Phytotoxic potential of secondary metabolites and semisynthetic compounds from endophytic fungus xylaria feejeensis strain SM3e-1b isolated from sapium macrocarpum

Article abstract of DOI:10.1021/acs.jafc.6b01111

Bioactivity-directed fractionation of the combined culture medium and mycelium extract of the endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium macrocarpum, led to the isolation of three known natural products: (4S,5S,6S)-4-hydroxy-3-methoxy-5-methyl-5,6-epoxycyclohex-2-enone or coriloxine, 1; 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 2; and 2,6-dihydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione or fumiquinone B, 3. This is the first report of compound 3 being isolated from this species. Additionally, four new derivatives of coriloxine were prepared: (4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone, 4; 6-hydroxy-5-methyl-3-(methylamino)cyclohexa-2,5- diene-1,4-dione, 5; (4R,5R,6R)-4,5-dihydroxy-3-methoxy-5-methyl-6-(phenylamino)cyclohex-2-enone, 6; and 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 7. X-ray analysis allowed us to unambiguously determine the structures and absolute configuration of semisynthetic derivatives 4, 5, and 6. The phytotoxic activity of the three isolated natural products and the coriloxine derivatives is reported. Germination of the seed, root growth, and oxygen uptake of the seedlings of Trifolium pratense, Medicago sativa, Panicum miliaceum, and Amaranthus hypochondriacus were significantly inhibited by all of the tested compounds. In general, they were more effective inhibiting root elongation than suppressing the germination and seedling oxygen uptake processes as shown by their IC₅₀ values.

Full text of DOI:10.1021/acs.jafc.6b01111

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Article
Phytotoxic Potential of Secondary Metabolites and Semisynthetic
Compounds from Endophytic Fungus Xylaria feejeensis
Strain SM3e-1b Isolated from Sapium macrocarpum
Marbella Claudia García-Méndez, Norma Angelica Macias-Ruvalcaba, Patricia
Lappe-Oliveras, Simón Hernandez-Ortega, and Martha Lydia Macias Rubalcava
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01111 • Publication Date (Web): 09 May 2016
Downloaded from http://pubs.acs.org on May 13, 2016
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Page 1 of 37
Journal of Agricultural and Food Chemistry
Phytotoxic Potential of Secondary Metabolites and Semisynthetic Compounds from
Endophytic Fungus Xylaria feejeensis Strain SM3eꢀ1b Isolated from Sapium
macrocarpum
Marbella Claudia GarcíaꢀMéndez†,‡, , Norma Angélica MacíasꢀRuvalcaba§, Patricia Lappeꢀ
∇
Oliveras#, Simón HernándezꢀOrtega⊥, Martha Lydia MacíasꢀRubalcava*,†,‡
†
‡
⊥
Instituto de Química, Departamento de Productos Naturales. Laboratorio de Rayos X.
Universidad Nacional Autónoma de México (UNAM). Ciudad Universitaria, Coyoacán,
México, D.F., 04510.
§Facultad de Química, Departamento de Fisicoquímica. Universidad Nacional Autónoma
de México (UNAM). Ciudad Universitaria, Coyoacán, México, D.F., 04510.
#Instituto de Biología, Departamento de Botánica, Universidad Nacional Autónoma de
México (UNAM). Ciudad Universitaria, Coyoacán, México, D.F., 04510.
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ABSTRACT: Bioactivityꢀdirected fractionation of the combined culture medium and
mycelium extract of the endophytic fungus Xylaria feejeensis strain SM3eꢀ1b, isolated from
Sapium macrocarpum, led to the isolation of three known natural products: (4S,5S,6S)ꢀ4ꢀ
hydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylꢀ5,6ꢀepoxycyclohexꢀ2ꢀenone, or coriloxine, 1, 2ꢀhydroxyꢀ5ꢀ
methoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 2, and 2,6ꢀdihydroxyꢀ5ꢀmethoxyꢀ3ꢀ
methylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione or fumiquinone B, 3. This is the first report of
compound 3 being isolated from this species. Additionally, four new derivatives of
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5
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coriloxine
were
prepared:
(4R,5S,6R)ꢀ6ꢀchloroꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀ
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methylcyclohexꢀ2ꢀenone, 4, 2ꢀhydroxyꢀ3ꢀmethylꢀ5ꢀ(methylamino)cyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀ
dione, 5, (4R,5R,6R)ꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylꢀ6ꢀ(phenylamino)cyclohexꢀ2ꢀ
enone, 6, and 2ꢀ((4ꢀbutylphenyl)amino)ꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀ
dione, 7. Xꢀray analysis allowed us to unambiguously determine the structures and absolute
configuration of semisynthetic derivatives 4, 5 and 6. The phytotoxic activity of the three
isolated natural products and the coriloxine derivatives is reported. Germination of the seed,
root growth, and oxygen uptake of the seedling of Trifolium pratense, Medicago sativa,
Panicum miliaceum, and Amaranthus hypochondriacus were significantly inhibited by all
of the tested compounds. In general, they were more effective inhibiting root elongation
than suppressing the germination and seedling oxygen uptake processes as shown by their
IC50.
