American Journal of Plant Sciences, 2011, 2, 245-254
doi:10.4236/ajps.2011.22026 Published Online June 2011 (http://www.SciRP.org/journal/ajps)
Copyright © 2011 SciRes. AJPS
Analogs of Antifungal Indoles Isolated from
Aporpium Caryae with Activity against
Sudden-Death Syndrome of Soybean
Brenda Bertinetti1, Mercedes Scandiani2, Gabriela Cabrera1
1Organic Chemistry Dept and UMYMFOR (CONICET), Buenos Aires University, Ciudad Universitaria, Buenos Aires, Argentina;
2Agricultural Laboratory Río Paraná, San Pedro, Buenos Aires, Argentina.
Email: gabyc@qo.fcen.uba.ar
Received March 1st, 2011; revised April 28th, 2011; accepted May 8th, 2011.
ABSTRACT
Based on the precedent discovery of a weak antifungal indole isolated from Aporpium caryae, which increased its ac-
tivity when changing the N-alkyl chain, nineteen N-alkyl indoles, with alkyl chains from one to ten carbons and one or
two hydroxyls, one amine or bromine functional groups, were prepared and fully characterized by spectroscopic meth-
ods. The aim of this study is the search for new synthetic agrochemical leads derived from natural products. The anti-
fungal activity of the synthesized compounds against three fungal strains was measured in vitro. Six compounds pre-
sented good activity against Fusarium virguliforme, the causal agent of sudden-death syndrome (SDS) in soybean, in a
bioautography assay. Four of them were tested in a germination test and in a greenhouse experiment. All tested com-
pounds, applied as seed treatment, showed antifungal properties being effective to control SDS when there was low
level of fungal contamination. Results indicate that some of the tested compounds are acting as growth inhibitors and
represent new leads for the treatment of SDS for which no specific treatment has been previously reported.
Keywords: N-Alkyl Indole, Soybean Phytopathogen, Fusarium Virguliforme, Sudden-Death Syndrome
1. Introduction
Fungal infections are one of the main limiting factors in
the production of soybean and other crops, affecting
yield, and the quality of seeds and byproducts. Approxi-
mately 50 fungal diseases are known, although only
some of them are harmful, and only under specific condi-
tions. Among the important fungal pathogens of soy-
beans are Sclerotinia sclerotiorum (Sclerotinia stem rot),
Fusarium virguliforme and F. tucumaniae [1] (sudden-
death syndrome, SDS), Cercospora kikuchii (purple seed
stain), Colletotrichum truncatum (anthracnose), Macro-
phomina phaseolina (charcoal rot) and Rhizoctonia so-
lani (damping-off).
Most of the commercial, extended-use fungicides used
to control these diseases are simple synthetic compounds
with disadvantages, such as their possible contamination
of the environment and the introduction of risks to worker
health [2-4].
The development of sustainable alternatives to syn-
thetic fungicides has attracted growing interest as the use
of natural products. Some examples of natural microbial
agrochemicals are griseofulvin, isolated from Penicillium
griseofulvum, cycloheximide, isolated from Streptomyces
griseus, strobilurin A, isolated from Strobilurus tenacel-
lus and oudemansin A, isolated from Oudemansiella
mucida [5].
The use of microorganisms to produce agrochemicals
has been limited [6], mainly because agrochemical use
requires large amounts of product, and because of the
comparative costs of producing simple synthetic com-
pounds versus cultured natural products. Even though the
low-cost production of natural agrochemicals by fermen-
tation cannot be achieved in many cases, the use of these
compounds as templates for the production of synthetic
agrochemicals derived from natural products is a viable
alternative, as in the case of strobilurins. Strobilurin A
and oudemansin A were the lead compounds in the de-
velopment of azoxystrobin from Zeneca and kresoxim-
methyl from Basf, which are employed as fungicides for
several crops [7].
In this context, the goal of this study was the discovery
of new leads based on a weakly antifungal indole, iso-
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against
246
Sudden-Death Syndrome of Soybean
lated from Aporpium caryae and designated compound 1
[8].
In this work, we describe the performance in antifun-
gal activity tests of new synthetic analogs of 1.
2. Materials and Methods
2.1. General Procedures
Optical rotation was recorded on a Perkin Elmer po-
larimeter 343. FTIR spectra were recorded on a Nicolet
Magna-IR 550. NMR spectra were recorded on a Bruker
Avance II instrument at 500.13 MHz for 1H (referenced
to TMS, δ = 0) and at 125.13 MHz for 13C NMR (refer-
enced to the center line of CDCl3, δ 77.0). High per-
formance liquid chromatography used a variable-wave-
length UV detector coupled with a refractive index de-
tector (RefractoMonitor IV, Thermo Separation Prod-
ucts). Accurate ESI MS was carried out on a Bruker Mi-
crOTOF-Q II, whereas EIMS employed a mass spec-
trometer Trio-2 VG Masslab (Manchester, UK). All
chemicals were purchased from standard commercial
suppliers. Methyl 3-indole-carboxylate was purchased
from Acros Organics and all the alkyl halides (including
(R)-(-)-3-chloro-1,2-propanediol and (S)-(+)-3-chloro-
1,2-propanediol) were purchased from Sigma-Aldrich.
2.2. Bioassays
2.2.1. Bioautography on Silicagel
Direct bioautography on TLC was employed as a method
for detecting fungitoxic substances [9,10]. Fusarium
virguliforme O’Donnell & T. Aoki NRRL 34551, Fusa-
rium lateritium Nees ex Link (BAFC 759), Macro-
phomina phaseolina (Tassi) Goid (BAFC 3428) and Bo-
trytis cinerea Pers.: Fr. (BAFC 535) were employed as
fungal targets.
