Methyl and p-Bromobenzyl Esters of Hydrogenated Kaurenoic Acid

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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, Lavras-MG, CEP 37.200-000, Brazil

‡

Departamento de Química, Universidade Federal de Lavras, Lavras-MG, CEP 37.200-000, Brazil

§

Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Franca-SP, CEP

14.404-600, Brazil §§

Department of Chemistry, University of Nairobi, Nairobi 00100, Kenya

*Fax number: 55-35-3829-1271 Telephone number: 55-35-3829-1623 E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Kaurenoic acid derivatives were prepared and submitted to in vitro assays with the

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fungus Colletotrichum lindemuthianum, which causes anthracnose disease in the common bean.

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The most active substances were found to be methyl and p-bromobenzylesters, 7 and 9 respectively,

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of the hydrogenated kaurenoic acid, which presented a minimum inhibitory concentration (MIC) of

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0.097 and 0.131 mM, respectively, while the commercial fungicide methyl thiophanate (MT)

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presented a MIC of 0.143 mM. Substances 7 (1.401 mM) and 9 (1.886 mM) reduced the severity of

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anthracnose in common bean to values statistically comparable to MT (2.044 mM). According to an

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in silico study, both compounds 7 and 9 are inhibitors of the ketosteroid isomerase (KSI) enzyme

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produced by other organisms, the amino acid sequence of which could be detected in fungal

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genomes. These substances appeared to act against C. lindemuthianum by inhibiting its KSI.

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Therefore, substances 7 and 9 are promising for the development of new fungicides.

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KEYWORDS: Colletotrichum lindemuthianum, ent-kauran-18-oic acid methyl ester, ent-kauran-

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18-oic acid p-bromobenzyl ester, ketosteroid isomerase, fungicide

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INTRODUCTION

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The common bean (Phaseolus vulgaris L.) is a basic food source in developing countries, as it

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provides part of the necessary dietary protein, carbohydrate and iron, especially for low-income

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populations.1 According to the Food and Agriculture Organization of the United Nations, about

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22.5 million tons of this bean was produced in 2013.2 However, this value could have been much

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higher if problems like those caused by phytopathogenic fungi could be better controlled. One of

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the fungal diseases of major economic importance for the production of common bean is

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anthracnose, which can cause production losses of up to 100%.1 This disease is caused by the

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fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, which has wide

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pathogenic variability. Therefore, the development of strains resistant to this pathogen is very

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difficult.3 Consequently, commercial fungicides are currently the main method used to control

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anthracnose in common bean plants.4 However, the continued use of such fungicides may result in

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the emergence of resistant fungus populations, and the harmful substances used can contaminate

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human populations and the environment.5 As a result, there is growing demand for new products

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that more efficiently control anthracnose with fewer unwanted side effects.6

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One possibility to circumvent the problems associated with the use of commercial

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fungicides is the development of naturally-derived substances as fungicides. Kaurenoic acid appears

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to be an interesting starting point, as it has demonstrated in vitro activity against the fungi

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Aspergillus nidulans G. Winter,7 Sclerotinium sclerotiorum (Lib.) de Bary, Verticillium dahlia

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Kleb.,8 Trichophyton rubrum(Castell.) Sabour. and Trichophyton mentagrophytes Priestley and

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Epidermophyton floccosum Langeron et Milochevitch.9 Moreover, this diterpene is known to

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present several other activities, such as antimicrobial, anti-inflammatory, cytotoxic and

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trypanocidal.9 However, there do not appear to be any reports in the literature regarding the activity

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of this substance or its derivatives against C. lindemuthianum. Therefore, to assist in the

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development of new fungicides to control anthracnose in the common bean, the present study aimed 3



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to prepare kaurenoic acid derivatives, then screen these substances for their in vitro antifungal

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activity against C. lindemuthianum. An assay on common bean plants was then carried out with the

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most active substances to determine their ability to reduce anthracnose severity. Finally, an in silico

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study was performed to identify the enzyme in the fungus targeted by the most active substance.

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MATERIALS AND METHODS

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Instruments. Nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 (concentration

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of the substances ranged from 8–16 mg/mL) at 298 K on an Avance DRX 400 spectrometer

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(Bruker, Billerica, MA) (400 MHz for 1H and 100 MHz for 13C), using tetramethylsilane (TMS) as

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the internal standard. Infrared (IR) spectra were obtained using an FT-IR Spectrum Two

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spectrometer (Perkin-Elmer, Waltham, MA). The optical rotations were determined with a P-2000

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digital polarimeter (Jasco, Portland, OR). High resolution mass spectrometry (HRMS) analyses

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were performed using a micrOTOF-QH hybrid instrument (Bruker, Billerica, MA). Melting points

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were measured with a Fisher-Johns melting point apparatus equipped with a 10-250 °C

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thermometer, coupled to a monocular microscope.

