Metabolism of the phytoalexins camalexins, their bioisosteres and analogues in the plant pathogenic fungus Alternaria brassicicola

Article abstract of DOI:10.1016/j.bmc.2013.05.026

The metabolism of the phytoalexins camalexin (1), 1-methylcamalexin (10) and 6-methoxycamalexin (11) by Alternaria brassicicola and their antifungal activity is reported. This work establishes that camalexins are slowly biotransformed (ca. six days) to the corresponding indole-3-thiocarboxamides, which are further transformed to the indole-3-carboxylic acids. These metabolites are substantially less inhibitory to A. brassicicola than the parent camalexins, indicating that these enzyme-mediated transformations are detoxifications. In addition, analyses of the metabolism of synthetic isomers and bioisosteres of camalexin (1) indicate that isomers of camalexin in the thiazole ring are not metabolized. Based on these results, the potential intermediates that lead to formation of indole-3-thiocarboxamides are proposed.

Full text of DOI:10.1016/j.bmc.2013.05.026

Bioorganic & Medicinal Chemistry 21 (2013) 4541–4549
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Metabolism of the phytoalexins camalexins, their bioisosteres and
analogues in the plant pathogenic fungus Alternaria brassicicola
⇑
M. Soledade C. Pedras , Abbas Abdoli
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
The metabolism of the phytoalexins camalexin (1), 1-methylcamalexin (10) and 6-methoxycamalexin
(11) by Alternaria brassicicola and their antifungal activity is reported. This work establishes that camal-
exins are slowly biotransformed (ca. six days) to the corresponding indole-3-thiocarboxamides, which
are further transformed to the indole-3-carboxylic acids. These metabolites are substantially less inhib-
itory to A. brassicicola than the parent camalexins, indicating that these enzyme-mediated transforma-
tions are detoxifications. In addition, analyses of the metabolism of synthetic isomers and bioisosteres
of camalexin (1) indicate that isomers of camalexin in the thiazole ring are not metabolized. Based on
these results, the potential intermediates that lead to formation of indole-3-thiocarboxamides are
proposed.
Received 3 April 2013
Revised 10 May 2013
Accepted 18 May 2013
Available online 30 May 2013
Keywords:
Antifungal
Brassicaceae
Biotransformation
Camalexin
Ó 2013 Elsevier Ltd. All rights reserved.
Crucifer
Detoxification
6-Methoxycamalexin
1-Methylcamalexin
Alternaria brassicicola
Indole-3-thiocarboxamide
Thiazolyl-3-indole
Oxadiazolyl-3-indole
Thiadiazolyl-3-indole
Phytoalexin
1. Introduction
Interestingly, fungal species specific to cruciferous crops such as
Leptosphaeria maculans ((Desm.), Ces. et de Not., anamorph Phoma
As part of complex defense programs against microbial patho-
gens, cruciferous plants produce phytoalexins having unique struc-
tures that are biosynthetically derived from (S)-tryptophan.1,2
Camalexin (1) is a cruciferous phytoalexin, that is antimicrobial
plant metabolite produced de novo in response to stress conditions
that include microbial attack, UV irradiation or certain metal salts
(CuCl2).1 Recently, the fungal plant pathogen Botrytis cinerea (Pers.
Fr., teleomorph Botryotinia fuckeliana (de Bary) Whetzel) was re-
ported to detoxify camalexin (1) to indole-3-carboxylic acid (9)
lingam (Tode ex Fr.) Desm.) and Alternaria brassicae ((Berk.) Sacc.)
did not metabolize camalexin (1).9 Although it has been shown
that various other fungal pathogens can overcome plant chemical
defenses using detoxification reactions, the number of fungal spe-
cies investigated to date is rather small.1,4 Nonetheless, because
phytoalexin detoxifying enzymes can be selectively inhibited, a
molecular understanding of these processes is essential to design
selective crop protectants, namely paldoxins.10,11
Camalexin (1), first reported as a phytoalexin of Camelina sativa
(false flax) and later on also from Arabidopsis thaliana (thale cress),
occurs only in wild crucifers.1 The significance and role of camalex-
in (1) in the interaction of A. thaliana with various microbial patho-
gens appears to depend on the particular pathogen. For example,
an ecotype of A. thaliana (Columbia) that produces camalexin (1)
is highly resistant to the fungal pathogen Alternaria brassicicola
((Schwein.) Wiltshire), whereas mutants deficient in camalexin
biosynthesis are highly susceptible.12,13 On the other hand, cama-
lexin (1) was not thought to be involved in the resistance of A. tha-
liana to B. cinerea.14 Our previous work3 also suggested that
camalexin (1) was not a prominent factor in the resistance of A.
via indole-3-thiocarboxamide (7) and indole-3-carbonitrile (8),3
a
pathway different from those used by the fungal pathogens Rhizoc-
tonia solani and Sclerotinia sclerotiorum ((Lib) de Bary) (Scheme 1).4
S. sclerotiorum detoxified camalexin (1) to 6-hydroxycamalexin (2)
and 6-b-D
-glucosylcamalexin (3),5,6 whereas R. solani detoxified
camalexin (1) to 5-hydroxycamalexin (4), which was further trans-
formed to metabolites 5 and 6.7,8
⇑
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966 4730.
E-mail addresses: s.pedras@usask.ca, soledade.pedras@usask.ca (M. Soledade C.
Pedras).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.026

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M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
Scheme 1. Biotransformations of camalexin (1) by plant pathogenic fungi: (i) Sclerotinia sclerotiorum; (ii) Rhizoctonia solani; (iii) Botrytis cinerea.
thaliana to B. cinerea because it was quickly detoxified by this path-
ogen. To date no such a correlation has been established between
camalexin (1) and A. brassicicola. Considering that A. brassicicola
can cause one of the most economically important diseases of Bras-
sica species,13,15 and that camalexin (1) appears to be involved in
the disease resistance of some wild plant species to this pathogen,
it is of great interest to determine if camalexin is resistant to trans-
formation by A. brassicicola.
Toward this end, the metabolism of the naturally occurring
phytoalexins1 camalexin (1), 1-methylcamalexin (10) and 6-meth-
oxycamalexin (11) by A. brassicicola was investigated for the first
time. In this work it was established that camalexins 1, 10 and
11 were biotransformed, but the rates of transformation were sub-
stantially slower than those observed in B. cinerea, (12 h vs 6–
8 days). In addition, analyses of the metabolism of synthetic cam-
alexin isomers 12 and 13 and camalexin bioisosteres 14–19 were
carried out. Based on results of these metabolic transformations
by A. brassicicola, the potential intermediates that lead to formation
of indole-3-thiocarboxamides are proposed as well as the substrate
range of the enzyme(s) involved in the biotransformations.