A concentration level of 50 μg/spot of each assayed
compound was used. Benomyl and Maxim® XL (50 μg
of total fludioxonil plus metalaxyl active ingredients),
were used as test compounds. Benomyl was tested at a
conc. level of 25 μg/spot. When big inhibitory halos were
observed, minimum inhibitory concentrations (MIC) were
measured by the same method [9]. The experiments were
repeated 3 times or 5 times for the most active com-
pounds.
2.2.2. See d T reatment
Soybean untreated seeds (50 g) were treated with an
aqueous suspension (Tween 80, 2 drops; water, 0.5 ml)
of the test compound (100 mg) with agitation for 60 sec-
onds. These treated (C) seeds were employed in the bio-
assays.
For a positive control, the abovementioned treatment
was repeated employing Maxim XL (fludioxonyl +
metalaxyl, 0.1 ml) instead of test compound. These
treated (M) seeds were employed in the following bioas-
says.
2.2.3. Germination Test in the Laboratory. Blotter
Paper Technique
400 treated (C), treated (M) and untreated seeds (U) were
used to estimate seed fungal incidence of pathogens [11]
and compound efficacy.
The seeds were plated on trays (16 × 20 × 5 cm, 50
seeds per tray) and incubated at 25˚C ± 1˚C under alter-
nating periods of 12 h fluorescent cool daylight (Osram
18 W/765) for 7 days [12]. The seeds were then exam-
ined under stereomicroscope at 40x magnification and
the identification of the fungi was based on the presence
of conidiophores and conidia of the pathogen at 20 – 40x
[13]. The experiment was repeated three times.
Data from this experiment were subjected to analysis
of variance (ANOVA). Treatment means were compared
by least significant differences at P = 0.05.
2.2.4. Greenhouse Experiment
Ninety eight disinfected untreated seeds and ninety eight
treated (C) seeds were added to pots containing field soil
and covered with another 2 cm of soil. Half of them were
inoculated with Fusarium virguliforme NRRL 34551 [1].
Pots were then placed on a greenhouse bench and grown
under natural photoperiod at 25˚C ± 2˚C for 4 - 5 weeks.
Soil was watered to saturation after planting and main-
tained at near field capacity throughout the study.
Plants were rated for incidence of SDS-like symptoms
on the foliage, plant height and shoot fresh weight. Dis-
ease incidence (DI) of plants was based on the percent-
age of plants with foliar symptoms typical of SDS [1].
Symptoms ranged from leaf curling and rugosity, mar-
ginal cupping, mottling, chlorotic interveinal spots, in-
terveinal chlorosis and necrosis, to leaf drop and stunt-
ing.
Foliar disease severity (DS) was rated during 5 weeks
after planting based on a scale of 1 to 5, where 1 = no
symptoms; 2 = light symptom development with mottling
and mosaic (1% - 20% foliage affected); 3 = moderate
symptom development with interveinal chlorosis and
necrosis (21% - 50% foliage affected); 4 = heavy symp-
tom development (51% - 80% foliage affected); and 5 =
severe symptom development with interveinal chlorosis
and necrosis and/or dead plants (81% - 100% foliage
affected) [14].
At the end of the experiment, all plants were rated for
height and the fresh shoot weight was determined. Data
from this experiment were subjected to analysis of vari-
ance (ANOVA). Treatment means were compared by
Copyright © 2011 SciRes. AJPS
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against
Sudden-Death Syndrome of Soybean
Copyright © 2011 SciRes. AJPS
247
least significant differences at P = 0.05. The experiment
was repeated three times.
3. Results and Discussion
During the structural elucidation of indole derivatives
from Aporpium caryae [8], compounds 7 and 8 were
prepared to compare the sign of the optical rotation and
the absolute stereochemistry. Their antifungal activity
against the phytopathogen Cladosporium cucumerinum
was weak, although higher than the natural compound 1.
For this reason, a new set of indole derivatives (Figure 1)
with a variety of N-alkyl chains was prepared to explore
the antifungal activity of this type of compound against
fungal strains of economical importance for soybean
production. The preparation of these compounds was
easily achieved in basic media using the corresponding
alkyl halide (Suppl. Mat.). All compounds were purified,
fully characterized and structurally assigned by 1D and
2D NMR (HSQC, HMBC, COSY) and mass spectrome-
try (Suppl. Mat.). To the best of our knowledge, com-
pounds 3-13, 16 and 18-20 have not been previously iso-
lated or described. Compound 15 was claimed in a patent
as an intermediate in the synthesis of triazoles [15], and
compounds 2 and 17 were previously reported as a
by-product and an intermediate, respectively, in the syn-
thesis of alkyl indoles [16,17].
As a preliminary step in detecting antifungal properties,
the effect of each compound was evaluated in an in vitro
test against three fungal species, F. virguliforme, F. lat-
eritium and M. phaseolina. These species represent
strains of significant economic importance, and they
generally have different responses to antifungal com-
pounds. As stated above, F.virguliforme is one of the
causal agents of SDS in soybeans, and M. phaseolina
causes charcoal rot; F. lateritium is a phytopathogen of
Cucurbitaceae. Botrytis cinerea, a phytopathogen of
grapevine, was also tested in some cases. The antifungal
activity of the synthesized compounds is presented in
Table 1. Compounds 2-4, 10, 14 and 17 showed anti-
fungal activity in this screen against F. virguliforme, F.
lateritium and M. phaseolina. Their inhibitory halos were
from 10 to 17 mm but since halo diameter is dependent
on diffusion and other physical properties of the com-
pounds, the absolute value may be not very relevant. The
minimum inhibitory concentration (MIC) [9] for com-
pounds 2, 3, 4 and 10 against F. virguliforme was 5
μg/point.