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Isolation of compounds from plants. ent-kaur-16-en-19-oic acid, 1, was isolated from a

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commercial sample of Mikania glomerata Spreng.,10 whereas ent-16,17-dihydroxykauran-20-oic

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acid, 15, and ent-18,19-dihydroxykaur-16-en-2-one, 16, were isolated from the African plant

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Psiadia punctulata (DC.) Vatke.11 For compound 1, dried and pulverized leaves of M. glomerata

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(1.0 kg) were extracted with CH2Cl2 (3.5 L) at room temperature to give 42.0 g of crude extract.

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This extract was suspended in 300 mL of a MeOH/H2O (9:1, v/v) solution, to result in a suspension

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that was filtered and partitioned with hexane (4x150 mL). The hexane fraction (6.0 g) was then

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fractionated by silica gel 60 (0.070-0.230 mm) column chromatography (hexane with increasing

65

amounts of EtOAc). The second fraction (1.5 g) was washed with cold MeOH to afford 800 mg of 4


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1. To obtain compounds 15 and 16, fresh leaves of P. punctulata (394 g) were extracted with a 2 L

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portion of acetone to give 22.0 g of crude extract. This extract was subjected to column

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chromatography over silica gel 60 (0.070-0.230 mm) using hexane/CH2Cl2 and CH2Cl2/MeOH with

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constant increase of polarity. This process furnished 150 mg of 15 and 290 mg of 16.

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Semi-synthetic preparation of ent-16-methoxykauran-19-oic acid, 2. Compound 2 was

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obtained from 3.0 mL of a methanolic solution of compound 1 (50.0 mg), which was stirred at room

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temperature for 18 h in the presence of four drops of concentrated H2SO4. The reaction mixture was

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poured into 20 mL water and extracted with EtOAc (3x20 mL). Solvent was removed under

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reduced pressure to afford 35.0 mg of 2.12

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Hydrogenation of the C16-C17 double bond of ent-kaur-16-en-19-oic acid, 1. In the

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vessel of a hydrogenation apparatus, 200 to 300 mg of compound 1 was dissolved in 20 mL of

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absolute ethanol. To this mixture, a catalytic amount of palladium on activated carbon was added.

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The vessel containing the reaction mixture was positioned inside the reactor, which was then

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carefully and tightly closed. The atmosphere inside the reactor was changed to hydrogen, and

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pressure was set to 4 atm. After it had been kept for 2 h at room temperature, the reaction mixture

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was filtered through Celite® 545 (Synth, Diadema, SP, Brazil), and the solvent was removed by

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rotary evaporation. ent-kauran-19-oic acid, dihydro-1, was used directly without further

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purification.

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General procedure for the preparation of esters 3 to 14. The procedure used was based

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on the method published by Boeck and co-workers,9 with some slight modifications in order to use

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compounds 1, 2 and ent-kauran-19-oic acid, dihydro-1, as the starting materials. Initially, 0.032 g of

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KOH was added to 2.0 mL of anhydrous acetone, and the resulting mixture was stirred for 5 min at

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25ºC. The substrate (0.05 g) was then added to this mixture, followed by the addition of alkyl halide

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(0.17 mmol). The reaction mixture was stirred at 25ºC. This mixture was then poured into 20 mL

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H2O, and extracted with EtOAc (3x20 mL). The combined organic extracts were washed three 5



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times with 10 mL water, dried over anhydrous MgSO4, filtered, then concentrated to dryness by

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rotary evaporation. The product was purified by silica gel 60 (15.0 g, 0.070-0.230 mm) column

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chromatography, using a hexane/EtOAc gradient (100% hexane; 8:2; 6:4; 1:1; 4:6; 2:8; 100%

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EtOAc) as the eluent. This fractionation was monitored by thin layer chromatography. Reaction of 1

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with iodomethane (2 h); benzyl bromide (2 h); bromobutane (2.5 h); or p-Cl benzyl bromide (25 h)

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gave the esters 3-6 in 55, 75, 56, and 34% yields, respectively. Reaction of dihydro-1 with

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iodomethane (3.5 h); benzyl bromide (4 h); or p-Br benzyl bromide (23 h) gave the esters 7-9 in 42,