2. Results and discussion
2.1. Chemical synthesis and antifungal activity of camalexins
and analogues
Camalexins 1 and 11 and analogue 17 were synthesized and
purified as previously reported,16 and camalexin 10 was prepared
by methylation of 1.8 Compounds 14,17 1818 and 1918 were pre-
pared as previously reported. Compounds 12, 13, 15 and 16 are
new and were prepared as shown in Schemes 2–4 and described
in the Sections 4.2.1–4.2.4. In brief, preparation of 40-methylcama-
lexin (12) was achieved in quantitative yield by coupling indole-3-
thiocarboxamide with chloroacetone,17 whereas 50-methylcama-
lexin (13) was prepared in modest yield by coupling indole-3-thio-
carboxamide with 2-bromopropanal19 (Scheme 2). Reaction of 2-
chloro-1-(3-indolyl)ethanone with sodium azide in acetone20
yielded the corresponding azide 21, which after standard hydroge-
nation followed by condensation with ethyl formate and thiation
using Lawesson’s reagent yielded camalexin isomer 15 (Scheme 3).
Thiazolyl-3-indole 16 was obtained following a procedure similar
to that used for preparation of various 4-arylisothiazoles.21 First,
indolyl-3-acetic acid (22) was allowed to react with DMF/POCl3 fol-
lowed by base catalyzed hydrolysis to yield enol-aldehyde 23,
which was chlorinated and condensed with ammonium thiocya-
nate in DMF to yield the desired product 16 (Scheme 4).
The antifungal activities of phytoalexins 1, 10, and 11 and com-
pounds 12–19 against A. brassicicola were determined employing
the mycelial radial growth assay described in Section 4; results
of these assays are shown in Table 1. Camalexin (1) and 1-methyl-
camalexin (10) caused complete growth inhibition of mycelial
growth at 0.20 mM, whereas 6-methoxycamalexin (11) caused
Scheme 2. Syntheses of 40-methylcamalexin (12) and 50-methylcamalexin (13).

M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
4543
HPLC-DAD-ESI-MS. Samples were withdrawn from cultures imme-
diately after addition of each compound and then up to 10 days.
The samples were subjected to neutral, acidic and basic extrac-
tions, and the extracts were analyzed by HPLC, as reported in Sec-
tion 4. Samples of media incubated with phytoalexins or synthetic
compounds (control solutions) were analyzed similarly to deter-
mine the chemical stability of each compound during incubation
experiments.
The HPLC chromatograms of neutral extracts of cultures incu-
bated with camalexin (1) showed that it was metabolized slowly
(Fig. 1A). The chromatograms of neutral extracts showed the
presence of camalexin and additional peaks that were due to in-
dole-3-thiocarboxamide (7, tR = 4.6 0.2 min), indole-3-carboni-
trile (8, tR = 7.8 0.2 min) and indole-3-carboxylic acid (9,
tR = 3.6 0.1 min), by direct comparison with authentic synthetic
samples. No metabolites were detected in the acidic or basic ex-
tracts of cultures. Next, to establish the sequence of steps of cam-
alexin (1) transformation, each metabolite was administered
separately to cultures of A. brassicicola. The cultures were incu-
bated, extracted and analyzed as described above for camalexin
(1). As expected, indole-3-thiocarboxamide (7) was transformed
to the nitrile 8, which in turn was transformed to indole-3-carbox-
ylic acid (9) (Scheme 5). Similar biotransformation experiments
using camalexins 10 and 11 indicated that they were metabolized
at similar rates by A. brassicicola to the corresponding thiocarbox-
amides 24 and 25, nitriles 26 and 27 and carboxylic acids 28 and
29, respectively (Fig. 1A and B). As previously reported, thiocarbox-
amide 7 was not stable in media, however its transformation in
culture was much faster, indicating that enzyme-mediated trans-
formation occurred simultaneously. Progress curves of transforma-
tions of: thiocarboxamides 7, 24 and 25 in culture and in control
medium, camalexins 1, 10 and 11 in culture and in control medium
and formation of their metabolic products are provided in Supple-
mentary Data (Figs. S1–S6).
Scheme 3. Synthesis of camalexin isomer 15.
Scheme 4. Synthesis of camalexin isomer 16.
To obtain mechanistic clues on the biotransformations of the
thiazolyl rings of camalexins 1, 10 and 11, structures were de-
signed to contain methyl groups at C-40 and C-50 of thiazolyl (12
and 13) to test if its oxidative degradation could be blocked. In
addition, structural isomers 14–16 and bioisosteres 17–19 were
planned to probe the selectivity of the oxidative enzymes of A.
brassicicola. Compounds 12–19 were synthesized as summarized
above and incubated with cultures of A. brassicicola; samples of
cultures incubated for different times were extracted and the ex-
tracts were analyzed by HPLC-DAD-ESI-MS, as described for cama-
lexin (1). Except for the camalexin structural isomers 14–16, all
tested compounds were biotransformed. The chromatograms of
only ca. 70% inhibition at identical concentration. Among the cam-
alexin analogues, 40-methylcamalexin (12) and 50-methylcamalex-
in (13) displayed growth inhibitory activities similar to those of
camalexins 1 and 10, whereas 19 was substantially less inhibitory
than any of the compounds tested.
