Methyl and p-Bromobenzyl Esters of Hydrogenated Kaurenoic Acid for Controlling Anthracnose in Common Bean Plants

Article abstract of DOI:10.1021/acs.jafc.6b05159

Kaurenoic acid derivatives were prepared and submitted to in vitro assays with the fungus Colletotrichum lindemuthianum, which causes anthracnose disease in the common bean. The most active substances were found to be methyl and p-bromobenzylesters, 7 and

Full text of DOI:10.1021/acs.jafc.6b05159

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Article
Methyl and p-Bromobenzyl Esters of Hydrogenated Kaurenoic
Acid for Controlling Anthracnose in Common Bean Plants
Suellen F. Mota, Denilson Ferreira Oliveira, Vladimir Constantino Gomes Heleno,
Ana Carolina Ferreira Soares, Jacob Ogweno Midiwo, and Elaine A. Souza
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05159 • Publication Date (Web): 05 Feb 2017
Downloaded from http://pubs.acs.org on February 8, 2017
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Journal of Agricultural and Food Chemistry
Methyl and p-Bromobenzyl Esters of Hydrogenated Kaurenoic Acid for Controlling
Anthracnose in Common Bean Plants
Suellen F. Mota,† Denilson F. Oliveira,*,‡ Vladimir C. G. Heleno,§ Ana Carolina F. Soares, § Jacob
O. Midiwo,§§ and Elaine A. Souza†
† Departamento de Biologia, Universidade Federal de Lavras, LavrasꢀMG, CEP 37.200ꢀ000, Brazil
‡ Departamento de Química, Universidade Federal de Lavras, LavrasꢀMG, CEP 37.200ꢀ000, Brazil
§ Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, FrancaꢀSP, CEP
14.404ꢀ600, Brazil
§§ Department of Chemistry, University of Nairobi, Nairobi 00100, Kenya
*Fax number: 55ꢀ35ꢀ3829ꢀ1271
Telephone number: 55ꢀ35ꢀ3829ꢀ1623
Eꢀmail: denilson@dqi.ufla.br
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ABSTRACT: Kaurenoic acid derivatives were prepared and submitted to in vitro assays with the
fungus Colletotrichum lindemuthianum, which causes anthracnose disease in the common bean.
The most active substances were found to be methyl and pꢀbromobenzylesters, 7 and 9 respectively,
of the hydrogenated kaurenoic acid, which presented a minimum inhibitory concentration (MIC) of
0.097 and 0.131 mM, respectively, while the commercial fungicide methyl thiophanate (MT)
presented a MIC of 0.143 mM. Substances 7 (1.401 mM) and 9 (1.886 mM) reduced the severity of
anthracnose in common bean to values statistically comparable to MT (2.044 mM). According to an
in silico study, both compounds 7 and 9 are inhibitors of the ketosteroid isomerase (KSI) enzyme
produced by other organisms, the amino acid sequence of which could be detected in fungal
genomes. These substances appeared to act against C. lindemuthianum by inhibiting its KSI.
Therefore, substances 7 and 9 are promising for the development of new fungicides.
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KEYWORDS: Colletotrichum lindemuthianum, entꢀkauranꢀ18ꢀoic acid methyl ester, entꢀkauranꢀ
18ꢀoic acid pꢀbromobenzyl ester, ketosteroid isomerase, fungicide
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INTRODUCTION
The common bean (Phaseolus vulgaris L.) is a basic food source in developing countries, as it
provides part of the necessary dietary protein, carbohydrate and iron, especially for lowꢀincome
populations.1 According to the Food and Agriculture Organization of the United Nations, about
22.5 million tons of this bean was produced in 2013.2 However, this value could have been much
higher if problems like those caused by phytopathogenic fungi could be better controlled. One of
the fungal diseases of major economic importance for the production of common bean is
anthracnose, which can cause production losses of up to 100%.1 This disease is caused by the
fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, which has wide
pathogenic variability. Therefore, the development of strains resistant to this pathogen is very
difficult.3 Consequently, commercial fungicides are currently the main method used to control
anthracnose in common bean plants.4 However, the continued use of such fungicides may result in
the emergence of resistant fungus populations, and the harmful substances used can contaminate
human populations and the environment.5 As a result, there is growing demand for new products
that more efficiently control anthracnose with fewer unwanted side effects.6
One possibility to circumvent the problems associated with the use of commercial
fungicides is the development of naturallyꢀderived substances as fungicides. Kaurenoic acid appears
to be an interesting starting point, as it has demonstrated in vitro activity against the fungi
Aspergillus nidulans G. Winter,7 Sclerotinium sclerotiorum (Lib.) de Bary, Verticillium dahlia
Kleb.,8 Trichophyton rubrum(Castell.) Sabour. and Trichophyton mentagrophytes Priestley and
Epidermophyton floccosum Langeron et Milochevitch.9 Moreover, this diterpene is known to
present several other activities, such as antimicrobial, antiꢀinflammatory, cytotoxic and
trypanocidal.9 However, there do not appear to be any reports in the literature regarding the activity
of this substance or its derivatives against C. lindemuthianum. Therefore, to assist in the
development of new fungicides to control anthracnose in the common bean, the present study aimed
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to prepare kaurenoic acid derivatives, then screen these substances for their in vitro antifungal
activity against C. lindemuthianum. An assay on common bean plants was then carried out with the
most active substances to determine their ability to reduce anthracnose severity. Finally, an in silico
study was performed to identify the enzyme in the fungus targeted by the most active substance.