The results in Table 1 also indicated that for these de-
rivatives the presence of three to four carbon atoms with
only one hydroxyl group in the alkyl chain attached to
the indole nitrogen led to strongly positive result. The
presence of a larger chain in 9 or a bulky chain in 20
caused a complete loss of activity. The results with
compounds 4, 16 and 18 (Table 1) indicated that the
N
OO
R1
R2
CH3
Compound
OH
OH
CH3OH
CH3
CH3OH
OH
CH3OOH
CH3OOH
CH3OH
OH
CH3OH
OH
CH38
CH3OAc
1
2
3
4
5
6
7
8
9
10
R1 R2
CH3CH3
CH3Br
CH3Br
CH3
CH3
CH3
14
15
16
17
18
19
NH2
N
CH3
9
20 NCO2CH3
8 8
13
Compound R1 R2
H
OH
H8
11
12
2
3
43a
77a
8
1 3
24
Figure 1. Tested compounds.
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against
248
Sudden-Death Syndrome of Soybean
Table 1. Antifungal activities of synthetic compounds 2 - 20.
Compound Fusarium virguliforme Fusarium lateritium Macrophomina phaseolina Botrytis cinerea
2 17 +/ 2(5) 16 +/ 2(5) 22 +/ 1 19 +/ 1
3 15 +/ 1(5) 12 +/ 2(5) 6 +/ 1
4 17 +/ 2(5) 12 +/ 2(5) 15 +/ 2 20 +/ 2
5 6 +/ 1 10 +/ 1 4 +/ 1
7 3 +/ 1 14 +/ 1
8 4 +/ 1 4 +/ 2
9 10 +/ 1
10 14 +/ 2(5) 16 +/ 2(5) 16 +/ 2 nd
11
12 7 +/ 1 7 +/ 1
13
14 10 +/ 1 13 +/ 1 15 +/ 1 nd
15 5 +/ 1 9 +/ 1 9 +/ 1 nd
16 5 +/ 1 8 +/ 1 9 +/ 1 nd
17 11 +/ 1 15 +/ 1 17 +/ 2 nd
18 nd
19 5 +/ 1 7 +/ 1 10 +/ 1 nd
20 nd
Benomyl 27 +/ 2 30 +/ 1 30 +/ 2 25 +/ 1
Maxim XL 12 +/ 2 12 +/ 1 18 +/ 1 nd
Diameter of inhibition zone in mm (MIC μg/pt). 50 μg/spot was used except benomyl (25 μg/spot); nd: not determined.
replacement of the hydroxyl group by a primary amine or
bromine resulted in a loss of activity against all strains.
Initially, compound 6 gave approximately the same re-
sults as 4, and NMR showed that 6 decomposed rapidly
to 4. Therefore, 6 was not further examined and is not
shown in Table 1. The responses of the tested com-
pounds against F.virguliforme and F. lateritium were
similar, but some differences were observed when active-
ties against M. phaseolina and B. cynerea were compared.
For example, compound 3 was inactive or weakly active,
and 7, 14 and 17 gave more activity against these strains
than against the Fusarium species.
Four of the active compounds, 2, 4, 10 and 14 were
selected for further analysis.
A germination test was performed, in order to deter-
mine the fungal incidence of pathogens [11] and com-
pound efficacy on natural contaminated seeds. The use of
different seed batches also allows the comparison of di-
verse natural situations, with dissimilar type and degree
of pathogen contamination. The incidence of fungal in-
fection in untreated seeds and seeds treated with the se-
lected compounds is shown in Table 2, and the fre-
quency of fungal pathogens is listed in Table 3 .
Table 2. Effect of compound 2, 4, 10 and 14 on fungal inci-
dence (%).
Treatment % incidence
U 41.7 a
C2 13.4 b
C4 9.8 b
C10 19.9 b
C14 13.8 b
M 2.5 c
U = untreated soybean seeds, Cx = treated soybean seeds with compound x,
M = treated soybean seeds with Maxim XL, SD ranged from 0.3 to 1.1 (n =
4) Values within a column followed by the same letter are not significantly
ifferent. Tukey analysis test at P = 0.05. d
Copyright © 2011 SciRes. AJPS
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against 249
Sudden-Death Syndrome of Soybean
Table 3. Effect of compounds on frequency (%) of pathogens.
Treatment Fusarium spp.i Phomopsis spp.i Fusarium spp.ii Phomopsis spp.ii Cercospora kikuchiiiii
U 26.5 a 7.8 a 81.9 a 28.8 a 6.7 a
C2 4.9 c 3.5 ab 64.9 b 9.3 d 3.5 a
C4 2.7 cd 3.5 ab 71.0 ab 10.9 bcd 6.9 a
C10 10.6 b 4.3 a 61.1 b 20.7 a 5.2 a
C14 6.9 bc 5.3 a 62.0 b 19.4 ab 6.3 a
M 0.0 d 0.9 b 33.9 c 10.0 cd 0.4 b
i-iii experiments using different seed batches, U = untreated soybean seeds, Cx = treated soybean seeds with compound x, M = treated soybean seeds with
Maxim XL, SD ranged from 0.1 to 1.2 (n = 4), Values within a column followed by the same letter are not significantly different. Tukey analysis test at P =
0.05.
Significant differences were observed between the un-
treated seeds (treatment U, Table 2), which showed a
high percent of fungal incidence (41.7% +/ 0.6%), seeds
treated with any of the tested compounds 2, 4, 10 or 14,
which showed from 9.8 % to 19.9 % (treatment Cx), and
seeds treated with Maxim XL, which showed the small-
est percentage of fungal incidence (2.5% +/ 0.4%,
treatment M). There were no significant differences be-
tween the tested compounds. When the frequency of
fungal pathogens was analyzed, some differences were
observed, depending on the pathogen. Compounds 2, 4,
10 or 14 showed to be effective in the inhibition of Fusa-
rium spp. with different significance between them, be-
ing the order 4, 2, 14, 10 from higher to smaller response
(Table 3). The tendency was less pronounced against
Phomopsis spp. and a lack of activity was observed in the
case of the pathogen Cercospora kikuchii. When the seed
batch was naturally very contaminated (Table 3, entries
ii), the same tendency remains although the percentages
of frequency were high. Furthermore, compound 2 was
as active as Maxim in this situation.