2.2. Metabolism of camalexins 1, 10, and 11 and compounds 12–
19 and structure determination of metabolic products
Cultures of A. brassicicola were incubated with each phytoalexin
separately and their transformations were monitored by
Table 1
Antifungal activitya of camalexins 1, 10 and 11 and compounds 12–19 against Alternaria brassicicola
Compound
Inhibition SDa (%)
0.50 mM
0.20 mM
0.10 mM
0.05 mM
Camalexin (1)
1-Methylcamalexin (10)
—
—
100 0b
100 0b
71 0d
100 0b
100 0b
45 3f
76 0b,c,d
71 1c,d
28 5g,h
80 0b
25 5d
43 3b,c
14 5e
47 3b
37 3c
—
6-Methoxycamalexin (11)
100 0b
—
40-Methylcamalexin (12)
50-Methylcamalexin (13)
—
65 2d
33 5f,g
53 5e
1H-3-(40-Thiazolyl)indole (14)
1H-3-(50-Thiazolyl)indole (15)
1H-3-(40-Isothiazolyl)indole (16)
1H-3-(20-Oxazolyl)indole (17)
1H-3-(20-Thia-30,40-diazolyl)indole (18)
1H-3–20-Oxa-30,40-diazolyl)indole (19)
100 0b
—
100 0b
46 2f
23 2d
—
100 0b
100 0b
100 0b
53 1c
23 2h
38 1f
60 0e
92 1c
30 3g
—
—
—
72 1b,c,d
24 1h
a
The percentage of inhibition was calculated using the formula: % inhibition = 100 ꢀ [(growth on amended/growth in control) ꢁ 100]; values are averages of three
independent experiments conducted in triplicate.
b
For statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted
a set at 0.05; n = 3; different letters in the same column (b–h)
indicate significant differences (P < 0.05).

4544
M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
0.060
0.050
0.040
0.030
0.020
0.010
0.000
A
0.050
0.040
0.030
0.020
0.010
0.000
0
24
48
72
96
0
24
48
72
96
120
Incubation time (h)
Incubation time (h)
Figure 2. Progress curves of biotransformations of camalexin analogues 12 (d) and
13 (j) and by Alternaria brassicicola and formation of products 30 (N) and 31 (j).
0.010
0.008
0.006
0.004
0.002
0.000
B
0
24
48
72
96
120
Incubation time (h)
Figure 1. Progress curves of biotransformations of: (A) camalexins 1 (j), 10 (N) and
11 (d) by Alternaria brassicicola and, (B) formation of thiocarboxamides 7 (j), 24
(N) and 25 (d).
Scheme 6. Biotransformations of camalexin analogues 12 and 13 by Alternaria
brassicicola.
bated with 13 showed that it was almost completely metabolized
within 96 h (Fig. 2), but no metabolites could be detected in the ex-
tracts of cultures incubated with 13. Compound 13 was stable in
culture media. Eventually, HPLC–ESI-MS data of freeze-dried sam-
ples of cultures incubated with 13 in water indicated the presence
of a polar metabolite whose structure was proposed to be carbox-
ylic acid 31 (Scheme 6). This compound was not soluble in ethyl
acetate, hence was not present in extracts of any cultures. The
structure of the new compounds 30 and 31 were confirmed by syn-
thesis, as described in Section 4. These compounds were not
metabolized by A. brassicicola, remaining intact in cultures for at
least 120 h.
Analyses of the extracts of culture samples incubated with the
camalexin bioisosteres 17–19 indicated these compounds were
transformed by A. brassicicola. The HPLC-DAD-ESI-MS chromato-
grams of neutral extracts of cultures incubated with 17 showed
that it was slowly metabolized (>120 h, Fig. 3) to indole-3-carbox-
amide (32, tR = 3.6 0.1 min), as determined by comparison with
an authentic sample. Indole-3-carboxamide (32) was not detected
either in control cultures or in media incubated with 17 and was
not metabolized further by A. brassicicola. No other metabolites
were detected in either the acidic or basic extracts of either culture.
The HPLC-DAD chromatograms of neutral extracts of cultures of A.
brassicicola incubated with 18 showed that it was metabolized via
indole-3-thiocarboxamide (7) to indole-3-carbonitrile (8) and in-
dole-3-carboxylic acid (9) (Scheme 7). None of these metabolites
Scheme 5. Biotransformations of camalexins 1, 10 and 11 by Alternaria brassicicola
and metabolic products.
neutral extracts of cultures of A. brassicicola incubated with 12
showed that it was metabolized at faster rate than camalexin (1)
(within 96 h, Fig. 2) to a compound with tR = 10.3 0.1 min. Isola-
tion of this metabolite from larger scale cultures, as described in
Section 4, and analyses of spectroscopic data allowed the identifi-
cation of the metabolite with tR = 10.3 as (2-(1H-indol-3-yl)thia-
zol-4-yl)methanol (30) (Scheme 6). Metabolite 30 was not
detected either in control cultures or in media incubated with
12. No other metabolites were detected in either the acidic or basic
extracts of any cultures. Similarly, cultures of A. brassicicola incu-

M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
4545
complete >120 h) were slower. Considering that the products of
these transformations are (thio)carboxamides likely to be formed
via similar intermediates, it would be tempting to speculate
whether these reactions are mediated by the same redox en-
zyme(s); nonetheless the current evidence is insufficient to make
such suggestions.
A
0.050
0.040
0.030
0.020
0.010
0.000
To understand the biodegradation pathway of the thiazolyl
rings of camalexins in A. brassicicola, it was important to uncover
potential precursor(s) of indole-3-thiocarboxamides 7, 24 and 25.
Thus, biotransformation experiments were conducted under differ-
ent conditions, including incubation of cultures with higher con-
centration of camalexin (1) (0.10 mM vs 0.050 mM) and at higher
temperature (30 °C vs 23 °C) and incubations in different growth
media (potato dextrose vs chemically defined media). However,
HPLC-DAD-ESI-MS analyses of extracts of these cultures did not
show the presence of new camalexin metabolites. In a last attempt,
it was reasoned that the precursor(s) of indole-3-thiocarboxamide
(7) might be relatively more polar than thiocarboxamide 7 and
thus not extractable with ethyl acetate. Hence a different approach
to isolate such polar metabolites was used; mycelia was grown in
chemically defined media and were then transferred to sterile
water and incubated with camalexin (1) for various periods. The
mycelia were filtered off, the filtrate was extracted with ethyl ace-
tate and the aqueous phase was freeze-dried. HPLC-DAD-ESI-MS
analyses of the ethyl acetate extracts and the aqueous residues
showed that the ethyl acetate extracts contained the expected
camalexin metabolites 7–9, while no camalexin metabolites were
detected in the aqueous residue. These results indicated that cam-
alexin (1) was biotransformed similarly in water and media, but
did not show the presence of potential precursor(s) of indole-3-
thiocarboxamide (7).
0
24
48
72
96
120
Incubation time (h)
B
0.020
0.015
0.010
0.005
0.000
0
24
48
72
96
120
Incubation time (h)
Figure 3. Progress curves of biotransformations of: (A) camalexin analogues 17 (N),
18 (j) and 19 (d) by Alternaria brassicicola and (B) formation of products 7 (d
resulting from 18), 32 (N resulting from 17) and 32 (d resulting from 19).