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64, and 33% yields, respectively. Reaction of 2 with iodomethane (3.5 h); benzyl bromide (2.5 h);

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bromobutane (2 h); p-Br benzyl bromide (20 h); or p-Cl benzyl bromide (25 h) gave the esters 10-

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14 in 54, 33, 44, 26, and 27% yields, respectively. Only the spectroscopic data for the six new

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diterpenes (8, 9 and 11–14) are presented below. The NMR data for the known diterpenes (1–7, 10,

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15 and 16) were compared to previously published NMR data.9,11,13–16

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Kauran-19-oic acid benzyl ester, 8. Pale yellow oil; [α]24

= -61.4 (c 0.03, CH2Cl2); IR

D

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(neat) νmax 2932, 2855, 1724, 1464, 1144, 696 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.40-7.27 (m, 5,

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H-Ar), 5.12 (d, 1, J = 12.4 Hz, H-1'a), 5.02 (d, 1, J = 12.4 Hz, H-1'b), 2.20-0.70 (m, 22, CH and

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CH2 groups), 1.18 (s, 3, CH3-18), 0.98 (d, 3, J = 7.1 Hz, CH3-17), 0.78 (s, 3, CH3-20);

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(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,

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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]+

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(calculated for C27H38O2Na, m/z 417.2769).

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C NMR

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Kauran-19-oic acid p-bromobenzyl ester, 9. White solid; melting point 110ºC; [α]24

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40.2 (c 0.006, CH2Cl2); IR (KBr) νmax 3414, 2923, 2858, 1907, 1716, 1590, 1462, 1368, 1156, 1012,

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842, 799, 534 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.50 (m, 2, H-Ar), 7.25 (m, 2, H-Ar), 5.10 (d, 1,

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J = 12.6 Hz, H-1'a), 4.99 (d, 1, J = 12.6 Hz, H-1'b), 2.20-0.70 (m, 22, CH and CH2 groups), 1.17 (s,

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3, CH3-18), 1.00 (d, 3, J = 7.1 Hz, CH3-17), 0.77 (s, 3, CH3-20);

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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, 6


D

=-

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C NMR (100 MHz, CDCl3) δ

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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

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C27H37BrO2H, m/z 473.2055).

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16-Methoxykauran-19-oic acid benzyl ester, 11. Pale yellow oil; [α]24

D

= -45.0 (c 0.007,

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CH2Cl2); IR (neat) νmax 2934, 1721, 1454, 1371, 1234, 1144, 1074, 753, 698 cm-1; 1H NMR (400

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MHz, CDCl3) δ 7.40-7.27 (m, 5, H-Ar), 5.14 (d, 1, J = 12.5 Hz, H-1'a), 5.07 (d, 1, J = 12.5 Hz, H-

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1'b), 3.12 (s, 3, OCH3), 2.20-0.70 (m, 21, CH and CH2 groups) 1.27 (s, 3, CH3-17), 1.16 (s, 3, CH3-

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18), 0.82 (s, 3, CH3-20);

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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,

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18.4, 18.2, 15.5; HRMS m/z 447.2888 [M + Na]+ (calculated for C28H40O3Na, m/z 447.2875).

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C NMR (100 MHz, CDCl3) δ 177.3, 136.2, 128.4, 128.2, 128.0, 83.9,

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16-Methoxykauran-19-oic acid butyl ester, 12. White solid; melting point 51ºC; [α]24 D = -

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74.4 (c 0.01, CH2Cl2); IR (KBr) νmax 3431, 2936, 1724, 1468, 1377, 1229, 1160, 1073, 947, 773,

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587 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.06 (dt, 1, J1 = 10.8 Hz, J2 = 6.6 Hz, H-1'a), 4.00 (dt, 1, J1

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= 10.8 Hz, J2 = 6.6 Hz, H-1'b), 3.12 (s, 3, OCH3), 2.20-0.70 (m, 25, CH and CH2 groups), 1.27 (s, 3,

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CH3-17), 1.16 (s, 3, CH3-18), 0.95 (t, 3, J = 7.4 Hz, CH3-4'), 0.85 (s, 3, CH3-20);

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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,

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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]+

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(calculated for C25H42O3Na, m/z 413.3032).