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KEYWORDS: Xylaria feejeensis; endophytic fungi; quinones; coriloxine; phytotoxicity;
Sapium macrocarpum.
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INTRODUCTION
Endophytic fungi are microorganisms that live in the tissues of living plants, causing
no apparent symptoms of disease for the host.1,2 Some endophytes protect the host plant
from other organisms, such as pathogens, herbivores and insects; and can also improve the
drought tolerance and nutrients uptake of the plant.3 Besides, as part of the colonization
process, endophytes have the ability to produce a wide diversity of phytotoxic secondary
metabolites and exoenzymes. Also, the endophyte is capable of producing phytotoxic
metabolites in vitro with activity against different invading organisms, including algae and
plants.4 For this reason, endophytic fungi have been identified as potential sources of active
secondary metabolites that could be useful in agriculture, as fungicides, herbicides,
insecticides, among others.5,6 This is particularly important since the excessive use of
agrochemical products has promoted a faster evolution of resistant forms of pests. Studying
these metabolites may lead to the discovery and design of new herbicides with effective
action towards targets for pest control.7–11
It is remarkable that almost every known plant species contains at least one species of
endophyte fungus. Strobel and Daisy2 have suggested strategies to identify those plants
hosting endophytic fungi that produce bioactive secondary metabolites. The main strategies
contemplate plants living in uncommon conditions with novel survival skills, plants that
have a history being used by indigenous peoples, ancient and endemic plants, and,
particularly interesting for this investigation, plants from areas of great biodiversity. Plants
selected under these considerations are more likely to lodge endophytic fungi producing
novel bioactive products than other plants.
The Reserva de la Biósfera Sierra de Huautla “REBIOSH” is a region rich in water
resources, with a significant forest cover, consisting of a medium low oak forest and a low
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deciduous forest.12 According to the aforementioned criteria from Strobel and Daisy,2 the
reserve is an ideal environment for the collection of plants. For this reason, the tree Sapium
macrocarpum Mull. Arg. (Euphorbiaceae) was randomly selected at the “REBIOSH”
reserve as potential source of interesting endophytes.
The largest genus of the family Xylariaceae Tul.&C. Tul. (Xylariales,
Sordariomycetes), Xylaria Hill ex Schrank, comprises more than 300 species,13 including
endophytes that can be found in a variety of tropical plants. A wide range of bioactive
secondary metabolites, mainly with antimicrobial, antimalarial, and cytotoxic properties,
are produced by members of the genus Xylaria.14,15 In particular, from Xylaria feejeensis
the following compounds have been isolated: integric acid,16 xylaropyrone,17 nonenolide
xyolide,18 pestalotin 4 ′ ꢀOꢀmethylꢀ
3S,4Sꢀ(+)ꢀ4ꢀhydroxymellein, 3Sꢀ(+)ꢀ8ꢀmethoxymellein,
methylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione,
β ꢀmannopyranoside 3S,4Rꢀ(+)ꢀ4ꢀhydroxymellein,
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2ꢀhydroxyꢀ5ꢀmethoxyꢀ3ꢀ
4S,5S,6Sꢀ4ꢀhydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylꢀ5,6ꢀ
epoxycyclohexꢀ2ꢀenone, and 4R,5Rꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylcyclohexenꢀ2ꢀenꢀ1ꢀ
one.19
The variety of biological activities, and the structural diversity of the secondary
metabolites from Xylaria, offer the opportunity to find new chemical skeletons that may be
used as templates for the development of compounds potentially bioactive in the fields of
medicine, agriculture, and industry.2,5,15 However, only a few studies have been focused on
the evaluation of the secondary metabolites of Xylaria fejeensis as promising naturalꢀlike
herbicides. There is only one report describing the phytotoxic effect of coriloxine on
Lactuca sativa seeds.20
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In this context, we report the evaluation of the phytotoxic potential of three major
secondary metabolites, obtained from the combined culture medium and mycelium extracts,
of the fungus Xylaria feejeensis strain SM3eꢀ1b isolated from S. macrocarpum, collected at
“REBIOSH”. The three compounds strongly inhibited the germintaion of the seed, root
growth, and the oxygen uptake of the seedlings on four weeds: T. pratense, M. sativa, P.
miliaceum, and A. hypochondriacus. Looking to find new compounds with improved
activity, four new synthetic derivatives of coriloxine were prepared and their phytotoxic
potential was also evaluated. Our results, show that all the molecules, natural and
semisynthetic, were potent inhibitors of the three physiological process tested on the four
weeds.