2.3. Synthesis and antifungal activity of metabolites
were detected either in control cultures or in media incubated with
18. No metabolites were detected in either the acidic or basic ex-
tracts of any culture. Similarly, the HPLC-DAD chromatograms of
neutral extracts of culture of A. brassicicola incubated 19 showed
that it was metabolized to indole-3-carboxamide (32), which was
not metabolized further. No additional metabolites were detected
in either the acidic or basic extracts of any culture.
The biotransformations of methylcamalexins 12 and 13 (Fig. 2,
t1/2 ca. 36 h, complete ca. 96 h) by A. brassicicola were somewhat
faster than the rate of biotransformation of camalexin 1 (Fig. 1,
t1/2 ca. 40 h, complete >120 h), whereas the rates of biotransforma-
tion of oxazole 17 and diazoles 18 and 19 (Fig. 3, t1/2 ca. 72 h,
The compounds (25, 27, 30 and 31) that resulted from biotrans-
formations of camalexins 10 and 11 and analogues 12 and 13 by A.
brassicicola were synthesized as described in Section 4 and summa-
rized in Schemes 8 and 9. 6-Methoxyindole-3-carbonitrile (27), ob-
tained by dehydration of oxime 34,22 was treated with
thioacetamide, as previously reported for 7,3 to yield 25 (Scheme 8).
1-Methylindole-3-thiocarboxamide (24) was prepared similarly
from the corresponding 1-methylindole-3-carbonitrile (26).3 The
new compound 1H-3-(4-Hydroxymethyl-2-thiazolyl)indole (30)
was obtained by treatment of thiocarboxamide 7 with ethyl bro-
mopyruvate17 followed by reduction of the resulting ethyl ester
35 with LiAlH4 (Scheme 9). The carboxylic acid 31, also a new com-
pound, was prepared by condensation of 7 with methyl-2-chloro-
oxopropanoate followed by alkaline hydrolysis of the resulting
methyl ester (Scheme 9).
Scheme 7. Biotransformation of camalexin bioisosteres 17–19 by Alternaria
brassicicola.
Scheme 8. Synthesis of new compounds 25 and 27.

4546
M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
against A. brassicicola of all metabolites resulting from transforma-
tions of the camalexins, their bioisosteres and analogues were
determined as described in Section 4; results of these assays are
shown in Table 2. Thiocarboxamides 7, 24, 25 and carboxamide
32 were substantially less inhibitory than the parent compounds;
however, the carbonitriles 8, 26, 27 were more inhibitory causing
complete mycelial growth inhibition at 0.50 mM. As with other
fungal species,3 the corresponding acids had the lowest growth
inhibitory activity among the compounds tested. Based on these
results it is concluded that the complete fungal transformations
of phytoalexins 1, 10 and 11 and related compounds 12, 13 and
17–19 are detoxifications.
3. Conclusion
This investigation has shown for the first time that the plant
pathogen A. brassicicola was able to slowly detoxify the phytoalex-
ins camalexins 1, 10 and 11 to thiocarboxamides, which were fur-
ther transformed to the corresponding carboxylic acids, the least
inhibitory among the metabolic products. The first intermediates
isolated, thiocarboxamides 7, 24 and 25 resulted from the degrada-
tion of the thiazole rings of camalexins 1, 10 and 11, respectively.
Scheme 9. Synthesis of new compounds 30 and 31.
To establish if the biotransformations of camalexins 1, 10 and
11 and compounds 12, 13 and 17–19 were detoxifications, myce-
lial radial growth assays were carried out. The antifungal activities
Table 2
Antifungal activitya of compounds 7–9 and 24–32 against Alternaria brassicicola
Compound
Inhibition SDa (%)
0.50 mM
0.20 mM
0.10 mM
Indole-3-thiocarboxamide (7)
Indole-3-carbonitrile (8)
Indole-3-carboxylic acid (9)
53 3f
100 0b
26 2h
60 3e
40 3g
100 0b
100 0b
26 2h
34 0g
90 2c
85 1c
68 1d
31 3e,f
92 1b
11 2i
24 3c,d
73 3b
0
0g
1-Methylindole-3-thiocarboxamide (24)
6-Methoxyindole-3-thiocarboxamide (25)
1-Methylindole-3-carbonitrile (26)
6-Methoxyindole-3-carbonitrile (27)
1-Methylindole-3-carboxylic acid (28)
6-Methoxyindole-3-carboxylic acid (29)
1H-3-(4-Hydroxymethyl-2-thiazolyl)indole (30)
2-(1H-Indol-3-yl)-4-thiazolecarboxylic acid (31)
Indole-3-carboxamide (32)
35 3d,e
21 3g,h
35 3d,e
52 3c
21 3d,e
0
8
0g
3f
31 0c
16 2h,i
26 2f,g
41 4d
38 3d,e
41 2d
0
0
0g
0g
13 2f
11 2f
15 3e,f
a
The percentage of inhibition was calculated using the formula: % inhibition = 100 ꢀ [(growth on amended/growth in control) ꢁ 100]; values are averages of three
independent experiments conducted in triplicate.
b
For statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted
a set at 0.05; n = 3; different letters in the same column (b–i)
indicate significant differences (P < 0.05).
Scheme 10. Proposed biotransformation pathway of camalexins 1, 10 and 11 and bioisostere 17.

M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
4547
While the detoxification pathway of camalexin (1) in A. brassicicola
was identical to that used by B. cinerea,3 the rate of transformation
in A. brassicicola is much slower than that observed in B. cinerea.
Importantly, the methyl analogues of camalexin 12 and 13 were
oxidized faster than camalexins, but without degradation of the
thiazole ring. That is, the presence of a methyl group at either C-
40 or C-50 of thiazolyl-3-indoles 12 and 13 blocked oxidative degra-
dation of the thiazole, but did not prevent oxidation of the methyl
groups. Furthermore, because compounds 1, 10, 11, and 17–19
were degraded and 14–16 were not, it is concluded that oxidative
degradation of the thiazole ring requires it to be attached to indole
by C-20. These inferences together with previous proposals on the
metabolic transformations of thiabendazole23 and other thiazoles
in mice, which were metabolized to thioacetamides and alkyl/aryl
glyoxals,24,25 lead us to propose the pathway shown in Scheme 10.