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C NMR (100

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16-Methoxykauran-19-oic acid p-bromobenzyl ester, 13. Pale yellow oil; [α]24 D = -48.6 (c

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0.01, CH2Cl2); IR (neat) νmax 2934, 1725, 1594, 1465, 1370, 1143, 1073, 1012, 804, 530 cm-1; 1H

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NMR (400 MHz, CDCl3) δ 7.47 (m, 2, H-Ar), 7.23 (m, 2, H-Ar), 5.09 (d, 1, J = 12.5 Hz, H-1'a),

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5.00 (d, 1, J = 12.5 Hz, H-1'b), 3.12 (s, 3, OCH3), 2.20-0.70 (m, 21, CH and CH2 groups), 1.26 (s, 3,

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CH3-17), 1.17 (s, 3, CH3-18), 0.77 (s, 3, CH3-20);

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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,

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28.8, 26.6, 22.2, 19.1, 18.4, 18.2, 15.5; HRMS m/z 525.1930 [M + Na]+ (calculated for

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C28H39BrO3Na, m/z 525.1980).

13

C NMR (100 MHz, CDCl3) δ 177.1, 135.1,

7



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16-Methoxykauran-19-oic acid p-clorobenzyl ester, 14. White solid; melting point 71ºC;

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[α]24 D = -55.7 (c 0.02, CH2Cl2); IR (KBr) νmax 3431, 2932, 1720, 1599, 1494, 1466, 1369, 1228,

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1072, 1015, 808, 534, 426 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.52 (m, 2, H-Ar), 7.31 (m, 2, H-

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Ar), 5.10 (d, 1, J = 12.5 Hz, H-1'a), 5.02 (d, 1, J = 12.5 Hz, H-1'b), 3.12 (s, 3, OCH3), 2.20-0.70 (m,

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21, CH and CH2 groups), 1.26 (s, 3, CH3-17), 1.17 (s, 3, CH3-18), 0.77 (s, 3, CH3-20); 13C NMR

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(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,

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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 +

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Na]+ (calculated for C28H39ClO3Na, m/z 481.2485).

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Inoculum of Colletotrichum lindemuthianum. Monosporic strains belonging to races 65

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(LV134) and 89 (LV228), which had been preserved in M3 culture medium (10 g sucrose, 20 g

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agar, 2 g KH2PO4, 1 g MgSO4.7H2O, 6 g peptone, 1 g yeast extract and 1 L water) at 22°C in the

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dark, were used in the present study. Each strain was inoculated in bean pods that were maintained

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in M3 medium, then incubated at 22°C for 15 days in the dark. Conidial suspensions were obtained

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after adding 7 mL of sterile water and filtering the suspension through Miracloth. The concentration

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of the final suspension was adjusted to 1.2 × 106 conidia/mL.

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In vitro assay. Kaurenoic acid and its derivatives were dissolved in DMSO to a

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concentration of 20,000 µg/mL, then further diluted to 1000 µg/mL with M3S culture medium (10 g

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sucrose, 2.7 g KH2PO4, 2.5 g MgSO4.7H2O, 1 g peptone, 1 g yeast extract and 1 L water). Aliquots

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(100 µL) of each resulting solution and an aqueous suspension (100 µL) containing 1.2 × 106 C.

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lindemuthianum conidia/mL were poured into each cell (350 µL) of a 96-cell polypropylene plate.

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Each experiment was carried out with three replicates, with 55 mg/mL DMSO in M3S culture

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medium and 0.7 mg/mL methyl thiophanate (a fungicide marketed under the name Cerbonin 700

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WP) as the negative and positive controls, respectively. To obtain the commercial fungicide

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solution, the methyl thiophanate was dissolved in DMSO to a concentration of 55 mg/mL, then

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diluted with M3S. Following 72 h under constant shaking (110 rpm) at 22°C, substances that 8


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appeared to prevent fungal development from visual observation were considered to be active. This

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study was carried out separately for both fungal strains 65 (LV134) and 89 (LV228).

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Minimum inhibitory concentration. Adapting the method described in the literature17

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solutions of the substances (1000 µg/mL) found to be active against both strains of the fungus in the

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in vitro assay (7, 9 and 13) were prepared as described above for use in two-fold serial dilutions, in

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order to obtain solutions with concentrations ranging from 3.90-1000 µg/mL. All dilutions were

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performed with M3S culture medium in cells (350 µL) of a 96-cell polypropylene plate. To 100 µL

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of each resulting solution, 100 µL of an aqueous suspension containing 1.2 × 106 C.

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lindemuthianum conidia/mL was added. This experiment was carried out with three replicates,

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using DMSO (55 mg/mL) dissolved in M3S and the commercial fungicide methyl thiophanate

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(final concentrations ranging from 15.3 to 980 µg/mL) as controls. Plates were kept under constant

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shaking (110 rpm) for 72 h at 22°C. The minimum inhibitory concentration (MIC) was defined as

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the lowest concentration of each substance that prevented the fungal development, according to

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visual observation. This study was carried out with both 65 (LV134) and 89 (LV228) fungal strains.