MATERIALS AND METHODS
Instruments. Nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 (concentration
of the substances ranged from 8–16 mg/mL) at 298 K on an Avance DRX 400 spectrometer
(Bruker, Billerica, MA) (400 MHz for 1H and 100 MHz for 13C), using tetramethylsilane (TMS) as
the internal standard. Infrared (IR) spectra were obtained using an FTꢀIR Spectrum Two
spectrometer (PerkinꢀElmer, Waltham, MA). The optical rotations were determined with a Pꢀ2000
digital polarimeter (Jasco, Portland, OR). High resolution mass spectrometry (HRMS) analyses
were performed using a micrOTOFꢀQH hybrid instrument (Bruker, Billerica, MA). Melting points
were measured with a FisherꢀJohns melting point apparatus equipped with a 10ꢀ250 °C
thermometer, coupled to a monocular microscope.
Isolation of compounds from plants. entꢀkaurꢀ16ꢀenꢀ19ꢀoic acid, 1, was isolated from a
commercial sample of Mikania glomerata Spreng.,10 whereas entꢀ16,17ꢀdihydroxykauranꢀ20ꢀoic
acid, 15, and entꢀ18,19ꢀdihydroxykaurꢀ16ꢀenꢀ2ꢀone, 16, were isolated from the African plant
Psiadia punctulata (DC.) Vatke.11 For compound 1, dried and pulverized leaves of M. glomerata
(1.0 kg) were extracted with CH2Cl2 (3.5 L) at room temperature to give 42.0 g of crude extract.
This extract was suspended in 300 mL of a MeOH/H2O (9:1, v/v) solution, to result in a suspension
that was filtered and partitioned with hexane (4x150 mL). The hexane fraction (6.0 g) was then
fractionated by silica gel 60 (0.070ꢀ0.230 mm) column chromatography (hexane with increasing
amounts of EtOAc). The second fraction (1.5 g) was washed with cold MeOH to afford 800 mg of
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1. To obtain compounds 15 and 16, fresh leaves of P. punctulata (394 g) were extracted with a 2 L
portion of acetone to give 22.0 g of crude extract. This extract was subjected to column
chromatography over silica gel 60 (0.070ꢀ0.230 mm) using hexane/CH2Cl2 and CH2Cl2/MeOH with
constant increase of polarity. This process furnished 150 mg of 15 and 290 mg of 16.
Semi-synthetic preparation of ent-16-methoxykauran-19-oic acid, 2. Compound 2 was
obtained from 3.0 mL of a methanolic solution of compound 1 (50.0 mg), which was stirred at room
temperature for 18 h in the presence of four drops of concentrated H2SO4. The reaction mixture was
poured into 20 mL water and extracted with EtOAc (3x20 mL). Solvent was removed under
reduced pressure to afford 35.0 mg of 2.12
Hydrogenation of the C16-C17 double bond of ent-kaur-16-en-19-oic acid, 1. In the
vessel of a hydrogenation apparatus, 200 to 300 mg of compound 1 was dissolved in 20 mL of
absolute ethanol. To this mixture, a catalytic amount of palladium on activated carbon was added.
The vessel containing the reaction mixture was positioned inside the reactor, which was then
carefully and tightly closed. The atmosphere inside the reactor was changed to hydrogen, and
pressure was set to 4 atm. After it had been kept for 2 h at room temperature, the reaction mixture
was filtered through Celite® 545 (Synth, Diadema, SP, Brazil), and the solvent was removed by
rotary evaporation. entꢀkauranꢀ19ꢀoic acid, dihydro-1, was used directly without further
purification.
General procedure for the preparation of esters 3 to 14. The procedure used was based
on the method published by Boeck and coꢀworkers,9 with some slight modifications in order to use
compounds 1, 2 and entꢀkauranꢀ19ꢀoic acid, dihydro-1, as the starting materials. Initially, 0.032 g of
KOH was added to 2.0 mL of anhydrous acetone, and the resulting mixture was stirred for 5 min at
25ºC. The substrate (0.05 g) was then added to this mixture, followed by the addition of alkyl halide
(0.17 mmol). The reaction mixture was stirred at 25ºC. This mixture was then poured into 20 mL
H2O, and extracted with EtOAc (3x20 mL). The combined organic extracts were washed three
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times with 10 mL water, dried over anhydrous MgSO4, filtered, then concentrated to dryness by
rotary evaporation. The product was purified by silica gel 60 (15.0 g, 0.070ꢀ0.230 mm) column
chromatography, using a hexane/EtOAc gradient (100% hexane; 8:2; 6:4; 1:1; 4:6; 2:8; 100%
EtOAc) as the eluent. This fractionation was monitored by thin layer chromatography. Reaction of 1
with iodomethane (2 h); benzyl bromide (2 h); bromobutane (2.5 h); or pꢀCl benzyl bromide (25 h)
gave the esters 3ꢀ6 in 55, 75, 56, and 34% yields, respectively. Reaction of dihydroꢀ1 with
iodomethane (3.5 h); benzyl bromide (4 h); or pꢀBr benzyl bromide (23 h) gave the esters 7ꢀ9 in 42,
64, and 33% yields, respectively. Reaction of 2 with iodomethane (3.5 h); benzyl bromide (2.5 h);
bromobutane (2 h); pꢀBr benzyl bromide (20 h); or pꢀCl benzyl bromide (25 h) gave the esters 10ꢀ
14 in 54, 33, 44, 26, and 27% yields, respectively. Only the spectroscopic data for the six new
diterpenes (8, 9 and 11–14) are presented below. The NMR data for the known diterpenes (1–7, 10,
15 and 16) were compared to previously published NMR data.9,11,13–16
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Kauranꢀ19ꢀoic acid benzyl ester, 8. Pale yellow oil; [α]24 = ꢀ61.4 (c 0.03, CH2Cl2); IR
D
(neat) νmax 2932, 2855, 1724, 1464, 1144, 696 cmꢀ1; 1H NMR (400 MHz, CDCl3) δ 7.40ꢀ7.27 (m, 5,
HꢀAr), 5.12 (d, 1, J = 12.4 Hz, Hꢀ1'a), 5.02 (d, 1, J = 12.4 Hz, Hꢀ1'b), 2.20ꢀ0.70 (m, 22, CH and
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CH2 groups), 1.18 (s, 3, CH3ꢀ18), 0.98 (d, 3, J = 7.1 Hz, CH3ꢀ17), 0.78 (s, 3, CH3ꢀ20); C NMR
(100 MHz, CDCl3) δ 177.4, 136.3, 128.4, 128.2, 128.0, 65.9, 57.3, 56.5, 48.7, 44.8, 44.0, 42.2, 40.8,
40.7, 40.0, 39.5, 38.2, 34.4, 28.9, 25.9, 22.2, 19.2, 19.0, 15.6, 15.5; HRMS m/z 417.2722 [M + Na]+
(calculated for C27H38O2Na, m/z 417.2769).
