141.2, 128.5, 121.1, 116.8, 116.6, 97.6, 69.3, 63.2, 30.8,
25.8 and 19.4; FTIR nmax 3456±3200 (br ), 2945, 1580,
1515, 1340, 1284 and 1035 cm 1; EIMS, m/z 252.0209
[M+ 84] (23), (calculated for C10H8N2O4S, 252.0204);
CIMS m/z 337 [M++1] (25).
(2-tetrahydropyranyloxy)-2-aminophenyl-2'-thiazolyl
ketone (18, 26 mg, 96%). 1H-NMR (CD3CN) d 8.73
(1H, d, J = 2.8 Hz), 8.05 (1H, d, J = 3 Hz), 7.83
(1H, d, J = 3 Hz), 7.14 (1H, dd, J = 2.8, 9.0 Hz),
6.75 (1H, d, J = 9 Hz), 6.48 (2H, br s ), 5.22 (1H, m ),
3.89 (1H, m ), 3.56 (1H, m ) and 1.86±1.60 (6H, m );
13C-NMR (CD3CN) d 185.0, 171.3, 150.3, 148.1,
145.7, 128.2, 127.4, 122.4, 119.1, 116.2, 99.5, 63.7,
31.70, 26.4 and 20.6; FTIR nmax, 3463, 3342, 2941,
1580, 1544, 1390, 1253, 1219, 1200 and 1034 cm
EIMS m/z 304.0876 [M+] (7) (calculated for
C15H16N2O3S, 304.0881), 220 (100), 192 (76), 191 (33)
166 (5), 148 (15), 136 (6), and 85 (24); CIMS m/z 305
[M++1] (100).
1
;
A solution of 18 (30 mg, 0.098 mmol) in CH2Cl2
was added with stirring to a hot mixture (heated to
60±708C for 1 h) of acetic anhydride (48 mg, 0.47
mmol) and formic acid (23 mg, 0.5 mmol) (Furstner
& Ernst, 1995). The mixture was allowed to cool
and quenched with a saturated solution of NaHCO3.
The aqueous layer was extracted with CH2Cl2, the
combined organic phases were dried (Na2SO4) and
concentrated under vacuum to yield 5-(2-tetrahydro-
pyranyloxy)-2-formamidophenyl-2'-thiazolylketone
1
(32.0 mg, 98%). H-NMR spectral data (CD3CN) d
10.0 (1H, br s ), 8.32 (3H, m ), 8.11 (1H, d, J = 3
Hz), 7.96 (1H, d, J = 2.9 Hz), 7.33 (1H, dd, J =
2.9, 9.0 Hz), 5.43 (1H, m ), 3.83 (1H, m ), 3.55 (1H,
m ) and 1.80±1.60 (6H, m ); 13C-NMR spectral data
(CD3CN) d 187.0, 169.1, 161.3, 153.6, 146.4, 134.3,
129.0, 125.9, 124.6, 123.7, 122.7, 98.5, 63.5, 31.3,
26.2 and 20.2; FTIR nmax 3305, 3104, 2943, 1694,
1627, 1584, 1514, 1480, 1384, 1286 and 1035 cm
EIMS m/z 248.0253 [M+ 84] (95) (calculated for
C11H8N2O3S, 248.0255), 220 (10), 192 (100), 148
(5), 106 (5) and 85 (71); CIMS m/z 333 [M++1]
(100).
Pyridinium dichromate (PDC, 335 mg, 0.89 mmol)
was added with stirring to a solution of 17 (150 mg,
0.44 mmol) in CH2Cl2 (6 ml) at room temperature
under Ar atmosphere (Furstner & Ernst, 1995). After
3 h the reaction mixture was ®ltered through a short
silica gel pad and the inorganic residue was washed
with CH2Cl2. The combined organic phases were con-
centrated under vacuum to yield 5-(2-tetrahydropyra-
nyloxy)-2-nitrophenyl-2'-thiazolylketone (111.3 mg,
1
;
A solution of 5-(2-tetrahydropyranyloxy)-2-formami-
dophenyl-2'-thiazolylketone (10 mg, 0.03 mmol) in
ethanol acidi®ed 1.5 ml (EtOH, 10 and 0.1 ml of 0.1
M hydrochloric acid) was re¯uxed for 3 h (Boom &
Herschied, 1973). The reaction mixture was then
cooled, concentrated under vacuum and the residue
was dissolved in EtOAc. The solution was washed
with a saturated solution of NaHCO3, and then with
H2O, dried (Na2SO4), and concentrated under vacuum.