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In vivo bioassay. Only substances 7 and 9 were included in this experiment, which followed

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a completely randomized design with five replicates, each plot corresponding to a 1 L pot

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containing a 15-day old common bean plant (Phaseolus vulgaris 'Pérola') known to be susceptible

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to anthracnose. The first trifoliate (phenological stage V3) developed by each plant was inoculated.

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The substances were dissolved in an aqueous 0.01 g/mL Tween 80® solution to the concentration

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(C) calculated according to the following: C = (A × B)/D. Where A represents the MIC of the

186

substance; B is the concentration of methyl thiophanate (positive control) used in this experiment;

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and D is the MIC of methyl thiophanate. Three controls were used in this study: water, aqueous

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0.01 g/mL Tween 80® solution, and methyl thiophanate (Cerbonin 700 WP) at a concentration of

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0.7 mg/mL. All solutions (1.3 mL) were applied to plant leaves with a brush, and after 5 h an

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aqueous suspension containing 1.2 × 106 C. lindemuthianum (race 65) conidia/mL was sprayed onto 9



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leaves until they began to drip. Plants were maintained in a greenhouse at 23°C under 80% relative

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humidity. The anthracnose severity was evaluated 7 days after fungal inoculation using the scoring

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scale developed by Schoonhoven and Pastor-Corrales.18 The resulting values were statistically

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analyzed by analysis of variance (ANOVA), and means were compared using the Scott-Knott test

195

(P ≤ 0.05),19 using R® software. 20

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Enzymes complexed to ligands similar to the methyl and p-bromobenzyl esters of

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hydrogenated kaurenoic acid. The structures of compounds 7 and 9 (Figure 1) were submitted to

198

a conformational search using Open3Dalign 2.103 software.21 For each chemical structure, 1000

199

molecular dynamics simulations were carried out at 1000 K, with a 1 femtosecond (fs) time step for

200

1 picosecond (ps) using the MMFF94 force field, and treating the solvent (water) implicitly using

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the Generalized Born-surface area model (GBSA). The most stable conformation of each substance,

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and those up to 10 kcal/mol more energetic than the most stable conformation, were optimized with

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MOPAC 2012 software,22 using the Hamiltonian PM7 and treating the solvent (water) implicitly.

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The most stable conformation for each substance was used in a pharmacophoric search using Align-

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it 1.0.4 software.23 The database used for this search was the Ligand Expo database,24 which was

206

downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data

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bank. The protein structures found to be complexed to ligands with Tanimoto scores24 equal to or

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above 0.50 were selected for use in the subsequent analyses.

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Ketosteroid isomerase. The pdb and fasta files for ketosteroid isomerases were downloaded

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from the RCSB protein data bank,25 and their amino acid sequences were aligned using the Ugene

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1.12.3 software26 with the ClustalO 1.2.0 algorithm,27 using the default parameters. Following this

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step, the amino acid sequences of the ketosteroid isomerases 1BUQ, 1C7H, 2Z76, 3D9R, 3F8X,

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3HX8, 3KE7, and 4CDL25 were used to search for similar amino acid sequences of fungi in the

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non-redundant (nr) database of the National Center for Biotechnology Information (NCBI), using

215

the software BLASTP 2.2.30+28,29 with DELTA-BLAST30 set to the default parameters. The amino 10


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acid sequences of the above-mentioned enzymes downloaded from the RCSB protein data bank and

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NCBI databases with query coverage and identity higher than 90% and 20%, respectively,

218

underwent alignment using the ClustalO 1.2.0 algorithm, as described above.

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Docking of substances to ketosteroid isomerases. Each of the substances presented in

220

Figure 3 were used in this step of the study, which was initially carried out by determining their

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chemical structures, as described above for compounds 7 and 9. The 20 conformations of the 1ISK

222

enzyme31 were separated using OpenBabel 2.3.0 software,32 then the Makemultimer.py Python

223

script was used to generate a pdb file containing the homodimeric KSI enzyme in the 8CHO

224

complex.33 The resulting 21 tridimensional structures, as well as those from the enzymes 1E3V34

225

and 1OH0,35 were aligned using the software Lovovalign 1.1.0,36 underwent hydrogen additions

226

with VMD 1.9.2b1,37 and were converted to the pdbqt format using the Autodock Tools 1.5.6 rc2