Other fungal pathogens were also present although
their frequency was too small to be statistically consid-
ered.
All these results would indicate that compounds 2, 4,
10 and 14 are acting as fungal growth inhibitors. This
outcome is in accordance with other studies which have
reported that indoles like indole-3-acetic acid possess
fungistatic activity [18]; at the same time, indole also has
been shown to act synergistically with other known anti-
fungal compounds to enhance their fungistatic properties
[19].
A greenhouse experiment was performed with these
compounds to see their effect on the incidence of SDS on
soybean plants inoculated with Fusarium virguliforme
(Suppl. Mat., Table 1). All the tested compounds pre-
vented plant infection comparing with plants derived
from untreated seeds (95% +/ 2% infected). It is note-
worthy that the infected plants had light symptoms of
foliar disease severity, with a development of chlorotic
mottling as the main infection.
In another experiments, which resulted in a more se-
vere global infection, carried out only with 2, it was
shown that this compound did not affect plant growth,
and both height and fresh shoot weight of treated plants
were not significantly different from untreated plants
(Table 4). A significant difference in height and fresh
shoot weights were seen between seeds inoculated with F.
virguliforme and left untreated (U + Fv), compared to
those treated with compound 2 (C + Fv). The percentage
of incidence of SDS for U + Fv was very high (85% ±
2%) from the first 15 dpi (days post-inoculation) to the
last measurement at 35 dpi (90% ± 2%), while in the case
of C + Fv a 48% ± 4% incidence at 15 dpi and 67% ± 4%
incidence at 35 dpi (Table 5) was observed. This differ-
ence is especially important when the severity results are
included (Figure 2), because the severity factor at 35 dpi
was near 5 for U + Fv, and less than 3 for C + Fv. These
results are in full agreement with previous tests and sup-
port the conclusion that compound 2 has a clear effect on
the incidence of F. virguliforme on soybean plants.
It is noteworthy that a good correlation between the in
vitro antifungal test using bioautography and the other
tests was observed in this work, probably because all the
compounds belong to the same structural family and
have similar physical properties.
As there are few data in the literature related to the
control of SDS, the results achieved in this work may not
be deeply compared with other works. In general, there
were no studies showing remarkable performance for
preventing SDS in soybean. For example the oligosac-
charide chitosan (poly-β-(1,4)-D-glucosamine) capability
to prevent SDS on soybean caused by Fusarium solani
was evaluated in greenhouse experiments [20]. Chitosan
Copyright © 2011 SciRes. AJPS
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against
250
Sudden-Death Syndrome of Soybean
Table 4. Effect of compound 2 on height and fresh shoot
weight of soybean plants inoculated with Fusarium virguli-
forme under greenhouse co nditions.
Height and fresh shoot weight
Treatment Height (cm) fresh shoot weight (g)
U 94 +/ 2 a 11 +/ 1 a
C2 90 +/ 2 a 12 +/2 a
U + Fv 22 +/ 2 c 1.4 +/ 0.2 c
C2 + Fv 31 +/ 5 b 4 +/ 1 b
U = untreated soybean seeds, C2 = treated soybean seeds with compound 2,
Fv = inoculation with Fusarium virguliforme, Values within a column fol-
lowed by the same letter are not significantly different. Tukey analysis test at
P = 0.05.
Table 5. Effect of compound 2 on incidence of Sudden-de ath
syndrome (SDS) on soybean plants inoculated with Fusa-
rium virguliforme under greenhouse conditions.
Incidence (%)
Treatment 15 dpi 18 dpi 24 dpi 32 dpi 35 dpi
U + Fv 85 ± 2 85 ± 2 90 ± 2 90 ± 2 90 ± 2
C + Fv 48 ± 4 63 ± 5 63 ± 5 67 ± 4 67 ± 4
15 20 25 30 35
2
3
4
5 % (FV)
% (FV-C)
Disease severity
Days after inoculation
Figure 2. Effect of compound 2 on foliar severity of Sud-
den-death syndrome (SDS) on soybean plants inoculated
with Fusarium virguliforme under greenhouse conditions.
showed certain ability to retard the SDS symptom ex-
pression in soybean leaves but it could not absolutely
protect the soybean from disease incidence. Strobilurins,
which are known fungicides used in crop protection,
were examined in field experiments in combination with
potassium chloride [21]. These studies revealed that stro-
bilurin fungicide treatments showed variable effects on
the severity of SDS disease and no significant yield re-
sponse to foliar application was displayed. It is worth
mentioning that the present work is the first one where
the activity of compounds against Fusarium virguliforme
is investigated and no similar studies about SDS in soy-
bean caused by this phytopathogen were found.
4. Conclusions
In summary, nineteen analogs of the natural compound 1
were prepared, characterized and screened for antifungal
activity. Six analogs, designated 2-4, 10, 14 and 17, were
determined to have antifungal activity against F. virguli-
forme in vitro and 2, 4, 10 and 14 also when applied as
seed treatments. Their antifungal activity is dependent on
the degree of contamination, being more effective in the
first period of infection or when low pathogen content is
present, and is more selective against Fusarium spp.
These substances represent new leads for the treatment of
SDS, for which no specific treatment has been reported,
and for which commercial fungicides have only limited
effects [22] and eventually would be useful for the de-
velopment of a combination treatment with known fun-
gicides, as a strategy to reduce their widespread use.
5. Acknowledgements
We thank Universidad de Buenos Aires (X029), ANPCYT
and CONICET for financial support and CONICET for
the fellowship to BVB.
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http://www.ces.purdue.edu/extmedia/BP/BP-58.pdf.