It is suggested that enzymatic oxidation of the thiazole ring of cam-
alexins 1, 10 and 11 could give intermediate I-1 that after hydroly-
sis to I-2 and further oxidation to I-3 and I-4 could lead to
thiocarboxamides 7, 24 and 25, respectively. Similar enzyme med-
iated transformations of 17 could lead to carboxamide 32. These
oxidative enzymes, contrary to those reported in mice,24,25 did
not appear able to oxidize the thiazoles rings of methylthiazoles,
however it is possible that oxidative degradations of 17–19 pro-
ceed similar to those of camalexins (Scheme 10).
It is noteworthy that these enzyme-mediated oxidative degra-
dations (detoxifications) of camalexins 1, 10 and 11 are rather
slow, unlike the metabolism of the phytoalexins brassinin (ca.
12 h at 0.10 mM)26 and cyclobrassinin (ca. 24 h at 0.10 mM)27 by
A. brassicicola. This slow biotransformation of camalexin (1) is con-
sistent with its importance in the resistance of the wild plant A.
thaliana to A. brassicicola.12 In conclusion, we suggest that since
camalexin (1) is transformed very slowly by A. brassicicola, engi-
neering the biosynthetic pathway of camalexins 1, 10 and 11 into
cultivated Brassica species could have a great positive impact in
obtaining resistance to black spot disease in agriculturally impor-
tant crops. This prospect is particularly interesting considering that
camalexin (1) might also be an important factor in plant resistance
to insects.28
i.d. ꢁ 150 mm), having an in-line filter, using two methods: (A)
mobile phase 50% H2O—50% CH3OH to 100% CH3OH for 25.0 min,
linear gradient, at a flow rate of 0.75 mL/min; (B) mobile phase
90% H2O—10% MeOH to 100% MeOH for 45.0 min, linear gradient,
at a flow rate 0.75 mL/min.
4.2. Synthesis and characterization of new compounds
Satisfactory spectroscopic data were obtained for all known
compounds synthesized as previously described (1,16 10,8 11,16
14,17 17,16 18,18 19,18 and 327). Compounds 8, 9, 28, 29 were com-
mercially available (Sigma–Aldrich).
4.2.1. 40-Methylcamalexin (12)
2-Chloropropanone (0.01 mL, 0.12 mmol) was added drop wise
to a solution of indole-3-thiocarboxamide3 (7, 10 mg, 0.060 mmol)
in EtOH (2 mL) and the reaction mixture was heated under reflux
at 80 °C for 1.5 h. Ethanol was removed in a rotary evaporator
and the residue was diluted with H2O, neutralized with NaHCO3
(satd sol.) and extracted with EtOAc. The combined extracts were
dried and the solvent was evaporated to afford 40-methylcamalexin
(12, 14 mg, quantitative) as a white powder, mp 168–169 °C.
1H NMR (500 MHz, CDCl3): d 9.42 (br s, 1H), 8.19 (d, J = 7.5 Hz,
1H), 7.88 (d, J = 2.5 Hz, 1H), 7.33 (d, J = 7.5 Hz, 1H), 7.25 (m, 2H),
6.79 (s, 1H), 2.50 (s, 3H). 13C NMR (125.8 MHz, CDCl3): d 163.0,
152.2, 136.6, 124.9, 124.8, 123.2, 121.5, 120.0, 112.1, 112.0,
110.8, 17.2. HPLC tR = 15.8 min. UV (HPLC, CH3OH–H2O) kmax
(nm): 220, 280, 320. FTIR (KBr) mmax cmꢀ1: 3119, 2222, 1534,
1445, 1242, 1116, 733. HRMS-EI m/z: measured 214.0561 ([M]+,
calcd 214.0565 for C12H10N2S) (100%).
4.2.2. 50-Methylcamalexin (13)
Bromine (2.7 mL, 0.06 mmol) was added drop wise to a solution
of propanal (4 mL, 0.06 mmol) in 1,4-dioxane (0.5 mL) in an ice
bath.19 The reaction mixture was stirred at 0 °C for an additional
1 h and the reaction mixture was subjected to FCC (EtOAc–hexane,
30:70) to yield 2-bromopropanal (6.5 mg, 90%). 2-Bromopropanal
(100 lL, 0.34 mmol) was added to a solution of indole-3-thiocarb-
oxamide (7, 30 mg, 0.17 mmol) in EtOH (1.5 mL) and the reaction
mixture was heated at 80 °C for 2 h. The reaction mixture was di-
luted with H2O, neutralized with NaHCO3, and extracted with
CH2Cl2. The combined extracts were dried, the solvent evaporated
and the residue subjected to FCC (EtOAc–hexane, 1:1) to yield
(20 mg, 55%) of 50-methylcamalexin (13) as a white powder, mp
170–172 °C.
4. Experimental
4.1. Materials and general procedures
Chemicals were purchased from Sigma–Aldrich Canada Ltd.,
Oakville, ON; solvents were HPLC grade and used as such. 95%
EtOH was used where EtOH is mentioned. Flash column chroma-
tography (FCC) was carried out using silica gel grade 60, mesh size
230–400 Å or WP C18 prepscale bulk packing 275 Å (J.T. Baker, NJ,
USA). Organic extracts were dried over Na2SO4 and concentrated
using a rotary evaporator.
Nuclear magnetic resonance (NMR) spectra were recorded on
Bruker 500 MHz Avance spectrometers, for 1H, 500.3 MHz and for
13C, 125.8 MHz; chemical shifts (d) are reported in parts per million
(ppm) relative to TMS; spectra were calibrated using the solvent
peaks; spin coupling constants (J) are reported to the nearest
0.5 Hz. Fourier transform infrared (FT-IR) data were recorded on
a spectrometer and spectra were measured by the diffuse reflec-
tance method on samples dispersed in KBr. MS [high resolution
(HR), electron impact (EI)] were obtained on a VG 70 SE mass spec-
trometer employing a solids probe.
1H NMR (500 MHz, CDCl3): d 8.60 (b, 1H), 8.22 (m, 1H), 7.83 (d,
J = 2.5 Hz 1H), 7.48 (s, 1H), 7.44 (m, 1H), 7.29 (m, 2H), 2.53 (s, 3H).
13C NMR (500 MHz, CDCl3): d 162.1, 139.7, 136.5, 130.9, 124.6,
124.3, 123.0, 121.3, 120.4, 112.3, 111.7, 12.0. HPLC tR = 16.3 min.