Kauranꢀ19ꢀoic acid pꢀbromobenzyl ester, 9. White solid; melting point 110ºC; [α]24 = ꢀ
D
40.2 (c 0.006, CH2Cl2); IR (KBr) νmax 3414, 2923, 2858, 1907, 1716, 1590, 1462, 1368, 1156, 1012,
842, 799, 534 cmꢀ1; 1H NMR (400 MHz, CDCl3) δ 7.50 (m, 2, HꢀAr), 7.25 (m, 2, HꢀAr), 5.10 (d, 1,
J = 12.6 Hz, Hꢀ1'a), 4.99 (d, 1, J = 12.6 Hz, Hꢀ1'b), 2.20ꢀ0.70 (m, 22, CH and CH2 groups), 1.17 (s,
13
3, CH3ꢀ18), 1.00 (d, 3, J = 7.1 Hz, CH3ꢀ17), 0.77 (s, 3, CH3ꢀ20); C NMR (100 MHz, CDCl3) δ
177.4, 136.3, 128.4, 128.2, 128.0, 65.9, 57.3, 56.5, 48.7, 44.8, 44.0, 42.2, 40.8, 40.7, 40.0, 39.5,
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38.2, 34.4, 28.9, 25.9, 22.2, 19.2, 19.0, 15.6, 15.5; HRMS m/z 473.2022 [M + H]+ (calculated for
C27H37BrO2H, m/z 473.2055).
16ꢀMethoxykauranꢀ19ꢀoic acid benzyl ester, 11. Pale yellow oil; [α]24 = ꢀ45.0 (c 0.007,
D
1
CH2Cl2); IR (neat) νmax 2934, 1721, 1454, 1371, 1234, 1144, 1074, 753, 698 cmꢀ1; H NMR (400
MHz, CDCl3) δ 7.40ꢀ7.27 (m, 5, HꢀAr), 5.14 (d, 1, J = 12.5 Hz, Hꢀ1'a), 5.07 (d, 1, J = 12.5 Hz, Hꢀ
1'b), 3.12 (s, 3, OCH3), 2.20ꢀ0.70 (m, 21, CH and CH2 groups) 1.27 (s, 3, CH3ꢀ17), 1.16 (s, 3, CH3ꢀ
13
18), 0.82 (s, 3, CH3ꢀ20); C NMR (100 MHz, CDCl3) δ 177.3, 136.2, 128.4, 128.2, 128.0, 83.9,
65.9, 57.1, 56.0, 54.5, 49.1, 44.7, 43.9, 43.3, 42.1, 40.7, 39.5, 38.1, 37.0, 28.8, 26.6, 22.1, 19.1,
18.4, 18.2, 15.5; HRMS m/z 447.2888 [M + Na]+ (calculated for C28H40O3Na, m/z 447.2875).
16ꢀMethoxykauranꢀ19ꢀoic acid butyl ester, 12. White solid; melting point 51ºC; [α]24 = ꢀ
D
74.4 (c 0.01, CH2Cl2); IR (KBr) νmax 3431, 2936, 1724, 1468, 1377, 1229, 1160, 1073, 947, 773,
587 cmꢀ1; 1H NMR (400 MHz, CDCl3) δ 4.06 (dt, 1, J1 = 10.8 Hz, J2 = 6.6 Hz, Hꢀ1'a), 4.00 (dt, 1, J1
= 10.8 Hz, J2 = 6.6 Hz, Hꢀ1'b), 3.12 (s, 3, OCH3), 2.20ꢀ0.70 (m, 25, CH and CH2 groups), 1.27 (s, 3,
13
CH3ꢀ17), 1.16 (s, 3, CH3ꢀ18), 0.95 (t, 3, J = 7.4 Hz, CH3ꢀ4'), 0.85 (s, 3, CH3ꢀ20); C NMR (100
MHz, CDCl3) δ 177.7, 83.9, 63.9, 57.0, 56.0, 54.5, 49.1, 44.7, 43.8, 43.3, 42.1, 40.8, 39.5, 38.1,
37.1, 30.6, 28.9, 26.6, 22.1, 19.5, 19.1, 18.4, 18.2, 15.5, 13.7; HRMS m/z 413.3008 [M + Na]+
(calculated for C25H42O3Na, m/z 413.3032).