The crude material was puri®ed by prep. TLC using
(CH2Cl2±MeOH, 95:5, v/v) to yield 5-hydroxy-2-
formamidophenyl-2'-thiazolylketone (8, 7 mg, 94%),
1
74.6%). H-NMR (CD3CN) d 8.00 (1H, d, J = 9 Hz),
7.73 (2H, m ), 7.12 (1H, dd, J = 2.7, 9.0 Hz), 7.02
(1H, d, J = 2.7 Hz), 5.44 (1H, m ), 3.55 (1H, m ), 3.40
(1H, m ) and 1.75±1.48 (6H, m ); 13C-NMR (CD3CN) d
186.5, 167.2, 163.2, 146.6, 142.2, 137.7, 129.0, 128.0,
119.5, 117.7, 98.2, 63.5, 30.9, 26.0 and 19.5; FTIR nmax
3110, 2946, 1679, 1580, 1517, 1339, 1233 and 1034
cm 1; EIMS m/z 250.0053 [M+ 84] (14) (calculated
for C10H6N2O4S, 250.0048), 217 (27), 204 (14), 191
(40), 159 (14), 130 (15) and 85 (100); CIMS m/z 335
[M++1] (15).
1
Rf 0.41 (CH2Cl2±MeOH, 95: 5, v/v). H-NMR spectral
5-(2-Tetrahydropyranyloxy)-2-nitrophenyl-2'-thiazo-
lylketone (30 mg, 0.089 mmol) in EtOAc was hydro-
genated (H2 1 atm) over 10% palladium on charcoal
(3 mg). After 10 h the orange solution was diluted
with EtOAc and ®ltered through a cotton plug. The ®l-
trate was evaporated under reduced pressure to give 5-
data in Table 1; 13C- NMR in Table 2; FTIR nmax
3314, 3090, 1672, 1522, 1383, 1304, 1234 and 827
cm 1; EIMS m/z 248.0254 [M+] (33) (calculated for
C11H8N2O3S, 248.0256), 220 (18), 192 (100), 148 (18),
149 (17) and 108 (12); CIMS m/z 249 [M++1]
(100).

68
M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
1
3.10. 5-Hydroxyindole-3-carbonitrile (9)
CH2Cl2±MeOH, 95: 5, v/v). H-NMR spectral data in
Table 1; 13C-NMR in Table 2; FTIR nmax 3075, 1646,
1451, 1245, 1175 and 1019 cm 1; EIMS m/z 186.0785
[M+] (100) (calculated for C11H10N2O, 186.0793), 156
(59), 144 (25), 130 (12), 116 (10), 78 (10); CIMS m/z
187 [M+ + 1] (100).
Phosphorus oxychloride (60 mg, 0.40 mmol, freshly
distilled) was added with stirring to freshly distilled
DMF (0.15 ml) under Ar atmosphere at room tem-
perature (Pedras & Khan, 1996). A solution of 5-
hydroxyindole (50 mg, 0.37 mmol) in DMF (0.1 ml)
was added dropwise with continuous stirring. The
reaction mixture was kept at room temperature for 45
min and then poured onto crushed ice. Aqueous
NaOH solution (3.8 g in 20 ml) was added to the reac-
tion mixture (slow addition exothermic reaction!), the
reaction mixture was acidi®ed to pH 2 with 10% HCl,
and was extracted with EtOAc. The organic phases
were combined, dried (Na2SO4) and concentrated
under vacuum. The residue obtained was separated by
FCC (CH2Cl2±MeOH, 93:7 v/v) to a€ord 5-hydroxyin-
dole-3-carboxaldehyde (50 mg, 84% yield). 1H-NMR
(CD3OD) d 9.79 (1H, s), 7.98 (1H, s), 7.57 (1H, d, J
= 2.4 Hz), 7.30 (1H, d, J = 8.7 Hz), 6.79 (1H, dd, J
= 2.4, 8.7 Hz); 13C-NMR (CD3OD) d 187.3, 154.9,
139.9, 133.4, 127.0, 119.8, 114.8, 113.8, 107.1; FTIR
nmax 3330, 3220, 1628, 1472 and 1200 cm 1; EIMS m/z
161.0473 [M+] (94) (calculated for C9H7NO2,
161.0476), 160 (100), 132 (24), 105 (7); CIMS m/z 162
[M+ + 1] (100).