UV (HPLC, CH3OH–H2O) kmax (nm): 220, 278, 320. FTIR (KBr) mmax
cmꢀ1: 2833, 1540, 1451, 1242, 1119, 740. HRMS-EI m/z: measured
214.0563 ([M]+, calcd 214.0565 for C12H10N2S) (100%).
4.2.2.1. 1H-3-(50-Thiazolyl)indole (15).
A mixture of 2-
chloro-1-(1H-indol-3-yl)ethanone (20, 300 mg, 1.5 mmol) and so-
dium azide (200 mg, 3 mmol) in acetone (20 mL) and H2O
(10 mL) was heated at 50 °C for 20 h. The reaction mixture was
cooled to room temperature, was diluted with H2O (20 mL) and
was extracted with CH2Cl2. The combined extracts were dried,
the solvent evaporated to dryness and the crude product was
washed with CH2Cl2 and used for the next step (250 mg, 81%). To
a solution of 2-azido-1-indolyl-3-ethanone (21, 50 mg, 0.23 mmol)
High performance liquid chromatography (HPLC) analysis was
carried out with Agilent high performance liquid chromatographs
equipped with quaternary pump, automatic injector, and photodi-
ode array detector (DAD, wavelength range 190–600 nm), degas-
in MeOH (3 mL) Pd/C (50 mg) was added followed by HCl (20 lL).
The reaction flask was connected to an H2-filled balloon and the
reaction mixture was stirred at room temperature under H2 atmo-
sphere for 3 h. The reaction mixture was filtered and the filtrate
ser, and a column Eclipse XDB-C18 (5 lm particle size silica, 4.6

4548
M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
was concentrated. The crude product was subjected to FCC
(MeOH–CH2Cl2, 1:4) to afford 2-amino-1-indolyl-3-ethanone
(40 mg, 85%). Ethyl formate (1.5 mL) was added to a solution of
2-amino-1-indolyl-3-ethanone (30 mg, 0.16 mmol) in dry THF
1582, 1457, 1243, 890. HRMS-EI m/z: measured 200.0407 ([M]+,
calcd 200.0408 for C11H8N2S) (100%).
4.2.3. 1-Methylindole-3-thiocarboxamide (24)
(2 mL) followed by Et3N (50
lL). The reaction mixture was heated
A mixture of 1-methylindole-3-carbonitrile (26, 100 mg,
at 50 °C for 8 h, concentrated using a rotary evaporator and diluted
with H2O. The mixture was neutralized with aqueous HCl (1 M)
and extracted with EtOAc. The combined extracts were dried, the
solvent evaporated to dryness and the residue was subjected to
FCC (MeOH–CH2Cl2, 1:9) to afford 30 mg of pure N-(2-(1H-indol-
3-yl)-2-oxoethyl)formamide (30 mg, 86%). Lawesson’s reagent
(50 mg) was added to a solution of N-(2-(1H-indol-3-yl)-2-oxoeth-
yl)formamide (30 mg, 0.15 mmol) in 1,4-dioxane (1.5 mL) while
the solution was heated at 120 °C under argon. After 30 min, 1,4-
dioxane was removed with a rotary evaporator and the residue
was diluted with H2O. The aqueous solution was neutralized using
NaHCO3 and extracted with EtOAc. The combined extracts were
dried, the solvent evaporated to dryness and the residue was sub-
jected to FCC (EtOAc–hexane, 1:1) to afford 1H-3-(50-thiazol-
yl)indole (15, 20 mg, 68%) as a white powder, mp 140–142 °C.
1H NMR (500 MHz, CD3CN): d 9.64 (b, 1H), 8.76 (s, 1H), 8.10 (s,
1H), 7.87 (d, J = 8 Hz, 1H), 7.60 (d, J = 2.5 Hz 1H), 7.50 (d, J = 8 Hz
1H), 7.25 (dd, J = 8, 7 Hz, 1H), 7.20 (dd, J = 8, 7 Hz, 1H). 13C NMR
(500 MHz, CD3CN): d 151.4, 139.1, 138.0, 134.1, 126.4, 125.4,
123.9, 121.8, 120.4, 113.3, 108.0. HPLC tR = 8.2 min. UV (HPLC,
CH3OH–H2O) kmax (nm): 210, 242, 262, 300. FTIR (KBr) mmax
cmꢀ1: 3204, 1566, 1416, 1253, 823. HRMS-EI m/z: measured
200.0413 ([M]+, calcd 200.0408 for C11H8N2S) (100%).
0.7 mmol) and thioacetamide (110 mg, 1.4 mmol) in 10% HCl–
DMF (1.5 mL) was stirred at 90 °C for 12 h. The reaction mixture
was then neutralized with NaHCO3 (satd sol.) and was extracted
with EtOAc. The combined extracts were dried, and the solvent
was evaporated. The residue was subjected to FCC (MeOH–CH2Cl2,
3:97) to afford 1-methylindole-3-thiocarboxamide (24, 82 mg, 80%
yield) as
a light yellowish powder, mp 125–128 °C. HPLC
tR = 6.3 min. UV (HPLC, CH3OH–H2O) kmax (nm): 220, 260, 290. Sat-
isfactory spectroscopic data were obtained for 24.17
4.2.4. 6-Methoxyindole-3-thiocarboxamide (25)
A
mixture of 6-methoxyindole-3-carbonitrile (27, 50 mg,
0.5 mmol) and thioacetamide (55 mg, 0.7 mmol) in 10% HCl–DMF
(1 mL) was stirred at 90 °C for 12 h. The reaction mixture was neu-
tralized with NaHCO3 (satd sol.), and was extracted with EtOAc.
The combined extracts were dried, and the solvent was evaporated.
The residue was subjected to FCC (MeOH–CH2Cl2, 3:97) to afford 6-
methoxyindole-3-thiocarboxamide (25, 24 mg, 40% yield) as a light
brownish powder, mp 133–135 °C.
1H NMR (500 MHz, CD3CN): d 9.68 (br s, 1H), 8.24 (d, J = 8.5 Hz,
1H), 7.83 (d, J = 2.5 Hz, 1H), 7.60 (br, 2H), 7.00 (d, J = 2.5 Hz, 1H),
6.86 (dd, J = 8.5, 2.5 Hz, 1H), 3.81 (s, 3H). 13C NMR (500 MHz,
CD3CN): d 195.3, 156.7, 137.9, 128.0, 121.9, 119.25, 111.4, 100.0,
95.1, 55.1. HPLC tR = 4.5 min. UV (HPLC, CH3OH–H2O) kmax (nm):
220, 250, 280. FTIR (KBr) mmax cmꢀ1: 3399, 3250, 1630, 1530,
1040. HRMS-EI m/z: measured 206.0515 ([M]+, calcd 206.0514 for
4.2.2.2. 1H-3-(40-Isothiazolyl)indole (16).