16ꢀMethoxykauranꢀ19ꢀoic acid pꢀbromobenzyl ester, 13. Pale yellow oil; [α]24 = ꢀ48.6 (c
D
1
0.01, CH2Cl2); IR (neat) νmax 2934, 1725, 1594, 1465, 1370, 1143, 1073, 1012, 804, 530 cmꢀ1; H
NMR (400 MHz, CDCl3) δ 7.47 (m, 2, HꢀAr), 7.23 (m, 2, HꢀAr), 5.09 (d, 1, J = 12.5 Hz, Hꢀ1'a),
5.00 (d, 1, J = 12.5 Hz, Hꢀ1'b), 3.12 (s, 3, OCH3), 2.20ꢀ0.70 (m, 21, CH and CH2 groups), 1.26 (s, 3,
CH3ꢀ17), 1.17 (s, 3, CH3ꢀ18), 0.77 (s, 3, CH3ꢀ20); 13C NMR (100 MHz, CDCl3) δ 177.1, 135.1,
131.6, 130.0, 122.1, 83.9, 65.2, 57.0, 55.9, 54.5, 49.1, 44.7, 43.9, 43.3, 42.1, 40.6, 39.5, 38.1, 37.0,
28.8, 26.6, 22.2, 19.1, 18.4, 18.2, 15.5; HRMS m/z 525.1930 [M + Na]+ (calculated for
C28H39BrO3Na, m/z 525.1980).
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16ꢀMethoxykauranꢀ19ꢀoic acid pꢀclorobenzyl ester, 14. White solid; melting point 71ºC;
[α]24 = ꢀ55.7 (c 0.02, CH2Cl2); IR (KBr) νmax 3431, 2932, 1720, 1599, 1494, 1466, 1369, 1228,
D
1
1072, 1015, 808, 534, 426 cmꢀ1; H NMR (400 MHz, CDCl3) δ 7.52 (m, 2, HꢀAr), 7.31 (m, 2, Hꢀ
Ar), 5.10 (d, 1, J = 12.5 Hz, Hꢀ1'a), 5.02 (d, 1, J = 12.5 Hz, Hꢀ1'b), 3.12 (s, 3, OCH3), 2.20ꢀ0.70 (m,
13
21, CH and CH2 groups), 1.26 (s, 3, CH3ꢀ17), 1.17 (s, 3, CH3ꢀ18), 0.77 (s, 3, CH3ꢀ20); C NMR
(100 MHz, CDCl3) δ 177.1, 131.7, 130.0, 129.7, 128.7, 83.9, 65.1, 57.1, 55.9, 54.6, 49.1, 44.7, 43.9,
43.3, 42.1, 40.7, 39.5, 38.1, 37.0, 28.8, 26.6, 22.1, 19.1, 18.4, 18.2, 15.5; HRMS m/z 481.2457 [M +
Na]+ (calculated for C28H39ClO3Na, m/z 481.2485).
Inoculum of Colletotrichum lindemuthianum. Monosporic strains belonging to races 65
(LV134) and 89 (LV228), which had been preserved in M3 culture medium (10 g sucrose, 20 g
agar, 2 g KH2PO4, 1 g MgSO4.7H2O, 6 g peptone, 1 g yeast extract and 1 L water) at 22°C in the
dark, were used in the present study. Each strain was inoculated in bean pods that were maintained
in M3 medium, then incubated at 22°C for 15 days in the dark. Conidial suspensions were obtained
after adding 7 mL of sterile water and filtering the suspension through Miracloth. The concentration
of the final suspension was adjusted to 1.2 × 106 conidia/mL.
In vitro assay. Kaurenoic acid and its derivatives were dissolved in DMSO to a
concentration of 20,000 µg/mL, then further diluted to 1000 µg/mL with M3S culture medium (10 g
sucrose, 2.7 g KH2PO4, 2.5 g MgSO4.7H2O, 1 g peptone, 1 g yeast extract and 1 L water). Aliquots
(100 µL) of each resulting solution and an aqueous suspension (100 µL) containing 1.2 × 106 C.
lindemuthianum conidia/mL were poured into each cell (350 µL) of a 96ꢀcell polypropylene plate.
Each experiment was carried out with three replicates, with 55 mg/mL DMSO in M3S culture
medium and 0.7 mg/mL methyl thiophanate (a fungicide marketed under the name Cerbonin 700
WP) as the negative and positive controls, respectively. To obtain the commercial fungicide
solution, the methyl thiophanate was dissolved in DMSO to a concentration of 55 mg/mL, then
diluted with M3S. Following 72 h under constant shaking (110 rpm) at 22°C, substances that
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appeared to prevent fungal development from visual observation were considered to be active. This
study was carried out separately for both fungal strains 65 (LV134) and 89 (LV228).