3.12. N(3-Hydroxyethyl)-indole-3-carboxamide (11)
Thionyl chloride (0.74 g, 6.2 mmol) was added to
indole-3-carboxylic acid (100 mg, 0.62 mmol) at room
temperature (Meyers, Temple, Haidukewych
&
Mihelich, 1974)1. The mixture was stirred for 6 h then
kept at room temperature overnight. The reaction mix-
ture was diluted with toluene and the excess of thionyl
chloride was removed in vacuo. The residue obtained
was dissolved in CH2Cl2 (5 ml), cooled to 08C, and
freshly distilled 2-aminoethanol (137 mg 2.2 mmol)
was added dropwise. The mixture was stirred for 2 h
at room temperature, was poured onto brine solution,
and extracted with EtOAc. The combined organic
phases were dried (Na2SO4), and the solvent was evap-
orated under reduced pressure. The residue was separ-
ated by FCC (CH2Cl2±MeOH, 93:7 v/v) to yield N(3-
hydroxyethyl)-indole-3-carboxamide (11) (100 mg;
79%, Rf 0.2, CH2Cl2±MeOH, 93:7, v/v). 1H-NMR
spectral data in Table 1; 13C-NMR spectral data in
Table 2; FTIR nmax 3300, 2950, 1610, 1544, 1455,
1318, 1207 and 1061 cm 1; EIMS m/z 204.0902 [M+]
(32) (calculated for C11H12N2O2, 204.0898), 160 (29),
144 (100), 116 (17); CIMS m/z 205 [M++1] (100).
Pyridine (0.01 ml in 0.5 ml of CHCl3) was added to
a re¯uxing mixture of HONH2ÁHCl (8.6 mg, 0.12
mmol) and 5-hydroxyindole-3-carboxaldehyde (20 mg,
0.12 mmol) in CHCl3±EtOH (7:3, v/v,
3
ml)
(Sosnovsky, Krogh & Umhoefer, 1979). After 3 h,
SeO2 (13.7 mg, 0.12 mmol) was added to the reaction
mixture and re¯uxing 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 ®ltered
and the solvent was removed under vaccum. The resi-
due was separated by prep. TLC (CH2Cl2±MeOH,
93:7 v/v, multiple development) to yield 9 as a white
powder (13 mg, 77%, Rf 0.30, CH2Cl2±MeOH 95:5, v/
3.13. Indole-3-carboxamide (12)
Indole-3-carboxylic acid (100 mg 0.62 mmol) was
treated with thionyl chloride as described above. The
residue obtained was dissolved in CH2Cl2, the solution
was cooled to 08C, and excess of ammonia was added
(ammonia gas was condensed at 708C). The reaction
mixture was stirred for 2 h at 08C, and then 1 h at
room temperature, the solvent was evaporated under
reduced pressure, and the crude material was separated
by prep. TLC (CH2Cl2±MeOH, 93:7 v/v, developed
twice) to yield indole-3-carboxamide (12) (70 mg, 90%,
Rf 0.3, CH2Cl2±MeOH, 93:7, v/v). 1H-NMR spectral
data in Table 1; 13C NMR spectral data in Table 2;
FTIR nmax 3382, 3325, 3170, 1647, 1618, 1532, 1453
and 749 cm 1; EIMS m/z 160.0638 [M+] (74) (calcu-
lated for C9H8N2O, 160.0366), 144 (100), 116 (23);
CIMS m/z 161 (M++1) (100).
v). 1H NMR in Table 1; 13C-NMR spectral data in
1
Table 2; FTIR nmax 3325, 3225, 2220, 1204 cm
;
EIMS m/z 158.0478 [M+] (100) (calculated for
C9H6N2O, 158.0480) 129 (11), 103 (11); CIMS m/z 176
[M+ + 18] (100).