MATERIALS AND METHODS
General. A FisherꢀJohns apparatus was used for the determination of the melting points,
the reported values are uncorrected. KBr disks were used to obtain the IR spectra on a 599ꢀ
B spectrophotometer (PerkinꢀElmer, Waltham, MA). COSY, NOESY, HMBC and HSQC
NMR spectra were recorded on a Bruker AVANCE IIIHD (Fällanden, Switzerland) using
tetramethylsilane (TMS) as an internal reference, in methan(olꢀd), at 500 (1H) and 125 (13C)
MHz, on a Bruker DMX500 (Billerica, MA), in chloroformꢀd, either at 400 (1H) or 125
(13C) MHz, and a Bruker 300 (Fällanden, Switzerland), in chloroformꢀd, at 300 (1H) or 75
(13C) MHz. Highꢀresolution mass spectra, HRMS (DARTꢀTOF+), were acquired with an
AccuTOFꢀJMSꢀT100LC (Jeol, Peabody, MA) spectrometer. Single crystals of compounds
4, 5 and 6 were obtained and their Xꢀray analysis was performed on a Bruker D8 Venture
κꢀgeometry diffractometer (Madison, WI) equipped with microꢀfocus Xꢀray source and
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Helios multilayer mirror as monochromator, Cu kα radiation (λ = 1.54178Å). For open
column chromatography, silica gel 60 (70–230 mesh) from MerckꢀMillipore was used
(Billerica, MA). For analytical and preparative thinꢀlayer chromatography separations, preꢀ
coated silica gel 60 F254 plates (Merck) were used. Lithium perchlorate (LiClO4, ACS
reagent, ≥98.0 %), anhydrous methanol (MeOH, 99.8%), anhydrous dichloromethane
(CH2Cl2 ≥ 99.8%), and anhydrous acetonitrile (MeCN, 99.8 %) were obtained from Sigmaꢀ
Aldrich (St. Louis, MO) and used as received. Aniline (ACS reagent ≥ 99.5%) and 97% 4ꢀ
butylaniline were obtained from SigmaꢀAldrich and distilled immediately before use. 35%
Methylamine solution in H2O was obtained from Merck. Sodium methoxide (NaOMe,
reagent grade, powder 95%) was used as received from SigmaꢀAldrich. Silica gel (SiO2 60ꢀ
120 mesh) was used as catalyst. Electrochemical grade tetrabutylammonium
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o
hexafluorophosphate (Bu4NPF6 ≥ 99.0%) from Fluka (St. Louis, MO), was dried at 60 C
under vacuum prior to use. An Autolab PGSTAT302N potentiostat Metrohm Autolab
(Kanaalweg, Utrecht, Netherlands) was used for the electrosynthesis of 2. An Ag/AgNO3
system was used as reference electrode (silver wire in 0.01 M silver nitrate / 0.10 M
Bu4NPF6/ acetonitrile).
Fungus Isolation. The endophytic fungus strain SM3eꢀ1b was isolated from
symptomless healthy leaves of S. macrocarpum, collected at the “REBIOSH” at Quilamula
(18° 30′ 4.1″ N 98° 51′ 52″ W and 18° 32′ 12.2″ N 99° 02′ 05″ W; 1080ꢀ1230 meters above
sea level), State of Morelos, Mexico, in September 2010.
Complete intact leaves were rinsed with running water, and then with distilled
water. Then, the surface was sterilized by soaking the leaves during 60 s in 75% ethanol
and subsequently in 3.4% NaClO solution (65% Clorox) also for 60 s, rinsing with sterile
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distilled water both between solutions and at the end.21 The sterilized leaves were dried
with sterile adsorbent paper and cut in 2 x 2 mm pieces at the central vein level.
Afterwards, four pieces were placed in a Petri dish containing potatoꢀdextroseꢀagar (PDA),
which was previously treated with 500 mg/L chloramphenicol (SigmaꢀAldrich) to prevent
bacterial growth. Plates were incubated at 28 °C with 12:12 h light:dark cycles using a
fluorescent light (T12 30W) (Philips, Chihuahua, Mexico). Petri dishes were observed daily
and the individual hypha tip of the emerging colonies were reꢀinoculated in new PDA
plates until pure cultures were obtained.22–24
The pure culture of the endophytic strain SM3eꢀ1b is preserved on PDA slants at the
Instituto de Química, and at the Instituto de Biología (Laboratorio de Micología C006),
UNAM both in water agar (0.2%) at 4 °C, and in 30% glycerol potatoꢀdextroseꢀbroth
(PDB) at – 80 °C. Dried cultures on PDA and oatmeal agar (OA) have been deposited, with
accession number MEXU 27802, in the fungal collection of the Herbario Nacional de
México (MEXU), UNAM.