Minimum inhibitory concentration. Adapting the method described in the literature17
solutions of the substances (1000 µg/mL) found to be active against both strains of the fungus in the
in vitro assay (7, 9 and 13) were prepared as described above for use in twoꢀfold serial dilutions, in
order to obtain solutions with concentrations ranging from 3.90ꢀ1000 µg/mL. All dilutions were
performed with M3S culture medium in cells (350 µL) of a 96ꢀcell polypropylene plate. To 100 µL
of each resulting solution, 100 µL of an aqueous suspension containing 1.2 × 106 C.
lindemuthianum conidia/mL was added. This experiment was carried out with three replicates,
using DMSO (55 mg/mL) dissolved in M3S and the commercial fungicide methyl thiophanate
(final concentrations ranging from 15.3 to 980 µg/mL) as controls. Plates were kept under constant
shaking (110 rpm) for 72 h at 22°C. The minimum inhibitory concentration (MIC) was defined as
the lowest concentration of each substance that prevented the fungal development, according to
visual observation. This study was carried out with both 65 (LV134) and 89 (LV228) fungal strains.
In vivo bioassay. Only substances 7 and 9 were included in this experiment, which followed
a completely randomized design with five replicates, each plot corresponding to a 1 L pot
containing a 15ꢀday old common bean plant (Phaseolus vulgaris 'Pérola') known to be susceptible
to anthracnose. The first trifoliate (phenological stage V3) developed by each plant was inoculated.
The substances were dissolved in an aqueous 0.01 g/mL Tween 80® solution to the concentration
(C) calculated according to the following: C = (A × B)/D. Where A represents the MIC of the
substance; B is the concentration of methyl thiophanate (positive control) used in this experiment;
and D is the MIC of methyl thiophanate. Three controls were used in this study: water, aqueous
0.01 g/mL Tween 80® solution, and methyl thiophanate (Cerbonin 700 WP) at a concentration of
0.7 mg/mL. All solutions (1.3 mL) were applied to plant leaves with a brush, and after 5 h an
aqueous suspension containing 1.2 × 106 C. lindemuthianum (race 65) conidia/mL was sprayed onto
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leaves until they began to drip. Plants were maintained in a greenhouse at 23°C under 80% relative
humidity. The anthracnose severity was evaluated 7 days after fungal inoculation using the scoring
scale developed by Schoonhoven and PastorꢀCorrales.18 The resulting values were statistically
analyzed by analysis of variance (ANOVA), and means were compared using the ScottꢀKnott test
(P ≤ 0.05),19 using R® software. 20
Enzymes complexed to ligands similar to the methyl and p-bromobenzyl esters of
hydrogenated kaurenoic acid. The structures of compounds 7 and 9 (Figure 1) were submitted to
a conformational search using Open3Dalign 2.103 software.21 For each chemical structure, 1000
molecular dynamics simulations were carried out at 1000 K, with a 1 femtosecond (fs) time step for
1 picosecond (ps) using the MMFF94 force field, and treating the solvent (water) implicitly using
the Generalized Bornꢀsurface area model (GBSA). The most stable conformation of each substance,
and those up to 10 kcal/mol more energetic than the most stable conformation, were optimized with
MOPAC 2012 software,22 using the Hamiltonian PM7 and treating the solvent (water) implicitly.
The most stable conformation for each substance was used in a pharmacophoric search using Alignꢀ
it 1.0.4 software.23 The database used for this search was the Ligand Expo database,24 which was
downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data
bank. The protein structures found to be complexed to ligands with Tanimoto scores24 equal to or
above 0.50 were selected for use in the subsequent analyses.
Ketosteroid isomerase. The pdb and fasta files for ketosteroid isomerases were downloaded
from the RCSB protein data bank,25 and their amino acid sequences were aligned using the Ugene
1.12.3 software26 with the ClustalO 1.2.0 algorithm,27 using the default parameters. Following this
step, the amino acid sequences of the ketosteroid isomerases 1BUQ, 1C7H, 2Z76, 3D9R, 3F8X,
3HX8, 3KE7, and 4CDL25 were used to search for similar amino acid sequences of fungi in the
nonꢀredundant (nr) database of the National Center for Biotechnology Information (NCBI), using
the software BLASTP 2.2.30+28,29 with DELTAꢀBLAST30 set to the default parameters. The amino
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acid sequences of the aboveꢀmentioned enzymes downloaded from the RCSB protein data bank and
NCBI databases with query coverage and identity higher than 90% and 20%, respectively,
underwent alignment using the ClustalO 1.2.0 algorithm, as described above.