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against
252
Sudden-Death Syndrome of Soybean
APPENDIX
1. Preparation of the N-Alkylindoles
The N-alkylindoles 2-4, 7-10, 15, 16 and 20 were pre-
pared by treatment of methyl 3-indole-carboxylate with
NaH in THF or DMSO, and subsequent substitution of
the corresponding alkyl halides. Typical conditions were
as follows: to a solution of methyl 3-indole-carboxylate
(200 mg, 1.14 mmol) in dry DMSO (2ml), 2 eq. NaH
were added and the mixture was stirred for about 20 min-
utes until it turned green-colored. The alkyl chloride
(1.2eq) was then added drop wise to the resulting solution.
The mixture was stirred overnight at room temperature.
Methanol was used to remove the excess of hydride, wa-
ter was added and the products were extracted with ethyl
acetate (3 × 5 ml). After concentration in vacuo, the resi-
due was purified by HPLC RP-18. Yields obtained were
between 40 to 95%.
Compounds 5, 6, 11, 12-13 and 17 were obtained as
minor by-products, in the preparation of 2, 4, 3, 9 and 15
respectively, and were separated by HPLC (YMC C18, 5
m, 22.5 × 2.5 cm, MeOH-H2O 6:4 (5, 11), MeOH-H2O
7:3 (6, 17) or preparative TLC (silicagel, cyclohexane-
CH2Cl2 1:1)(12, 13). Compounds 18 (and 19) were pre-
pared by treatment of 16 with NH3 (or dimethyl amine).
2. Spectroscopical Data of Previously
Undescribed Compounds
Compound 3. 1-(2-Hydroxy-p ropyl)-1H-indole-3-carboxylic
acid methyl ester
Oil. IR (KBr, cm–1) max: 3453 (OH), 2937 (CH), 1677
(C=O). 1H NMR (CDCl3): d 8.04 (m, 1H, H-4); 7.75 (s,
1H, H-2); 7.27 (m, 1H, H-7); 7.16 (m, 2H, H-5,6); 4.10
(m, 1H, H-2’); 4.04 (dd, J = 14.4 and 3.9 Hz, 1H, H-1’);
3.92 (dd, J = 14.4 and 7.7 Hz, 1H, H-1’); 3.69 (s, 3H,
CH3O); 2.40 (brs, 1H, OH); 1.16 (d, J = 6.2 Hz, 3H,
H-3’). 13C NMR (CDCl3): d 165.5 (C-8); 136.8 (C-7a);
135.2 (C-2); 126.5 (C-3a); 122.7, 121.9 (C-5, C-6); 121.7
(C-4); 110.0 (C-7); 107.1 (C-3); 66.6 (C-2’); 54.0 (C-1’);
50.9 (CH3O); 20.6 (C-3’). HR ESI-MS m/z: 234.1119
[M+H]+ (calcd for C13H16NO3, 234.1125, D 2.4 ppm).
EIMS (70 eV): m/z (%) 233 [M](52), 202 (15), 188
(100), 130 (43).
Compound 4. 1-(4-Hydroxy-butyl)-1H-indole-3-carboxy-
lic acid methyl ester
Oil. IR (KBr, cm–1) vma x : 3408 (OH), 2951 (CH), 2876
(CH), 1696 (C=O). 1H NMR (CDCl3): d 8.16 (m, 1H,
H-4); 7.81 (s, 1H, H-2); 7.34 (m, 1H, H-7); 7.25 (m, 2H,
H-5, 6); 4.13 (t, J = 7.1 Hz, 2H, H-1’); 3.88 (s, 3H,
CH3O); 3.60 (t, J = 6.2 Hz, 2H, H-4’); 1.96 (m, 2H, H-2’);
1.63 (brs, 1H, OH); 1.55 (m, 2H, H-3’). 13C NMR
(CDCl3): d 165.6 (C-8); 136.4 (C-7a); 134.2 (C-2); 126.6
(C-3a); 122.6, 121.7, 121.6 (C-4, C-5, C-6); 109.9 (C-7);
106.7 (C-3); 61.8 (C-4’); 50.7 (CH3O); 46.6 (C-1’); 29.6
(C-3’); 26.3 (C-2’). HR ESI-MS m/z: 248.1207 [M+H]+
(calcd for C14H18NO3, 248.1281, D 2.9 ppm). EIMS (70
eV): m/z (%) 247 [M](100), 216 (27), 188 (86), 144
(50).
Compound 5. 1-[3-(3-Hydroxy-propoxy)-propyl]- 1H-
indole-3-carboxylic acid methyl ester
Oil. IR (KBr, cm–1) vmax: 3417 (OH), 2928 (CH), 1696
(C=O), 1105 (C-O-C). 1H NMR (CDCl3): d 8.17 (m, 1H,
H-4); 7.84 (s, 1H, H-2); 7.38 (m, 1H, H-7); 7.27 (m, 2H,
H-5, 6); 4.29 (t, J = 6.6 Hz, 2H, H-1’); 3.91 (s, 3H,
CH3O); 3.81 (dd, J = 6.1, 5.0 Hz, 2H, H-7’); 3.56 (t, J =
5.70 Hz, 2H, H-3’ ); 3.33 (t, J = 5.70 Hz, 2H, H-5’); 2.15
(brs, 1H, OH); 2.11 (m, 2H, H-2’); 1.86 (m, 2H, H-6’).
13C NMR (CDCl3): d 165.6 (C-8); 136.5 (C-7a); 134.6
(C-2); 126.7 (C-3a); 122.7, 121.9, 121.8 (C-4, C-5, C-6);
109.9 (C-7); 107.1 (C-3); 69.7, 67.1 (C-5’, C-7’); 61.4
(C-3’); 50.9 (CH3O); 43.5 (C-1’); 32.2 (C-2’); 29.8
(C-6’). EIMS (70 eV): m/z (%) 291 [M](60), 260 (7),
189 (76), 188 (39), 130 (100).