227

software,38 which was also used to select the grid box (26.25 × 27.75 × 26.25 Å) centered over the

228

binding sites of the original ligand (deoxycholic acid) in the 1E3V complex.34 Each of the

229

substances presented in Figure 3 were converted to pdbqt files using the Autodock Tools 1.5.6 rc2

230

software, then docked to the enzymes using the software Autodock Vina 1.1.2,39 for which the

231

default values for most parameters were used, except for the exhaustiveness parameter which was

232

set to 128. The pdbqt files of the enzymes and the grid box (grid spacing = 0.375 Å) were used to

233

calculate the atomic affinity potential for each atom type with the Autogrid 4.2.3 software.38 The

234

results of this calculation were used by the Autodock 4.2.3 software38 to dock each of the

235

substances to the enzymes, with parameters set to the values described in the supporting

236

information. All files for the enzymes and substances were corrected using Spores software,40 then

237

submitted to docking using the Plants 1.2 software,41 using the default parameters. Regions within

238

15 Å of the binding site center of the original ligand (deoxycholic acid) in the 1E3V complex34

239

were used for docking. Using the same griding box described for docking with Autodock Vina

240

software, substances were also docked to the enzymes using Paradocks 1.0.1 software,42 using the 11



241

default parameters. Interactions of ligands with the enzymes were visually depicted using the

242

software VMD 1.9.2b1,37 Rasmol 2.7.5,43 Pymol (The PyMOL Molecular Graphics System, version

243

1.4.1, Schrödinger, LLC, New York, NY) and LigProt+ 1.4.5.44

244 245

RESULTS AND DISCUSSION

246 247

The natural compound 1 was easily isolated from the natural source, as noted in the literature,10 for

248

use as the starting point for the synthesis of compounds 2-14 through esterification at the carboxyl

249

group, hydrogenation of the C16-C17 double bond, or methoxyl addition to C16. The methoxyl

250

addition and hydrogenation reactions produced quantitative amounts of products that could be

251

easily separated and purified from the reaction mixtures. Although the esterification reaction has

252

been previously described in the literature,9 this proved to be the most challenging reaction,

253

requiring additional analysis to determine the best reaction time for each alkyl halide used. Among

254

the 13 synthesized compounds, six were new compounds (8, 9 and 11–14), for which the NMR and

255

mass spectrometric data obtained was consistent with their chemical structures. Regarding

256

compounds 15 and 16, these were able to be easily isolated from the natural sources, similar to that

257

described in the literature,11 for direct use in the present study.

258

Among the 16 substances screened for antifungal activity at 500 µg/mL, compounds 7, 9

259

and 13 were found to inhibit both strains of C. lindemuthianum (races 65 and 89). Substances 1–4,

260

11, 14 and 15 inhibited only one of the strains, while all remaining compounds tested presented no

261

antifungal activity. As compound 7 was found to have higher activity than compound 3,

262

hydrogenation of the carbon-carbon double bond appeared to increase the antifungal activity,

263

however, the opposite effect was observed for compounds 8 and 4. Esterification of compound 1

264

appeared to reduce the activity, as observed in compounds 5 and 6, although protecting the carboxyl

265

group with a methyl or a benzyl group, 3 and 4 respectively, did not appear to have any effect on 12


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266

the fungus. Interestingly, when compound 2 was converted to 10, its activity vanished. Likewise, by

267

making R3 equal to OMe, the activity of 11 increased in relation to 8, however, the opposite effect

268

was observed when 9 and 13 were compared. Therefore, rationalizing the effect of these

269

derivatizations on the antifungal activity is not simple.

270

As the derivatization increased the antifungal activity of compounds 7, 9 and 13 in relation

271

to kaurenoic acid, 1, these substances were selected to progress to the next step of the study, which

272

consisted of assays at different concentrations to calculate their MIC values, which were found to be

273

0.097, 0.131 and 0.496 mM, respectively. Under these same conditions, the commercial fungicide

274

methyl thiophanate (Cerbonin 700 WP) presented a MIC of 2.044 mM. As compounds 7 and 9 had

275

the lowest MIC values, these compounds were submitted to an assay with common bean plants

276

inoculated with C. lindemuthianum. Both of them reduced the anthracnose severity to values that

277

were not statistically different from those obtained for the commercial fungicide methyl thiophanate

278

(Figure 2). Therefore, both substances 7 and 9 can be considered promising for the development of

279

new fungicides to control anthracnose in the common bean.