Docking of substances to ketosteroid isomerases. Each of the substances presented in
Figure 3 were used in this step of the study, which was initially carried out by determining their
chemical structures, as described above for compounds 7 and 9. The 20 conformations of the 1ISK
enzyme31 were separated using OpenBabel 2.3.0 software,32 then the Makemultimer.py Python
script was used to generate a pdb file containing the homodimeric KSI enzyme in the 8CHO
complex.33 The resulting 21 tridimensional structures, as well as those from the enzymes 1E3V34
and 1OH0,35 were aligned using the software Lovovalign 1.1.0,36 underwent hydrogen additions
with VMD 1.9.2b1,37 and were converted to the pdbqt format using the Autodock Tools 1.5.6 rc2
software,38 which was also used to select the grid box (26.25 × 27.75 × 26.25 Å) centered over the
binding sites of the original ligand (deoxycholic acid) in the 1E3V complex.34 Each of the
substances presented in Figure 3 were converted to pdbqt files using the Autodock Tools 1.5.6 rc2
software, then docked to the enzymes using the software Autodock Vina 1.1.2,39 for which the
default values for most parameters were used, except for the exhaustiveness parameter which was
set to 128. The pdbqt files of the enzymes and the grid box (grid spacing = 0.375 Å) were used to
calculate the atomic affinity potential for each atom type with the Autogrid 4.2.3 software.38 The
results of this calculation were used by the Autodock 4.2.3 software38 to dock each of the
substances to the enzymes, with parameters set to the values described in the supporting
information. All files for the enzymes and substances were corrected using Spores software,40 then
submitted to docking using the Plants 1.2 software,41 using the default parameters. Regions within
15 Å of the binding site center of the original ligand (deoxycholic acid) in the 1E3V complex34
were used for docking. Using the same griding box described for docking with Autodock Vina
software, substances were also docked to the enzymes using Paradocks 1.0.1 software,42 using the
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default parameters. Interactions of ligands with the enzymes were visually depicted using the
software VMD 1.9.2b1,37 Rasmol 2.7.5,43 Pymol (The PyMOL Molecular Graphics System, version
1.4.1, Schrödinger, LLC, New York, NY) and LigProt+ 1.4.5.44
RESULTS AND DISCUSSION
The natural compound 1 was easily isolated from the natural source, as noted in the literature,10 for
use as the starting point for the synthesis of compounds 2ꢀ14 through esterification at the carboxyl
group, hydrogenation of the C16ꢀC17 double bond, or methoxyl addition to C16. The methoxyl
addition and hydrogenation reactions produced quantitative amounts of products that could be
easily separated and purified from the reaction mixtures. Although the esterification reaction has
been previously described in the literature,9 this proved to be the most challenging reaction,
requiring additional analysis to determine the best reaction time for each alkyl halide used. Among
the 13 synthesized compounds, six were new compounds (8, 9 and 11–14), for which the NMR and
mass spectrometric data obtained was consistent with their chemical structures. Regarding
compounds 15 and 16, these were able to be easily isolated from the natural sources, similar to that
described in the literature,11 for direct use in the present study.
Among the 16 substances screened for antifungal activity at 500 µg/mL, compounds 7, 9
and 13 were found to inhibit both strains of C. lindemuthianum (races 65 and 89). Substances 1–4,
11, 14 and 15 inhibited only one of the strains, while all remaining compounds tested presented no
antifungal activity. As compound 7 was found to have higher activity than compound 3,
hydrogenation of the carbonꢀcarbon double bond appeared to increase the antifungal activity,
however, the opposite effect was observed for compounds 8 and 4. Esterification of compound 1
appeared to reduce the activity, as observed in compounds 5 and 6, although protecting the carboxyl
group with a methyl or a benzyl group, 3 and 4 respectively, did not appear to have any effect on
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the fungus. Interestingly, when compound 2 was converted to 10, its activity vanished. Likewise, by
making R3 equal to OMe, the activity of 11 increased in relation to 8, however, the opposite effect
was observed when 9 and 13 were compared. Therefore, rationalizing the effect of these
derivatizations on the antifungal activity is not simple.
As the derivatization increased the antifungal activity of compounds 7, 9 and 13 in relation
to kaurenoic acid, 1, these substances were selected to progress to the next step of the study, which
consisted of assays at different concentrations to calculate their MIC values, which were found to be
0.097, 0.131 and 0.496 mM, respectively. Under these same conditions, the commercial fungicide
methyl thiophanate (Cerbonin 700 WP) presented a MIC of 2.044 mM. As compounds 7 and 9 had
the lowest MIC values, these compounds were submitted to an assay with common bean plants
inoculated with C. lindemuthianum. Both of them reduced the anthracnose severity to values that
were not statistically different from those obtained for the commercial fungicide methyl thiophanate
(Figure 2). Therefore, both substances 7 and 9 can be considered promising for the development of
new fungicides to control anthracnose in the common bean.
No reports regarding the fungicidal properties of compounds 7 and 9 could be found in the
literature, therefore, an in silico study was carried out to identify the target enzyme of these
substances in the fungus. This study was performed with both diastereoisomers of each substance,
as the asymmetric carbon formed during the hydrogenation process could be either R or S. The
pharmacophore search using the most stable conformations of these compounds resulted in the
selection of five proteins with Tanimoto scores24 of equal to or greater than 0.50, among which only
one report suggested an enzyme important for fungi, identified to be a ketosteroid isomerase
(KSI).45 This enzyme, also known as 3ꢀoxoꢀꢁ5ꢀsteroid isomerase, catalyzes the isomerization of a
wide variety of 3ꢀoxoꢀꢁ5ꢀsteroids to their ꢁ4ꢀconjugated isomers. It is one of the fastest enzymes
known, with a reaction rate close to the diffusion limit.46 Unfortunately, no tridimensional structure
for a KSI produced by fungi could be found in the RCSB protein data bank.25 Therefore, the amino
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acid sequence of other KSIs, mainly produced by bacteria, were used to search the genome of fungi,
resulting in the selection of 24 similar sequences found to be produced by several fungi. These
sequences were very similar to those of KSIs deposited in the RCSB protein data bank (1BUQ,
1C7H, 2Z76, 3D9R, 3F8X, 3HX8, 3KE7 and 4CDL), especially those with accession numbers
ENH79813.1, KGO40240.1 and EFX01891.1 (similarities ≥ 70%), which corresponded to KSI
enzymes from the fungi Colletotrichum orbiculare, Penicillium expansum and Grosmannia
clavigera, respectively. Although these results are not sufficient to confirm the importance of KSIs
to fungi known to be pathogenic to plants, the ability of these microorganisms to produce such
enzymes is evident. Furthermore, owing to the aboveꢀmentioned similarity in amino acid sequences,
the corresponding tridimensional structures were expected to be similar. Therefore, KSIs produced
by other organisms appear to be good models for studying the same enzymes produced by fungi.