3.11. 2(3-Indolyl)-2-oxazoline (10)
Compound 11 (10 mg, 0.049 mmol) was heated to
1708C with anhydrous zinc acetate (89.7 mg, 0.49
mmol) under Ar atmosphere for 2 h (Ghera & Shoua,
1972). The mixture was diluted with CH2Cl2, ®ltered,
washed with brine, dried (Na2SO4), and the solvent
evaporated under reduced pressure. The crude material
was separated by prep. TLC (CH2Cl2±MeOH, 95:5 v/
v) to yield oxazoline 10 (7.7 mg, 84%, Rf 0.28,
3.13.1. 2-Formamidophenyl-2'-thiazolylketone (14)
1H-NMR spectral data (CD3OD) d 8.43 (1H, d, J =
8 Hz, H-4), 8.32 (1H, s, H-2), 8.26 (1H, d, J = 8 Hz,
H-7), 8.10 (1H, d, J = 3 Hz, H-4'), 8.03 (1H, d, 3 Hz,
H-5'), 7.64 (1H, m, H-5/6), and 7.20 (1H, m, H-6/5);

M.S.C. Pedras, A.Q. Khan / Phytochemistry 53 (2000) 59±69
69
13C-NMR spectral data (CD3OD) d 187.4 (C-3), 169.0
Acknowledgements
(C-2'), 162.2 (C-2), 145.9 (C-4'), 139.0 (C-3a), 135.0
(C-5/6), 134.4 (C-4), 128.5 (C-5'), 126.9 (C-7a), 125.0
Support for the authors' work was obtained in part
from the Natural Sciences and Engineering Research
Council of Canada and the University of
Saskatchewan. We would like to thank P.R. Verma
and D. Makenzie, Agriculture and Agri-Food Canada
Research Station (AAFC collection), Saskatoon SK,
for kindly providing isolates of Rhizoctonia solani.
(C-6/5) and 123.6 (C-7); FTIR nmax 3342, 3080, 1701,
1
1624, 1604, 1580, 1513, 1448, 1376 and 1271 cm
EIMS m/z
;
232.0306 [M+] (32) (calculated
C11H8N2O2S, 232.0306), 204 (10), 176 (100), 175 (60),
150 (11), 148 (11), 146 (13), 120 (15); CIMS, mass
(relative intensity) = 233 [M+ + 1] (100).
3.13.2. 3-Hydroxy-3-(2'-thiazolyl)-2-oxindole (15)
1H-NMR spectral data, (CD3OD) d 7.66 (1H, d, J
= 3 Hz, H-4'), 7.56 (1H, d, J = 3 Hz, H-5'), 7.30
(1H, m, H-5/6), 7.19 (1H, d, J = 7.4 Hz, H-4), 7.00
(1H, d, m, H-6/5) and 6.96 (1H, d, J = 7.7 Hz, H-7);
13C-NMR spectral data (CD3OD) d 178.6 (C-2), 173.8
(C-2'), 143.7 (C-4'), 143.3 (C-7a), 133.2 (C-3), 132.7
(C-3a), 131.3 (C-6/5), 125.8 (C-4), 123.9 (C-5/6), 121.3
(C-5') and 111.7 (C-7); FTIR nmax 3274, 3090, 1725,
1619, 1470, 1180 and 1056 cm 1; EIMS m/z 232.0298
[M+] (100) (calculated for C11H8N2O2S, 232.0306),
176 (32), 175 (32), 149 (27), 69 (22); CIMS m/z 233
[M+ + 1] (100).
References
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3.13.3. 2-Aminophenyl-2'-thiazolylketone (16)
2-Formamidophenyl-2'-thiazolylketone (14, 5 mg) in
NaOH±MeOH (5 g/100 ml, 2 ml) was re¯uxed for 2 h.
The solvent was removed under vacuum, the residue
dissolved in H2O, extracted with Et2O, the aqueous
layer neutralized with 5% HCl and extracted with
Et2O, the organic phases combined and dried
(Na2SO4). The solvent was removed under reduced
pressure, and the residue was separated by prep TLC
(acetone±hexane, 1:4 v/v, multiple development) to
yield 2-aminophenyl-2'-thiazolylketone (16) (4.0 mg,
91%). 1H-NMR spectral data (CD3OD) d 8.70 (1H,
dd, J = 1.4, 8.0 Hz, H-4), 8.04 (1H, d, J = 3 Hz, H-
4'), 7.90 (1H, d, J = 3 Hz, H-5'), 7.30 (1H, m, H-5/6),
6.80 (1H, dd, J = 1.4, 8 Hz, H-7) and 6.60 (1H, m, H-
6/5); 13C-NMR spectral data (CD3OD) d 186.0 (C-3),
171.3 (C-2'), 154.8 (C-3a), 145.2 (C-4'), 136.4 (C-5/6),
135.6 (C-4), 126.6 (C-5'), 118.1 (C-7), 116.5 (C-7a) and
116.2 (C-6/5); FTIR nmax 3468, 3343, 1616, 1578, 1542,
1299, 1256, 1161 and 898 cm 1; EIMS m/z 204. 0351
[M+] (68) (calculated for C10H8N2OS, 204.0357), 176
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(88), 175 (100), 150 (21), 120 (33); CIMS m/z [M+
1] (100).
+