280

No reports regarding the fungicidal properties of compounds 7 and 9 could be found in the

281

literature, therefore, an in silico study was carried out to identify the target enzyme of these

282

substances in the fungus. This study was performed with both diastereoisomers of each substance,

283

as the asymmetric carbon formed during the hydrogenation process could be either R or S. The

284

pharmacophore search using the most stable conformations of these compounds resulted in the

285

selection of five proteins with Tanimoto scores24 of equal to or greater than 0.50, among which only

286

one report suggested an enzyme important for fungi, identified to be a ketosteroid isomerase

287

(KSI).45 This enzyme, also known as 3-oxo-∆5-steroid isomerase, catalyzes the isomerization of a

288

wide variety of 3-oxo-∆5-steroids to their ∆4-conjugated isomers. It is one of the fastest enzymes

289

known, with a reaction rate close to the diffusion limit.46 Unfortunately, no tridimensional structure

290

for a KSI produced by fungi could be found in the RCSB protein data bank.25 Therefore, the amino 13



Page 14 of 28

291

acid sequence of other KSIs, mainly produced by bacteria, were used to search the genome of fungi,

292

resulting in the selection of 24 similar sequences found to be produced by several fungi. These

293

sequences were very similar to those of KSIs deposited in the RCSB protein data bank (1BUQ,

294

1C7H, 2Z76, 3D9R, 3F8X, 3HX8, 3KE7 and 4CDL), especially those with accession numbers

295

ENH79813.1, KGO40240.1 and EFX01891.1 (similarities ≥ 70%), which corresponded to KSI

296

enzymes from the fungi Colletotrichum orbiculare, Penicillium expansum and Grosmannia

297

clavigera, respectively. Although these results are not sufficient to confirm the importance of KSIs

298

to fungi known to be pathogenic to plants, the ability of these microorganisms to produce such

299

enzymes is evident. Furthermore, owing to the above-mentioned similarity in amino acid sequences,

300

the corresponding tridimensional structures were expected to be similar. Therefore, KSIs produced

301

by other organisms appear to be good models for studying the same enzymes produced by fungi.

302

Although more than 60 tridimensional structures of KSIs have been deposited in the RCSB

303

protein data bank, only a few have no missing residues or atoms close to the active site, or

304

mutations that can interfere with their biological activity. Therefore, only those enzyme structures

305

that did not present with either of these problems were selected for molecular docking, which was

306

carried out with both diastereoisomers of compounds 7 (7-S and 7-R) and 9 (9-S and 9-R),

307

inhibitors of KSIs (17–24)47 and antifungal substances (1 and 25–30)7,8,

308

structures similar to 7 or 9 (Figure 3). Although the results obtained from the four computer

309

programs used for molecular docking did not completely agree (Figure 3), there was no significant

310

difference observed between the diastereoisomers of compounds 7 or 9, suggesting that both of

311

these present the same biological activity. Furthermore, according to all calculations, compound 9

312

was found to bind more strongly to KSIs than any other substance evaluated in the present study,

313

including the enzyme inhibitors. Thus, compound 9 is a good candidate for use as a KSI inhibitor.

48–50

with chemical

314

Regarding compound 7, its affinity for the KSI enzymes was not as high as expected, as the

315

MIC value was lower than that observed for 9. However, the results for 7 were not so different from 14


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316

those obtained for the KSI inhibitors (17–24).47 According to calculations carried out with

317

Autodock 4.2.338 and Plants 1.2,41 the affinity of 7 for the enzymes did not differ significantly from

318

those calculated for 23 and 24. Therefore, compound 7 remains a potential KSI inhibitor. Similar

319

behavior was observed for some of the antifungal substances studied, among which compound 2648

320

was shown to have the highest enzyme affinities. Thus, it is possible that these substances act

321

against fungi by inhibiting their KSIs. However, for kaurenoic acid (1) the calculations obtained

322

from the computational programs Plants, Autodock and Paradocks,42 indicated very low affinities of

323

this substance for the enzymes, suggesting that this compound does not act against fungi by

324

inhibiting their KSIs. This result seems to be consistent with the previously demonstrated

325

association between the antifungal activity of compound 1 and its ability to interfere with the

326

intracellular Ca2+ gradient in fungal cells.7

327

It appeared that the affinities of the substances tested for amino acid residues in the active

328

sites of KSIs were predominantly hydrophobic interactions, as can be exemplified by substance 9.

329

The oxygen atoms of the carboxyl group did not even form hydrogen bonds with the amino acid

330

residues of the enzyme. A similar situation was observed for substance 7, for which many of the

331

hydrophobic interactions with the enzyme were more similar to those observed for substance 9.