Although more than 60 tridimensional structures of KSIs have been deposited in the RCSB
protein data bank, only a few have no missing residues or atoms close to the active site, or
mutations that can interfere with their biological activity. Therefore, only those enzyme structures
that did not present with either of these problems were selected for molecular docking, which was
carried out with both diastereoisomers of compounds 7 (7-S and 7-R) and 9 (9-S and 9-R),
48–50
inhibitors of KSIs (17–24)47 and antifungal substances (1 and 25–30)7,8,
with chemical
structures similar to 7 or 9 (Figure 3). Although the results obtained from the four computer
programs used for molecular docking did not completely agree (Figure 3), there was no significant
difference observed between the diastereoisomers of compounds 7 or 9, suggesting that both of
these present the same biological activity. Furthermore, according to all calculations, compound 9
was found to bind more strongly to KSIs than any other substance evaluated in the present study,
including the enzyme inhibitors. Thus, compound 9 is a good candidate for use as a KSI inhibitor.
Regarding compound 7, its affinity for the KSI enzymes was not as high as expected, as the
MIC value was lower than that observed for 9. However, the results for 7 were not so different from
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those obtained for the KSI inhibitors (17–24).47 According to calculations carried out with
Autodock 4.2.338 and Plants 1.2,41 the affinity of 7 for the enzymes did not differ significantly from
those calculated for 23 and 24. Therefore, compound 7 remains a potential KSI inhibitor. Similar
behavior was observed for some of the antifungal substances studied, among which compound 2648
was shown to have the highest enzyme affinities. Thus, it is possible that these substances act
against fungi by inhibiting their KSIs. However, for kaurenoic acid (1) the calculations obtained
from the computational programs Plants, Autodock and Paradocks,42 indicated very low affinities of
this substance for the enzymes, suggesting that this compound does not act against fungi by
inhibiting their KSIs. This result seems to be consistent with the previously demonstrated
association between the antifungal activity of compound 1 and its ability to interfere with the
intracellular Ca2+ gradient in fungal cells.7
It appeared that the affinities of the substances tested for amino acid residues in the active
sites of KSIs were predominantly hydrophobic interactions, as can be exemplified by substance 9.
The oxygen atoms of the carboxyl group did not even form hydrogen bonds with the amino acid
residues of the enzyme. A similar situation was observed for substance 7, for which many of the
hydrophobic interactions with the enzyme were more similar to those observed for substance 9.
Even kaurenoic acid, 1,7–9 presented no clear polar interaction of its carboxylate ion with the amino
acid residues of the enzyme, which may explain the lower calculated binding affinity for this
substance. These results suggest that replacing the carboxylate group of 9 with a less polar
functional group between the pꢀbromobenzyl group and the diterpene ring should increase the
binding affinity. Similarly, the presence of a polar group located further inside the active site of the
enzyme, such as the carboxylate ion in abietic acid, 25,48 can be a disadvantage for favorable
interactions between the ligand and the enzyme, however, a carbonyl group, like that in
progesterone, 17,47 can form a hydrogen bond with the Tyr14 amino acid residue, thereby
increasing the binding affinity.
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In conclusion, hydrogenation of kaurenoic acid, 1, and esterification with methyl or pꢀ
bromobenzyl groups resulted in two substances, 7 and 9 respectively, with demonstrated in vitro
and in vivo activity against the fungus C. lindemuthianum, which were comparable to those
observed for the commercial fungicide methyl thiophanate. Therefore, both substances 7 and 9 are
promising for the development of new fungicides. According to our in silico study, these
compounds inhibit the ketosteroid isomerase enzyme, the amino acid sequence of which is very
similar to sequences extracted from fungi genomes. Therefore, these substances appear to act
against C. lindemuthianum by inhibiting the ketosteroid isomerase enzyme.
ASSOCIATED CONTENT
Supporting information
Spectroscopic data for the known substances (1–7, 10, 15 and 16) studied (Item S1). Docking
parameter file for Autodock 4.2.3 (Item S2). Amino acid sequences in the fungi genome that are
similar to ketosteroid isomerases (Tables S1–S2); 2D and 3D representations of the interactions of
ligands docked to the ketosteroid isomerase enzyme from Comamonas testosterone (Figures S1–
S17). This material is available free of charge from http://pubs.acs.org.
FUNDING SOURCES
Financial support and fellowships were provided by Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (FAPEMIG), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP),
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq).
NOTES
The authors declare no competing financial interest.
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schematic diagrams of proteinꢀligand interactions. Protein Eng. 1995, 8, 127–134.
(45) Hunter, C. A.; Coyle, E.; Morse, F.; Dedi, C.; Dodd, H. T.; Koussoroplis, S. J.
Transformation of 5ꢀene steroids by the fungus Aspergillus tamarii KITA: mixed molecular fate in
lactonization and hydroxylation pathways with identification of a putative 3βꢀhydroxyꢀsteroid
dehydrogenase/ꢁ5 –ꢁ4 isomerase pathway. Biochim. Biophys. Acta 2009, 1791, 110–117.
(46) Pollack, R. M. Enzymatic mechanisms for catalysis of enolization: ketosteroid
isomerase. Bioorg. Chem. 2004, 32, 341–353.