Indolyl-3-acetic
acid (22, 220 mg, 1.25 mmol) in DMF (0.25 mL) was added to a
mixture of DMF (0.5 mL) in POCl3 (0.35 mL, 3.20 mmol) at 0 °C.
The reaction mixture was allowed to warm up to room tempera-
ture and was refluxed at 90 °C for 3 h. The reaction mixture was
cooled to room temperature and cracked ice was added to adjust
the volume to ca. 30 mL. The reaction mixture was extracted with
CH2Cl2, the combined extracts were dried, and the solvent was
evaporated to dryness to afford the crude diamine (315 mg) as a
yellowish powder in 91% yield. NaOH (25% aq, 2 mL) was added
to the diamine (100 mg, 0.40 mmol) in 1,4-dioxane (2 mL) and
the reaction mixture was refluxed for 8 h. 1,4-Dioxane was re-
moved with a rotary evaporator, the residue was diluted to ca.
30 mL by addition of cracked ice and acidified to pH 63 using aq
HCl (1:1). The resulting mixture was extracted with Et2O, the com-
bined extracts were dried, and the solvent was evaporated to yield
crude enolaldehyde 23 (69 mg, 90%). SOCl2 (0.35 mL, 4.8 mmol)
was added to crude enolaldehyde 23 (50 mg, 0.26 mmol) in THF
(1.5 mL) and the reaction mixture was cooled to ꢀ20 °C and stirred
for 10 min. The solvent was evaporated and the residue was dis-
solved in EtOAc and subjected to FCC (silica gel, EtOAc–Hexane,
7:3) to yield a fraction containing chloroacrolein 23a that was
immediately used in the next step. NH4SCN (30 mg, 0.40 mmol)
was added to the solution of chloroacrolein 23a (20 mg,
0.10 mmol) in DMF (1.5 mL), and the reaction mixture was heated
at 70 °C for 12 h with stirring (caution: in hood, NaOH trap for
HCN). The reaction mixture was diluted with brine (20 mL) and ex-
tracted with EtOAc. The combined extracts were dried, the solvent
evaporated, and the residue subjected to FCC (silica gel, EtOAc–
hexane, 3:7) to afford 1H-3-(40-isothiazolyl)indole (16, 16 mg,
56% yield) as a light yellowish powder, mp 124–125 °C.
C
10H10N2OS) (100%).
4.2.5. 6-Methoxyindole-3-carbonitrile (27)
A solution of 6-methoxyindole (33) in DMF (0.04 mL) was
added to an ice-cold mixture of DMF (0.07 mL) and POCl3
(0.07 mL) and the reaction mixture was stirred at room tempera-
ture for 2.5 h to yield 6-methoxyindol-3-carbaldehyde in 98%
yield.
A
solution of HONH2ꢂHCl (27 mg, 0.4 mmol), NaOAc
(31 mg, 0.38 mmol) and H2O (50
l
L) was added to 6-methoxyin-
dol-3-carbaldehyde (50 mg, 0.3 mmol) in EtOH (4 mL). The reac-
tion mixture was stirred at room temperature for 3 h, EtOH was
removed, the residue was diluted with H2O and was extracted with
EtOAc to yield 6-methoxyindol-3-oxime (34). Pyridine was added
to a solution of 6-methoxyindol-3-oxime (34, 50 mg, 0.25 mmol)
in THF (3 mL) and Ac2O (50 lL, 0.5 mmol) under argon and the
reaction mixture was refluxed for 9 h.22 Pyridine was removed in
a rotary evaporator using toluene to afford 6-methoxyindole-3-
carbonitrile (27, 43 mg, 96% yield) as a light yellow powder, mp
180 °C (decomposed; lit.29 174 °C, decomposed).
1H NMR (500 MHz, CD3CN): d 8.50 (br, 1H), 7.60 (s, 1H), 7.63 (m,
2H), 6.97 (dd, J = 8.5, 2 Hz 1H), 6.93 (s, 1H), 3.91 (s, 3H). 13C NMR
(500 MHz, CD3CN): d 157.6, 136.2, 132.4, 120.8, 119.4, 115.9,
112.2, 95.4, 85.4, 55.2. HPLC tR = 7.9 min. UV (HPLC, CH3OH–H2O)
kmax (nm): 220, 270, 290. FTIR (KBr) mmax cmꢀ1: 3383, 1612,
1451, 1122, 739. HRMS-EI m/z: measured 172.0638 ([M]+, calcd
172.0637 for C10H8N2O) (100%). Incomplete spectroscopic data
for this compound were reported.29
4.2.5.1. 1H-3-(4-Hydroxymethyl-2-thiazolyl)indole (30).
A
solution of indole-3-thiocarboxamide (7, 50 mg, 0.3 mmol) and
ethyl bromopyruvate (77 mg, 0.4 mmol) in EtOH (5 mL) was
heated under reflux for 1 h.17 Ethanol was removed using a rotary
evaporator and the residue (ethyl 2-(1H-indol-3-yl)thiazole-4-car-
boxylate, 35) was used in the next step. LiAlH4 (1.5 mg, 0.04 mmol)
was added to an ice-cold solution of 35 (10 mg) in dry THF (1 mL)
1H NMR (500 MHz, CDCl3): d 8.83 (s, 1H), 8.73 (s, 1H), 8.36 (br,
1H), 7.86 (d, J = 7.5 Hz, 1H), 7.48 (s, 2H), 7.25 (m, 3H). 13C NMR
(125.8 MHz, CDCl3): d 156.6, 140.6, 136.4, 133.3, 125.7, 122.9,
122.2, 120.8, 119.3, 111.6, 109.8. HPLC tR = 12.2 min. UV (HPLC,
CH3OH-H2O) kmax (nm): 225, 290. FTIR (KBr) mmax cmꢀ1: 3406,

M. Soledade C. Pedras, A. Abdoli / Bioorg. Med. Chem. 21 (2013) 4541–4549
4549
at 0 °C under argon and the reaction mixture was stirred at 0 °C for
10 min. The ice bath was removed and the reaction mixture was
stirred at room temperature for an additional 10 h. The reaction
mixture was quenched by addition of 25% aq NaOH (0.5 mL), was
filtered and concentrated to dryness. The residue was subjected
to FCC (EtOAc–hexane, 7:3) to afford (2–1H-3-(4-hydroxymethyl-
2-thiazolyl)indole (30, 7 mg, 83%) as a white powder, mp 196–
198 °C.