Morphological and Molecular Identification of the Strain SM3eꢀ1b. Based on
the colony and mycelium characteristics,25–27 the strain SM3eꢀ1b was identified as Xylaria
sp. In addition, the internal transcribed spacer region (ITS1ꢀ5.8SꢀITS2) was sequenced and
analyzed for identification of the species.22–24 Nucleotide BLAST analysis of the SM3eꢀ1b
sequence (Genbank accession number KR025539) revealed Xylaria feejeensis as the closest
match, with sequences identities ranging from 100 to 97%. The most similar sequence was
that of X. feejeensis E6912b (accession HM992808) followed by several other sequences of
X. feejeensis strains.
Fermentations and Extraction of Compounds from X. feejeensis. Three
Fernbach flasks (3 L capacity) with 1.2 L of PDB were used for the culture of endophytic
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fungus X. feejeensis strain SM3eꢀ1b. Each Fernbach flask was inoculated with five 2.0 cm2
agar plugs from a PDA stock culture of X. feejeensis, and were incubated for 30 days at 28
°C under static conditions with 12:12 h light:dark cycles. After this period, the mycelium
and the culture medium were separated by filtration through cheesecloth. Culture medium
was subsequently extracted with CH2Cl2 (× 6), and then with EtOAc (× 6). Both organic
phases were combined, dried with anhydrous sodium sulfate and concentrated under
vacuum, to yield 910 mg of a brown solid. The mycelium was macerated with 1.5 L of
CH2Cl2 (× 6) and 1.5 L of EtOAc (× 6). The dichloromethane and ethyl acetate phases were
mixed, dried with anhydrous sodium sulfate and concentrated under vacuum, to yield 3.58
g of a brown extract.24,28
Isolation of Compounds from X. feejeensis. The organic extracts from the culture
medium and mycelium (~4g) were combined. A silica gel chromatographic column (420 g
of SiO2, 70–230 mesh) with a gradient of nꢀhexaneꢀCH2Cl2 (100:0 to 0:100) and CH2Cl2ꢀ
MeOH (99.9:0.1 to 50:50) resulted in 200 fractions (50 mL each), all of which were
monitored by TLC; seven primary fractions (FI–FVII) were obtained from the combination
of the fractions with similar patterns.
Phytogrowthꢀinhibitory activity using a bioassay on a Petri dish showed that all
fractions were active (FI to FVII). From phytotoxic fraction IV (2.923 g), eluted with
CH2Cl2ꢀMeOH (99:1), the known epoxycyclohexenone derivative, (4S,5S,6S)ꢀ4ꢀhydroxyꢀ3ꢀ
methoxyꢀ5ꢀmethylꢀ5,6ꢀepoxycyclohexꢀ2ꢀenꢀ1ꢀone or coriloxine 1, was isolated. The pure
compound (930 mg) was obtained after exhaustive washing with a mixture of nꢀhexane and
dichloromethane (1:1), followed by recrystallization from CH2Cl2ꢀMeOH (99:1). Active
fractions IV (1.8 g) and V (0.29 g), eluted with CH2Cl2ꢀMeOH (96:4), were purified by
successive preparative TLC (CH2Cl2ꢀMeOH; 99:1 × 3) yielding the known quinone
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derivatives: 2ꢀhydroxyꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 2, (250 mg),
and 2,6ꢀhydroxyꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione or fumiquinone B, 3,
(100 mg). Coriloxine, 1, (170 mg ) was also obtained from these fractions.
Synthetic Derivatives of Coriloxine, 1.
Synthesis of (4R,5S,6R)ꢀ6ꢀChloroꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylcyclohexꢀ
2ꢀenone, 4. 100 mg (0.59 mmol) of coriloxine were dissolved in methanol (2 mL) and 26
mg (0.12 mmol) of indium(III) chloride were added to the solution. The mixture was
refluxed for 24 h, then 0.01 g (0.045 mmol) more of indium(III)chloride and 10 mg of silica
gel (60ꢀ120 mesh) were added; then, the solution was stirred under reflux for an additional
24 h. The reaction was allowed to cool down to room temperature, filtered to remove the
SiO2, and the solvent was evaporated.29 The residue was purified by chromatographic
column on silica gel (70–230 mesh) using an isocratic system of CH2Cl2ꢀMeOH (98:2), to
yield 21.5 mg of a colorless crystalline solid, 4.