(47) Batzold, F. H.; Benson, A. M.; Covey, D. F.; Robinson, C. H.; Talalay, P. The delta 5ꢀ3ꢀ
ketosteroid isomerase reaction: catalytic mechanism, specificity and inhibition. Adv. Enzyme Regul.
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(48) Spessard, G. O.; Matthews, D. R.; Nelson, M. D.; Rajtora, T. C.; Fossum, M. J.;
Giannini, J. L. Phytoalexinꢀlike activity of abietic acid and its derivatives. J. Agric. Food Chem.
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(49) Feio, S. S.; Gigante, B.; Roseiro, J. C.; MarceloꢀCurto, M. J. Antimicrobial activity of
diterpene resin acid derivatives. J. Microbiol. Methods 1999, 35, 201–206.
(50) Gigante, B.; Silva, A. M.; MarceloꢀCurto, M. J.; Feio, S. S.; Roseiro, J.; Reis, L. V.
Structural effects on the bioactivity of dehydroabietic acid derivatives. Planta Med. 2002, 68, 680–
684.
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Figure 1. Chemical structures of the studied substances.
Figure 2. Effect of kaurenoic acid derivatives on the disease caused by Colletotrichum
lindemuthianum on the common bean. W, water (control); T, Tween 80® (control); 7, methyl ester
of the hydrogenated kaurenoic acid (1.401 mM); 9, pꢀbromobenzyl ester of the hydrogenated
kaurenoic acid (1.886 mM); MT, commercial fungicide methyl thiophanate (2.044 mM). Mean
values with the same letter do not differ statistically according to the ScottꢀKnott test (P ≤ 0.05).
Figure 3. Affinity of ligands for ketosteroid isomerases according to calculations carried out with
Autodock Vina 1.1.2, Plant 1.2, Autodock 4.2.3 and Paradocks 1.0.1. Mean values annotated with
the same letter in each graphic do not differ statistically according to the ScottꢀKnott test (P ≤ 0.05).
7-R, (R)ꢀdiastereoisomer of the methyl ester of the hydrogenated kaurenoic acid; 7-S, (S)ꢀ
diastereoisomer of the methyl ester of the hydrogenated kaurenoic acid; 9-R, (R)ꢀdiastereoisomer of
the pꢀbromobenzyl ester of the hydrogenated kaurenoic acid; 9-S, (S)ꢀdiastereoisomer of the pꢀ
bromobenzyl ester of the hydrogenated kaurenoic acid; 17, progesterone; 18, 19ꢀnortestosterone;
19, ꢁ4ꢀestreneꢀ4,17ꢀdione; 20, 5αꢀdihyrotestosterone acetate; 21, testosterone acetate; 22, 17βꢀ
estradiol; 23, 5αꢀdihyrotestosterone; 24, testosterone; 25, abietic acid; 1, kaurenoic acid; 26,
abietinol; 27, 16ꢀoxoꢀ17ꢀnorkauranꢀ19ꢀoic acid; 28, 3β ꢀhydroxykaurenoic acid; 29, methyl cisꢀ7ꢀ
oxoꢀdeisopropyldehydroabietate; 30, 12ꢀhydroxyꢀ10αꢀmethylꢀ13ꢀdeisopropyldehydroabietinol.
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Figure 1. Chemical structures of the studied substances.
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Figure 2. Effect of kaurenoic acid derivatives on the disease caused by Colletotrichum lindemuthianum on
the common bean. W, water (control); T, Tween 80® (control); 7, methyl ester of the hydrogenated
kaurenoic acid (1.401 mM); 9, pꢀbromobenzyl ester of the hydrogenated kaurenoic acid (1.886 mM); MT,
commercial fungicide methyl thiophanate (2.044 mM). Mean values with the same letter do not differ
statistically according to the ScottꢀKnott test (P ≤ 0.05).
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Figure 3. Affinity of ligands for ketosteroid isomerases according to calculations carried out with Autodock
Vina 1.1.2, Plant 1.2, Autodock 4.2.3 and Paradocks 1.0.1. Mean values annotated with the same letter in
each graphic do not differ statistically according to the ScottꢀKnott test (P ≤ 0.05). 7-R, (R)ꢀdiastereoisomer
of the methyl ester of the hydrogenated kaurenoic acid; 7-S, (S)ꢀdiastereoisomer of the methyl ester of the
hydrogenated kaurenoic acid; 9-R, (R)ꢀdiastereoisomer of the pꢀbromobenzyl ester of the hydrogenated
kaurenoic acid; 9-S, (S)ꢀdiastereoisomer of the pꢀbromobenzyl ester of the hydrogenated kaurenoic acid;
17, progesterone; 18, 19ꢀnortestosterone; 19, ∆4ꢀestreneꢀ4,17ꢀdione; 20, 5αꢀdihyrotestosterone acetate;
21, testosterone acetate; 22, 17βꢀestradiol; 23, 5αꢀdihyrotestosterone; 24, testosterone; 25, abietic acid;
1, kaurenoic acid; 26, abietinol; 27, 16ꢀoxoꢀ17ꢀnorkauranꢀ19ꢀoic acid; 28, 3β ꢀhydroxykaurenoic acid; 29,
methyl cisꢀ7ꢀoxoꢀdeisopropyldehydroabietate; 30, 12ꢀhydroxyꢀ10αꢀmethylꢀ13ꢀdeisopropyldehydroabietinol.
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ToC Graphic
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