For experiments carried out in H2O, mycelia of cultures were fil-
tered off after 48 h of incubation and transferred into sterile H2O; a
solution of the compound to be tested (dissolved in CH3CN) was
added to the cultures (final concentration of 0.050 mM). Cultures
were incubated in a shaker at room temperature and treated as re-
ported above. The EtOAc residue was dissolved in CH3CN or
MeOH–H2O (1:1) and analyzed by HPLC-DAD-ESI-MS.
1H NMR (500 MHz, CD3OD): d 8.1 (d, J = 7 Hz, 1H), 7.9 (s, 1H),
7.4 (d, J = 7 Hz, 1H), 7.2 (m, 3H), 4.7 (s, 2H). 13C NMR (500 MHz,
CD3OD): d 164.6, 156.3, 136.9, 125.2, 124.4, 122.3, 120.5, 119.4,
111.6, 111.1, 110.7, 59.9. HPLC tR = 10.2 min. UV (HPLC, CH3OH–
H2O) kmax (nm): 220, 280, 320. FTIR (KBr) mmax cmꢀ1: 3191, 1534,
1444, 1241, 1014, 727. HRMS-EI m/z: measured 230.0513 ([M]+,
calcd 230.0514 for C12H10N2SO) (100%).
Acknowledgements
Financial support for the authors’ work was obtained from the
Natural Sciences and Engineering Research Council of Canada (Dis-
covery Grant to M.S.C.P.), the Canada Research Chairs program,
Canada Foundation for Innovation, the Saskatchewan Government,
and the University of Saskatchewan (graduate assistantship to
A.A.). We acknowledge the technical assistance of K. Brown
(NMR) and K. Thoms (MS) from the Department of Chemistry.
4.2.6. 2-(1H-Indol-3-yl)-4-thiazolecarboxylic acid (31)
Methyl 2-chloro-3-oxopropanoate30 was added to a solution of
indole-3-thiocarboxamide (7) (50 mg, 0.28 mmol) in EtOH (2 mL).
The reaction mixture was refluxed for 5 h, ethanol was removed
and the residue was subjected to FCC (EtOAc–Hexane, 3:7) to yield
methyl 2-(1H-indol-3-yl)thiazole-5-carboxylate (45 mg, 62%).
NaOH (25% aq, w/v, 0.3 mL) was added to a solution of 2-(1H-in-
dol-3-yl)thiazole-5-carboxylate (40 mg, 0.16 mmol) in THF (2 mL)
and the reaction mixture was refluxed for 4 h. THF was removed
and the residue was diluted with H2O (20 mL) and acidified (pH
63) with 37% HCl. The aqueous solution was extracted using
EtOAc, the organic layer was dried and was concentrated to afford
2-(1H-indol-3-yl)-4-thiazolecarboxylic acid (31, 38 mg, 100%) as a
yellowish powder, mp 215 °C (decomposed).
Supplementary data
Supplementary data (NMR spectra for new compounds 12, 13,
15, 16, 30 and 31 and progress curves of biotransformations of
compounds 1, 7, 10, 11, 24 and 25 by A. brassicicola and product
formation) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2013.05.026.
References and notes
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3. Pedras, M. S. C.; Hossain, S.; Snitynsky, R. B. Phytochemistry 2011, 72, 199.
4. Pedras, M. S. C.; Ahiahonu, P. W. K. Phytochemistry 2005, 66, 391.
5. Pedras, M. S. C.; Ahiahonu, P. W. K. Bioorg. Med. Chem. 2002, 10, 3307.
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8. Pedras, M. S. C.; Liu, J. Org. Biomol. Chem. 2003, 2, 1070.
9. Pedras, M. S. C.; Khan, A. Q.; Taylor, J. L. Plant Sci. 1998, 139, 1.
10. Pedras, M. S. C.; Sarma-Mamillapalle, V. K. Bioorg. Med. Chem. 2012, 20, 3991.
11. Pedras, M. S. C.; Minic, Z.; Hossain, S. Bioorg. Med. Chem. 2012, 20, 225.
12. Thomma, B. P. H. J.; Nelissen, I.; Eggermont, K.; Broekaert, W. F. Plant J. 1999,
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1H NMR (500 MHz, CD3OD): d 8.29 (s, 1H), 8.11 (m, 1H), 8.07 (s,
1H), 7.49 (m, 1H), 7.26 (m, 2H). 13C NMR (500 MHz, CD3OD): d
171.3, 164.7, 149.5, 138.6, 128.7, 127.3, 125.8, 124.2, 122.7,
121.2, 113.4, 112.1. HPLC tR = 4.5 min. UV (HPLC, CH3OH–H2O) kmax
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max cmꢀ1: 3199, 2392, 1637, 1526,
1220, 1081, 826. HRMS-EI m/z: measured 244.0307 ([M]+, calcd
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Alternaria brassicicola
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The antifungal activity of compounds against A. brassicicola was
investigated using a mycelial radial growth bioassay (PDA media
and DMSO solutions of each compound, 0.50, 0.20, 0.10, and
0.050 mM; control solutions contained 1% DMSO in PDA media)
previously reported.26
18. Pedras, M. S. C.; Minic, Z.; Sarma-Mamillapalle, V. K. J. Agric. Food Chem. 2009,
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The concentration that was moderately inhibitory of mycelial
growth (0.050 mM, except were noted otherwise) was selected
for biotransformation studies. Subsequently, fungal cultures of A.
brassicicola were initiated by inoculating A. brassicicola spores
(106/100 mL) in minimal media at 23 1 °C, under constant light,
on a shaker at 110 rpm. After 48 h of incubation, compounds to
be tested (dissolved in CH3CN) were added to fungal cultures and
to uninoculated media (final concentration in media 0.050 mM).
Control cultures of the fungus were grown separately. Cultures
were incubated in a shaker at room temperature, samples were
withdrawn at different time intervals, and extracted with EtOAc;
the aqueous phase was acidified and reextracted with EtOAc; the
remaining aqueous phase was basified and reextracted with CHCl3.
All extracts were concentrated and residues were dissolved in
CH3CN or MeOH–H2O (1:1) and analyzed by HPLC-DAD-ESI-MS.
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