2ꢀHydroxyꢀ3ꢀmethylꢀ5ꢀ(methylamino)cyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 5. In a
small vial, 57.5 mg (0.34 mmol) of coriloxine were dissolved in 500 µL of water, then 37
mg (0.35 mmol) of lithium perchlorate and 40 µL (0.41 mmol) of 35% aqueous
methylamine solution were added. The mixture was stirred for 40 min at room
temperature.30 A dark purple solution was obtained, the solvent was evaporated and the
residue was purified by silica gel (70–230 mesh) column chromatography eluting with an
isocratic system of EtOAc (100%), to yield 18.0 mg of a red crystalline solid, 5.
Synthesis
of
(4R,5R,6R)ꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylꢀ6ꢀ
(phenylamino)cyclohexꢀ2ꢀenone, 6. 44.9 mg (0.26 mmol) of coriloxine, 7.0 mg of silica
gel (60ꢀ120 mesh), and 2 mL of anhydrous dichloromethane were placed in a twoꢀneck
round bottom flask with a nitrogen inlet, and a reflux condenser attached to the flask. The
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reaction mixture was stirred at room temperature under N2 atmosphere during 15 min, then,
37 ꢁL (0.40 mmol) of freshly distilled aniline were added.31 After refluxing the reaction
mixture for 12 h, thin layer chromatography revealed that while the aniline was completely
consumed, there was coriloxine remaining. 20 ꢁL (0.22 mmol) more of freshly distilled
aniline were added and the mixture was refluxed for an additional period of 6 h, then stirred
during 10 h at room temperature. The dark yellow solution was filtered and the solvent was
evaporated. Extensive preparative TLC (nꢀhexaneꢀEtOAc (30:70) of the crude, yielded 20.0
mg of a pink crystalline solid, 6.
2ꢀ((4ꢀButylphenyl)amino)ꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 7.
88.0 mg (0.52 mmol) of coriloxine, 14.0 mg of silica gel (60ꢀ120 mesh), and 4 mL of
anhydrous dichloromethane were placed in a twoꢀneck round bottom flask equipped with a
reflux condenser and a nitrogen inlet. The reaction mixture was stirred at room temperature
under N2 atmosphere during 15 min; then, 120 ꢁL (0.76 mmol) of freshly distilled pꢀ
butylaniline were added to the mixture. This solution was refluxed during 15 h and stirred
at room temperature during additional 12 h.31 The dark brown solution was filtered, and the
solvent was evaporated. The crude product was purified by silica gel 60 (70–230 mesh)
column chromatography eluting with an isocratic system of EtOAc (100%) to afford 26.6
mg of a purple amorphous solid, 7.
2ꢀHydroxyꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 2. 84.9 mg (0.50
mmol) of coriloxine were dissolved in 1 mL anhydrous methanol, previously passed
through a short column of activated alumina; then, 32 mg (0.59 mmol) of sodium
methoxide were added and the reaction mixture was stirred during 2 h under nitrogen
atmosphere at room temperature.32 A dark brown solution was obtained, the solvent was
evaporated and the crude residue was purified by silica gel 60 (70–230 mesh) column
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chromatography eluting with an isocratic system of EtOAc (100%) to afford 10.2 mg of a
yellow crystalline solid, 2.
Electroꢀorganic Synthesis of 2ꢀHydroxyꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀ
dieneꢀ1,4ꢀdione, 2. 50 mL of anhydrous acetonitrile, containing 0.1 M of
tetrabutylammonium hexafluorphosphate as supporting electrolyte, were placed in a 75 mL
twoꢀcompartments electrolysis cell (Bioanalytical Systems West Lafayette, IN). Oxygen
was removed from the solution in the cell by bubbling nitrogen gas, previously saturated
with acetonitrile. Then, 150 mg of coriloxine were added and the solution was stirred under
N2 flow for additional 15 min. The working electrode consisted of a large area reticulated
vitreous carbon. The counter electrode (platinum wire) was placed in a separated
compartment using a frit glass; Ag/AgNO3 was used as reference electrode. A constant
potential of ꢀ2.1 V vs Ag/AgNO3 was applied under constant agitation at room temperature,
N2 flow continued throughout the experiment. The charge was monitored during the
electrolysis; when the total charge flow reached 2 Faradays/mole (two electron per
molecule), the reaction was stopped. The value of the potential applied during the
electrolysis was previously determined by cyclic voltammetry experiments of coriloxine,
and was set at approximately 100 mV after the peak potential of the reduction wave of
coriloxine. A purple solution was obtained after the electrolysis, acetonitrile was
evaporated and the residue was purified by column chromatography on silica gel eluting
with a gradient of nꢀhexaneꢀCH2Cl2 (100:0 to 0:100) and CH2Cl2ꢀMeOH (99.9:0.1 to 98:2).