Compound 6. 1-[4-(4-Hydroxy-butoxy)-butyl]-1H-indole-
3-carboxylic acid methyl ester
Oil. IR (KBr, cm–1) vma x : 3431 (OH), 2945 (CH), 2862
(CH), 1699 (C=O). 1H NMR (CDCl3): d 8.18 (m, 1H,
H-4); 7.85 (s, 1H, H-2); 7.37 (m, 1H, H-7); 7.27 (m, 2H,
H-5, 6); 4.19 (t, J = 7.1 Hz, 2H, H-1’); 3.91 (s, 3H,
CH3O); 3.64 (dd, J = 11.0 Hz and 5.6 Hz,, 2H, H-9’);
3.43 (m, 4H, H-4’ y 6’); 2.20 (brs, 1H, OH); 1.97 (m, 2H,
H-2’); 1.66 (m, 4H, H-7’, 8’); 1.60 (m, 2H, H-3’). 13C
NMR (CDCl3): d 165.6 (C-8); 136.4 (C-7a); 134.2 (C-2);
126.6 (C-3a); 122.6, 121.7, 121.6 (C-4, C-5, C-6); 109.9
(C-7); 106.7 (C-3); 71.0 (C-6’); 70.3 (C-4’); 62.8 (C-9’);
62.8 (C-9’); 50.9 (CH3O); 46.6 (C-1’); 30.3 (C-7’); 26.9
(C-2’, C-3’); 26.7 (C-8’). HR ESI-MS m/z: 320.1866
[M+H]+ (calcd for C18H26NO4, 320.1856, D -3.0 ppm).
EIMS (70 eV): m/z (%) 319 [M](39), 216 (37), 188
(100), 172 (73), 130 (83).
Compound 7. (S) 1-(2,3-Dihydroxy-propyl)-1H-indole
-3-carboxylic acid methyl ester
Mp 108˚C - 110˚C. [a]D
25= -20 (c 0.4, MeOH). IR
(KBr, cm-1) max: 3459 (OH), 2900 (CH), 1674 (C=O).
1H NMR (CDCl3): d 8.02 (m, 1H, H-4); 7.79 (s, 1H, H-2);
7.29 (m, 1H, H-7); 7.16 (m, 2H, H-5, 6); 4.15 (dd, J =
14.4 and 4.8 Hz, 1H, H-1’); 4.03 (dd, J = 14.4 and 7.3 Hz,
1H, H-1’); 3.94 (m, 1H, H-2’); 3.72 (s, 3H, CH3O); 3.53
(dd, J = 11.4 and 3.9 Hz, 1H, H-3’); 3.38 (dd, J = 11.4
and 5.7 Hz, 1H, H-3’); 3.05 (brs, 1H, OH). 13C NMR
(CDCl3): d 165.6 (C-8); 136.8 (C-7a); 135.2 (C-2); 126.6
(C-3a); 123.0, 122.1, 121.8 (C-4, C-5, C-6); 109.9 (C-7);
107.5 (C-3); 70.6 (C-2’); 63.7 (C-3’); 51.0 (CH3O); 49.1
(C-1’). HR ESI-MS m/z: 250.1061 [M+H]+ (calcd for
Copyright © 2011 SciRes. AJPS
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against 253
Sudden-Death Syndrome of Soybean
C13H16NO4, 250.1074, D 5.2 ppm). EIMS (70 eV): m/z
(%) 249 [M](63), 218 (20), 189 (22), 188 (100), 130
(24).
Compound 8. (R) 1-(2,3-Dihydroxy-propyl)-1H-indole
-3-carboxylic acid methyl ester
Mp 109˚C - 110˚C. [a]D
25= 16 (c 0.4, MeOH). IR (KBr,
cm-1) max: 3459 (OH), 2910 (CH), 1674 (C=O). 1H
NMR (CDCl3): d 8.06 (m, 1H, H-4); 7.86 (s, 1H, H-2);
7.37 (m, 1H, H-7); 7.21 (m, 2H, H-5, 6); 4.25 (dd, J =
14.4 and 5.0 Hz, 1H, H-1’); 4.10 (dd, J = 14.4 and 7.3 Hz,
1H, H-1’); 3.97 (m, 2H, H-2’); 3.83 (s, 3H, CH3O); 3.55
(dd, J = 11.4 and 4.3 Hz, 1H, H-3’); 3.43 (dd, J = 11.4,
5.5 Hz, 1H, H-3’); 3.05 (brs, 1H, OH). 13C NMR (CDCl3):
d 166.0 (C-8); 136.8 (C-7a); 135.5 (C-2); 126.4 (C-3a);
122.7, 121.8, 121.4 (C-4, C-5, C-6); 110.0 (C-7); 106.8
(C-3); 70.3 (C-2’); 63.4 (C-3’); 50.9 (CH3O); 49.0 (C-1’).
HR ESI-MS m/z: 250.1078 [M+H]+ (calcd for C13H16NO4,
250.1074, D -1.7 ppm). EIMS (70 eV): m/z (%) 249
[M](52), 218 (12), 189 (18), 188 (100), 130 (13).
Compound 9. 1-Decyl-1H-indole-3-carboxylic acid
methyl ester
Oil. IR (KBr, cm–1) vmax: 2920 (CH), 2851 (CH), 1702
(C=O). 1H NMR (CDCl3): d 8.10 (m, 1H, H-4); 7.74 (s,
1H, H-2); 7.28 (m, 1H, H-7); 7.19 (m, 2H, H-5, 6); 4.04
(t, J = 7.3 Hz, 2H, H-1’); 3.83 (s, 3H, CH3O); 1.78 (qi, J
= 7.1 Hz, 2H, H-2’); 1.15-1.23 (m, 14H, H-3’ to 9’); 0.80
(t, J = 6.8 Hz, 3H, H-10’). 13C NMR (CDCl3): d 165.5
(C-8); 136.6 (C-7a); 134.2 (C-2); 126.7 (C-3a); 122.6,
121.7 (C-4, C-5, C-6); 109.9 (C-7); 106.9 (C-3); 50.9
(CH3O); 47.0 (C-1’); 31.8 (C-2’); 29.8, 29.5, 29.4, 29.2,
29.1, 26.8, 22.6 (C-3’ to C-9’); 14.1 (C-10’). HR ESI-MS
m/z: 316.2261 [M+H]+ (calcd for C20H30NO2, 316.2271,
D 3.1 ppm). EIMS (70 eV): m/z (%) 315 [M](100), 284
(18), 188 (64), 130 (42).