332

Even kaurenoic acid, 1,7–9 presented no clear polar interaction of its carboxylate ion with the amino

333

acid residues of the enzyme, which may explain the lower calculated binding affinity for this

334

substance. These results suggest that replacing the carboxylate group of 9 with a less polar

335

functional group between the p-bromobenzyl group and the diterpene ring should increase the

336

binding affinity. Similarly, the presence of a polar group located further inside the active site of the

337

enzyme, such as the carboxylate ion in abietic acid, 25,48 can be a disadvantage for favorable

338

interactions between the ligand and the enzyme, however, a carbonyl group, like that in

339

progesterone, 17,47

340

increasing the binding affinity.

can form a hydrogen bond with the Tyr14 amino acid residue, thereby

15



341

In conclusion, hydrogenation of kaurenoic acid, 1, and esterification with methyl or p-

342

bromobenzyl groups resulted in two substances, 7 and 9 respectively, with demonstrated in vitro

343

and in vivo activity against the fungus C. lindemuthianum, which were comparable to those

344

observed for the commercial fungicide methyl thiophanate. Therefore, both substances 7 and 9 are

345

promising for the development of new fungicides. According to our in silico study, these

346

compounds inhibit the ketosteroid isomerase enzyme, the amino acid sequence of which is very

347

similar to sequences extracted from fungi genomes. Therefore, these substances appear to act

348

against C. lindemuthianum by inhibiting the ketosteroid isomerase enzyme.

349 350

ASSOCIATED CONTENT

351

Supporting information

352

Spectroscopic data for the known substances (1–7, 10, 15 and 16) studied (Item S1). Docking

353

parameter file for Autodock 4.2.3 (Item S2). Amino acid sequences in the fungi genome that are

354

similar to ketosteroid isomerases (Tables S1–S2); 2D and 3D representations of the interactions of

355

ligands docked to the ketosteroid isomerase enzyme from Comamonas testosterone (Figures S1–

356

S17). This material is available free of charge from http://pubs.acs.org.

357 358

FUNDING SOURCES

359

Financial support and fellowships were provided by Fundação de Amparo à Pesquisa do Estado de

360

Minas Gerais (FAPEMIG), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP),

361

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional

362

de Desenvolvimento Científico e Tecnológico (CNPq).

363 364

NOTES

365

The authors declare no competing financial interest. 16


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366 367 368

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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, p-bromobenzyl 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 Scott-Knott 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 Scott-Knott 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 p-bromobenzyl ester of the hydrogenated kaurenoic acid; 9-S, (S)-diastereoisomer of the pbromobenzyl ester of the hydrogenated kaurenoic acid; 17, progesterone; 18, 19-nortestosterone; 19, ∆4-estrene-4,17-dione; 20, 5α-dihyrotestosterone acetate; 21, testosterone acetate; 22, 17βestradiol; 23, 5α-dihyrotestosterone; 24, testosterone; 25, abietic acid; 1, kaurenoic acid; 26, abietinol; 27, 16-oxo-17-norkauran-19-oic acid; 28, 3β -hydroxykaurenoic acid; 29, methyl cis-7oxo-deisopropyldehydroabietate; 30, 12-hydroxy-10α-methyl-13-deisopropyldehydroabietinol.

23



ToC Graphic

24


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Figure 1. Chemical structures of the studied substances. 115x114mm (600 x 600 DPI)



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, p-bromobenzyl 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 Scott-Knott test (P ≤ 0.05). 54x60mm (300 x 300 DPI)


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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 Scott-Knott 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 p-bromobenzyl ester of the hydrogenated kaurenoic acid; 9-S, (S)-diastereoisomer of the p-bromobenzyl ester of the hydrogenated kaurenoic acid; 17, progesterone; 18, 19-nortestosterone; 19, ∆4-estrene-4,17-dione; 20, 5α-dihyrotestosterone acetate; 21, testosterone acetate; 22, 17β-estradiol; 23, 5α-dihyrotestosterone; 24, testosterone; 25, abietic acid; 1, kaurenoic acid; 26, abietinol; 27, 16-oxo-17-norkauran-19-oic acid; 28, 3β -hydroxykaurenoic acid; 29, methyl cis-7-oxo-deisopropyldehydroabietate; 30, 12-hydroxy-10α-methyl-13-deisopropyldehydroabietinol. 211x320mm (300 x 300 DPI)




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ToC Graphic 47x29mm (300 x 300 DPI)


Methyl and p-Bromobenzyl Esters of Hydrogenated Kaurenoic Acid

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