The fraction eluted with CH2Cl2 (100%) was purified by preparative TLC (CH2Cl2ꢀMeOH;
98:2) to yield 52 mg of a yellow crystalline solid, 2.
PhytogrowthꢀInhibitory Bioassays. The combined culture medium and mycelia
organic extracts from X. feejeensis, natural compounds and semisynthetic compounds were
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tested for their inhibitory effect on the germination of the seed, elongation of the root, and
the oxygen uptake (respiration) of the seedlings of three dicotyledonous species, Trifolium
pratense (Fabaceae) (red clover, peavine clover, and cow grass), Medicago sativa
(Fabaceae) (California clover and buffalo grass), and Amaranthus hypochondriacus
(Amaranthaceae) (amaranth); and one monocotyledonous plant, Panicum miliaceum
(Poacea) (red millet). Seeds were obtained from Casa Cobo, S.A. de C.V. (Central de
Abastos, Mexico City, Mexico).
A Petri dish bioassay was performed to evaluate the phytotoxic effects of different
treatments on seedling growth.23,24,28 The organic extract was evaluated at 50, 100, 200, and
400 µg/mL, and the natural compounds and semiꢀsynthetic derivatives were evaluated at
20, 50, 150, and 200 µg/mL by dilution in agar (1%). For each treatment, the extract was
dissolved in MeOH, not exceeding 0.5%, and added to ~40 °C sterile agar in 3ꢀcm Petri
dishes before its solidification. Rival® [glyphosate: Nꢀ(phosphonomethyl)glycine]
(Monsanto, Sao Paulo, Brasil) and ExterPro® [hexazinone: (3ꢀcyclohexylꢀ6ꢀ
(dimethylamino)ꢀ1ꢀmethylꢀ1,3,5ꢀtriazineꢀ2,4(1H,3H)ꢀdione)] (Comercial Solrac, S.A de
C.V., Mexico City, Mexico) at 20, 50, 100, 150, and 200 µg/mL were used as positive
control; agar (1% with 0.5% MeOH) and pure agar (1%) as negative controls. Thirty seeds
of every plant species were sown onto the agar in four replications for every treatment, in a
completely randomized design. The agar plates, sealed with parafilm foil, were placed in a
germination chamber at 28 °C under complete darkness. After treatment, germination of the
seed, root growth, and oxygen uptake of the seedling were measured for A.
hypochondriacus (24 h), and for T. pratense, M. sativa and P. miliaceum (48 h).
For the oxygen uptake experiments, a 10 mL glass chamber with 4 mL of air
saturated deionized water at 28 °C was used; seedlings were transferred into the chamber
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and different concentrations of either the extract or the pure compounds were added.
Oxygen consumption was polarographically measured during 3 min, using a Clarkꢀtype
electrode connected to a 5300A biological oxygen monitor (YSI Inc., Yellow Springs, OH).
Oxygen requirement was determined as a percentage, considering the control as 100%. For
each experiment, a certain number of seeds was selected so that appreciable change in O2
uptake could be detected.23,24,28
Statistical Analysis. The effect of the different treatments (extract or pure
compound) on the inhibition of the three physiological processes was analyzed by
ANOVA, and Tukey statistical tests.33 For each activity, Probit analysis34 was used to
determine the IC50 values (half maximal inhibitory concentration), based on the average
percentage of inhibition for each concentration of the organic extract, and the natural and
semisynthetic compounds. A p value of 0.05 or less (*) was used to denote statistical
significance. All the calculations were performed with GraphPad Prism statistical computer
software, ver. 6.0 (La Jolla, CA). Data are shown as mean ± standard deviation (SD).
Coriloxine, 1. Colorless crystals (EtOAc); mp 155–156 °C; IR (KBr) νmax 3450,
1
1665, 1612, 1234 and 1080 cmꢀ1; H NMR (CDCl3, 400 MHz) δ 1.66 (s, 3H, CH3ꢀ5), 2.59
(d, J= 6.0 Hz, 1H, OHꢀ4), 3.33 (d, J= 2.0 Hz, 1H, Hꢀ6), 3.77 (s, 3H, OCH3ꢀ3), 4.49 (d, J=
6.0 Hz, 1H, Hꢀ4); 5.26 (d, J= 1.6 Hz, 1H, Hꢀ2) ppm; 13CꢀNMR (CDCl3, 100 MHz), δ18.9
(CH3ꢀ5), 56.5 (OCH3ꢀ3), 59.8 (Cꢀ5), 60.6 (Cꢀ6), 69.1 (Cꢀ4), 97.7 (Cꢀ2), 171.3 (Cꢀ3), 193.3
(Cꢀ1) ppm.
2ꢀHydroxyꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione,
2.
Yellow
crystals (CH2Cl2ꢀCH3OH); mp 176ꢀ178 °C; IR (KBr) νmax 3284, 3074, 1646, 1599 and
1
1297 cmꢀ1; H NMR (CDCl3, 300 MHz) δ 1.95 (s, 3H, CH3ꢀ5), 3.86 (s, 3H, OCH3ꢀ3), 5.84
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(s, 1H, Hꢀ2), 7.23 (s, 1H, OHꢀ6); 13CꢀNMR (CDCl3, 75 MHz) 7.8 (CH3ꢀ5), 56.8 (OCH3ꢀ3),
102.2 (Cꢀ2), 114.8 (Cꢀ5), 151.6 (Cꢀ6), 161.4 (Cꢀ3), 181.9 (Cꢀ4), 182.6 (Cꢀ1) ppm.
Fumiquinone B, 3. Purple amorphous solid; mp 253ꢀ255 °C; IR (KBr) νmax 3387,
2961, 1678, 1605, 1370, 1284 and 1100; 1H NMR (CH3OD, 500 MHz) δ 1.79 (s, 3H, CH3ꢀ
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5), 3.81 (s, 3H, OCH3ꢀ3); 13CꢀNMR (CDCl3, 75 MHz); δ 8.18 (CH3ꢀ5), 57.1 (OCH3ꢀ3 ),
110.4 (Cꢀ5), 164.2 (Cꢀ2), 170.8 (Cꢀ6), 175.7 (Cꢀ1), 178.2 (Cꢀ3), 196.3 (Cꢀ4) ppm.
(4R,5S,6R)ꢀ6ꢀChloroꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylcyclohexꢀ2ꢀenone,
4.
Colorless crystals (CH2Cl2); mp 185ꢀ188 °C; IR (KBr) νmax 3592, 2942, 1677, 1621, 1241,
1188 and 848; 1H NMR (CDCl3, 300 MHz) δ 1.42 (s, 3H, CH3ꢀ5), 3.08 (s, 1H, OHꢀ4), 3.35
(s, 1H, OHꢀ5), 3.81 (s, 3H, OCH3ꢀ3), 4.40 (s, 1H, Hꢀ6), 4.57 (s, 1H, Hꢀ4), 5.44 (s, 1H, Hꢀ2);
13CꢀNMR (CDCl3, 75 MHz) δ 21.1 (CH3ꢀ5), 56.8 (s, 1H, OCH3), 65.3 (Cꢀ4), 60.5 (Cꢀ5),
72.3 (Cꢀ6), 99.8 (Cꢀ2), 172.7 (Cꢀ3), 189.9 (Cꢀ1) ppm. HRMS (ESI) [M + H]+ m/z
207.04223 (calcd for C8H12ClO4, 207.04241).
2ꢀHydroxyꢀ3ꢀmethylꢀ5ꢀ(methylamino)cyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 5. Red
crystalline solid (CH3OH); mp 195ꢀ198 °C; IR (KBr) νmax 3558, 3364, 2981, 2940, 1671,
1
1621, 1504, 1315, 1240, 1125, 1055, 1006 and 845; H NMR (CDCl3, 300 MHz); δ 1.92 (s,
3H, CH3ꢀ5), 2.92 (d, J=5.7 Hz, 3H, CH3ꢀ3), 5.35 (s, 1H, Hꢀ2), 5.51 (s, 1H, NHꢀ3), 6.43 (s,
1H, OHꢀ6), 13C RMN (CDCl3, 75 MHz); 9.0 (CH3ꢀ5), 31.2 (NꢀCH3ꢀ3), 111.4 (Cꢀ5), 112.4
(Cꢀ2), 151.1 (Cꢀ3), 155.2 (Cꢀ6), 182.0 (Cꢀ1), 183.4 (Cꢀ4) ppm. HRMS (ESI) [M + H]+ m/z
168.06659 (calcd for C8H10NO3, 168.06607).