Compound 10. 1-(4-Acetoxy-butyl)-1H-indole-3- car-
boxylic acid methyl ester
Oil. IR (KBr, cm–1) vmax: 2954 (CH), 1735 (C=O),
1696 (C=O), 1241 (C-O). 1H NMR (CDCl3): d 8.20 (t,
1H, H-4); 7.82 (s, 1H, H-2); 7.34 (t, 1H, H-7); 7.29 (m,
2H, H-5, 6); 4.19 (t, J = 7.0 Hz, 2H, H-1’); 4.06 (t, J =
6.4 Hz, 2H, H-4’); 3.92 (s, 3H, CH3O); 2.03 (s, 3H,
CH3CO); 1.92 (qi, J = 7.0 Hz, 2H, H-2’); 1.63 (m, 2H,
H-3’). 13C NMR (CDCl3): d 170.9 (CH3CO); 165.3 (C-8);
136.4 (C-7a); 134.2 (C-2); 126.7 (C-3a); 122.7, 121.8,
121.7 (C-4, C-5, C-6); 109.9 (C-7); 106.9 (C-3); 63.7
(C-4’); 51.1 (CH3O); 46.6 (C-1’); 26.7, 26.1 (C-2’, C-3’);
20.9 (CH3CO). HR ESI-MS m/z: 290.1403 [M+H]+
(calcd for C16H20NO4, 290.1387, D -4.53 ppm). EIMS
(70 eV): m/z (%) 289[M](99), 258 (27), 188 (100),
144 (22).
Compound 11. 1-(2-Hydroxy-propyl)-1H-indole -3-
carboxylic acid
Mp 150˚C - 151˚C. IR (KBr, cm–1) vmax : 3342 (OH),
2923 (CH), 1549.1 (C=O). 1H NMR (CDCl3-CD3OD 5%):
d 8.20 (m, 1H, H-4); 7.73 (s, 1H, H-2); 7.36 (m, 1H, H-7);
7.12 (m, 2H, H-5, 6); 4.13 (m, 2H, H-2’); 4.10 (m, 2H,
H-1’); 1.16 (d, J = 6.0 Hz, 3H, H-3’). 13C NMR
(CDCl3-CD3OD 5%): d 174.1 (C-8); 138.1 (C-7a); 134.6
(C-2); 128.6 (C-3a); 122.7, 122.4, 121.2 (C-4, C-5, C-6);
114.1 (C-3); 110.5 (C-7); 67.3 (C-2’); 54.4 (C-1’); 20.9
(C-3’). HR ESI-MS m/z: 220.0979 [M+H]+ (calcd for
C12H14NO3, 220.0968, D -4.8 ppm). EIMS (70 eV): m/z
(%) 219 [M](27), 175(35), 174 (39), 130 (100).
Compound 12. 1-Decyl-1H-indole-3-carboxylic acid
Mp 87˚C - 88˚C. IR (KBr, cm–1) vmax: 3239 (OH), 2917
(CH), 1663 (C=O). 1H NMR (CDCl3): d 8.13 (m, 1H,
H-4); 7.80 (s, 1H, H-2); 7.27 (m, 1H, H-7); 7.18 (m, 2H,
H-5, 6); 4.02 (t, J = 7.1 Hz, 2H, H-1’); 1.76 (qi, J = 7.1
Hz, 2H, H-2’), 1.15-1.23 (m, 14H, H-3’ to H-9’); 0.78 (t,
J = 6.8 Hz, 3H, H-10’). 13C NMR (CDCl3): d 168.9 (C-8);
136.6 (C-7a); 135.1 (C-2); 126.9 (C-3a); 122.6, 121.8,
121.7 (C-4, C-5, C-6); 109.9 (C-7); 106.3 (C-3); 46.9
(C-1’); 31.7 (C-2’); 29.7, 29.3, 29.3, 29.1, 29.0, 26.7,
22.5 (C-3’ to C-9’); 13.9 (C-10’). HR ESI-MS m/z:
302.2128 [M+H]+ (calcd for C19H28NO2, 302.2115, D
-4.6 ppm). EIMS (70 eV): m/z (%) 301 [M](100), 256
(14), 174 (80), 130 (56).
Compound 13. 1-Decyl-1H-indole-3-carboxylic acid
decyl ester
Oil. IR (KBr, cm–1) vmax: 2929 (CH), 1707 (C=O). 1H
NMR (CDCl3): d 8.17 (m, 1H, H-4); 7.82 (s, 1H, H-2);
7.36 (m, 1H, H-7); 7.26 (m, 2H, H-5, 6); 4.32 (t, J = 6.6
Hz, 2H, H-1’’); 4.13 (t, J = 7.3 Hz, 2H, H-1’); 1.80-1.87
(m, 4H, H-2’, 2’’); 1.25-1.47 (m, 28H, H-3’ to H-9’,
H-3’’ to H-9’’); 0.88 (t, J = 7.3 Hz, 3H, H-10’*); 0.87 (t,
J = 7.1 Hz, 3H, H-10’’*). 13C NMR (CDCl3): d 165.4
(C-8); 136.6 (C-7a); 134.2 (C-2); 126.7 (C-3a); 122.5,
121.8, 121.7 (C-4, C-5, C-6); 109.9 (C-7); 107.3 (C-3);
63.9 (C-4’); 47.0 (C-1’); 31.9, 29.9, 29.5, 29.3, 29.2, 29.0,
26.9, 22.7 (C-3’ to C-9’, C-3’’ to C-9’’); 14.1 (C-10’,
C-10’’). HR ESI-MS m/z: 442.3689 [M+H]+ (calcd for
C29H48NO2, 442.3680, D -2.1 ppm). EIMS (70 eV): m/z
(%) 441 [M](100), 301 (17), 284 (23), 174 (15), 130
(13).* may be interchanged.
Compound 16. 1-(4-Bromo-butyl)-1H-indole-3-carboxy-
lic acid methyl ester
Oil. IR (KBr, cm–1) vmax: 2942 (CH), 1691 (C=O), 747
(CBr). 1H NMR (CDCl3): d 8.18 (m, 1H, H-4); 7.82 (s,
1H, H-2); 7.37 (m, 1H, H-7); 7.29 (m, 2H, H-5, 6); 4.20
(t, J = 6.9 Hz, 2H, H-1’); 3.91 (s, 3H, CH3O); 3.39 (t, J =
6.5 Hz, 2H, H-4’); 2.06 (m, 2H, H-2’); 1.88 (m, 2H,
H-3’). 13C NMR (CDCl3): d 165.6 (C-8); 136.6 (C-7a);
134.1 (C-2); 126.9 (C-3a); 123.0, 122.1 (C-5, C-6); 122.0
(C-4); 109.9 (C-7); 107.4 (C-3); 51.2 (CH3O); 46.3
Copyright © 2011 SciRes. AJPS
Analogs of Antifungal Indoles Isolated from Aporpium Caryae with Activity against
Sudden-Death Syndrome of Soybean
Copyright © 2011 SciRes. AJPS
254
(C-1’); 32.8 (C-4’); 29.9 (C-3’); 28.6 (C-2’). HR ESI-MS
m/z: 310.0440 [M+H]+ (calcd for C14H17BrNO2, 310.0437,
D -0.9 ppm). EIMS (70 eV): m/z (%) 311 [M+2](48),
309 [M](44), 280 (13), 278 (13), 188 (100), 55 (49).
Compound 18. 1-(4-Amino-butyl)-1H-indole-3-carboxy-
lic acid methyl ester
Oil. IR (KBr, cm–1) vmax: 3459, 3423 (NH2), 2917 (CH),
1680 (C=O). 1H NMR (CDCl3-CD3OD 5%): d 8.14 (m,
1H, H-4); 7.88 (s, 1H, H-2); 7.39 (m, 1H, H-7); 7.29 (m,
2H, H-5, 6); 4.24 (t, J = 6.8 Hz, 2H, H-1’); 3.91 (s, 3H,
CH3O); 2.90 (t, J = 7.6 Hz, 2H, H-4’); 1.97 (m, 2H, H-2’);
1.68 (m, 2H, H-3’). 13C NMR (CDCl3-CD3OD 5%): d
165.9 (C-8); 136.2 (C-7a); 134.4 (C-2); 126.4 (C-3a);
123.1, 122.2 (C-5, C-6); 121.9 (C-4); 110.0 (C-7); 106.9
(C-3); 51.2 (CH3O); 46.2 (C-1’); 39.4 (C-4’); 26.8 (C-2’);
25.2 (C-3’). HR ESI-MS m/z: 247.1449 [M+H]+ (calcd
for C14H19N2O2, 247.1441, D -3.0 ppm). EIMS (70 eV):
m/z (%) 246 [M](15), 214 (16), 188 (21), 144 (36), 43
(100).
Compound 19. 1-(4-Dimethylamino-butyl)-1H-indole-
3-carboxylic acid methyl ester
Oil. IR (KBr, cm–1) vmax: 2942 (CH), 1702 (C=O). 1H
NMR (CDCl3): d 8.18 (m, 1H, H-4); 7.83 (s, 1H, H-2);
7.37 (m, 1H, H-7); 7.28 (m, 2H, H-5, 6); 4.18 (t, J = 7.1
Hz, 2H, H-1’); 3.91 (s, 3H, CH3O); 2.40 (t, J = 7.5 Hz,
2H, H-4’); 2.27 (s, 6H, NCH3); 1.91 (m, 2H, H-2’); 1.55
(m, 2H, H-3’). 13C NMR (CDCl3): d 165.6 (C-8); 136.6
(C-7a); 134.3 (C-2); 126.9 (C-3a); 122.9, 122.0 (C-5,
C-6); 121.9 (C-4); 110.0 (C-7); 107.2 (C-3); 51.1 (CH3O);
58.3 (C-4’); 46.9 (C-1’); 44.7 (NCH3); 27.7 (C-2’); 24.3
(C-3’). HR ESI-MS m/z: 275.1741 [M+H]+ (calcd for
C16H23N2O2, 275.1754, D 4.7 ppm). EIMS (70 eV): m/z
(%) 274 [M](4), 188 (2), 58 (100).
Compound 20. 1,3-di-[(3’-methoxycarbonyl)-1H -indol-
1-yl] propane
Mp 176˚C - 177˚C. IR (KBr, cm–1) max: 2920 (CH),
1691 (C=O). 1H NMR (CDCl3): d 8.20 (m, 2H, H-4);
7.76 (s, 2H, H-2); 7.29 (m, 2H, H-5); 7.26 (m, 2H, H-6);
7.19 (m, 2H, H-7); 4.16 (t, J = 6.9 Hz, 4H, H-1’); 3.91 (s,
6H, CH3O); 2.50 (qi, J = 6.9 Hz, 2H, H-2’).
13C NMR (CDCl3): d 165.4 (C-8); 136.4 (C-7a); 133.8
(C-2); 126.9 (C-3a); 123.3 (C-6); 122.4 (C-5); 122.2
(C-4); 109.8 (C-7); 107.9 (C-3); 51.2 (CH3O); 43.9
(C-1’); 29.8 (C-2’). HR ESI-MS m/z: 391.1669 [M+H]+
(calcd for C23H23N2O4, 391.1652, D -4.2 ppm). EIMS (70
eV): m/z (%) 390 [M](24), 359 (6), 189 (50), 188 (15),
130 (100).