(4R,5R,6R)ꢀ4,5ꢀDihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylꢀ6ꢀ(phenylamino)cyclohexꢀ2ꢀ
enone (6). Pink crystalline solid (CH2Cl2ꢀCH3OH); mp 289ꢀ294 °C; IR (KBr) νmax 3558,
1
3364, 2940, 1671, 1621, 1240, 1123, 1055, 1006 and 845; H NMR (CDCl3, 300 MHz) δ
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1.19 (s, 3H, CH3ꢀ5), 3.38 (s, 1H, ꢀNHꢀ6), 3.79 (s, 3H, OCH3ꢀ3), 4.29 (s 1H, Hꢀ4), 4.62 (s
1H, Hꢀ6), 5.46 (s, 1H, Hꢀ2), 6.80 (t, J= 6.6 Hz, 1H, Hꢀ4´), 6.90 (t, J= 8.0 Hz, 2H, Hꢀ2´, 6´),
7.20 (t, J= 8.0 Hz, 2H, Hꢀ3´, 5´). 13CꢀNMR (CDCl3, 75 MHz) δ 20.3 (CH3ꢀ5), 56.7 (OCH3ꢀ
3), 64.1 (Cꢀ6), 74.7 (Cꢀ5), 75.2 (Cꢀ4), 101.4 (Cꢀ2), 119.3 (Cꢀ4´), 114.8 (Cꢀ2´, 6´), 129.3 (Cꢀ
3´, 5´), 149.1 (Cꢀ1´), 172.6 (Cꢀ3), 194.7 (Cꢀ1) ppm. HRMS (ESI) [M + H]+ m/z 264.12342
(calcd for C14H18NO4, 264.12358).
2ꢀ((4ꢀButylphenyl)amino)ꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 7.
Purple amorphous solid; mp 290ꢀ295 °C; IR (KBr) νmax 3331, 2960, 2932, 1650, 1591,
1
1235, 1076 and 842; H NMR (CDCl3, 300 MHz) δ 0.93 (t, J= 7.4 Hz, 3H, CH3ꢀ4´´), 1.36
(m, 2H, CH2ꢀ3´´), 1.57 (s, 3H, CH3ꢀ5), 1.59 (m, 2H, CH2ꢀ2´´), 2.6 ( t, J= 7.7 Hz, 2H, CH2ꢀ
1´´), 3.85 (s, 3H, OCH3ꢀ3), 5.82 (s, 1H, Hꢀ2), 6.90 (d, J= 8.4 2H, Hꢀ2´, 6´), 7.13 (d, J= 8.4
2H, Hꢀ3´, 5´), 7.36 (s, 1H, NHꢀ6). 13CꢀNMR (CDCl3, 75 MHz) δ 12.6 (CH3ꢀ5), 13.9 (Cꢀ
4´´), 22.2 (Cꢀ3´´), 33.5 (Cꢀ2´´), 35.0 (Cꢀ1´´), 56.4 (OCH3ꢀ3), 102.9 (Cꢀ2), 111.2 (Cꢀ5),
123.1 (Cꢀ2´, 6´), 128.6 (Cꢀ3´, 5´), 136.7 (Cꢀ4´), 139.7 (Cꢀ1´), 141.1 (Cꢀ6), 161.3 (Cꢀ3),
181.1 (Cꢀ4), 184.2 (Cꢀ1) ppm. HRMS (ESI) [M + H]+ m/z 300.15997 (calcd for
C18H22NO3, 300.15997).
Xꢀray Crystallography of Compounds, 4ꢀ6. Colorless crystals of 0.26 × 0.20 ×
0.20 mm, and 0.376 × 0.148 × 0.094 mm dimensions were obtained from slow evaporation
of CH2Cl2 solutions for 4 and 6, and a red crystal of dimension 0.226 × 0.118 × 0.070 mm
for 5, with empirical formulas C8H11ClO4, C8H9NO3, and C14H17NO4, and Mr =206.62, Mr
= 167.16, and Mr = 293.28, for 4, 5, and 6, respectively. Compound 4 crystallized in a
orthorhombic crystal system, P212121 (No. 19), with cell parameters, a = 7.7512(4) Å, b =
3
3
327
10.3272(6) Å and c = 11.7465(7) Å, V = 940.29(9) Å Z = 4, Dcal=1.46 Mg/m , mꢀ 3.481
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mmꢀ1, F(000) = 432. Compound 5 crystallized in a triclinic crystal system, Pꢀ1 (No. 2) with
cell parameters a= 6.8367(5) Å, b= 7.4505(5) Å, c =8 2103 Å, α= 79.128(4)°, β=
81.026(4)°, γ = 71.417(4)°, V =387.23(5) Å3, Z =2, Dcal=1.434 Mg/m3, mꢀ 0.935 mmꢀ1,
F(000) = 176. Compound 6 crystallized in a monoclinic crystal system P21 (No. 4), with
cell parameters a = 12.4136(6) Å, b = 9.6126(5) Å, c = 22.0258(11) Å, β= 93.1066(19)°,V
= 2624.4(2)Å3, Z = 8, Dcal=1.333 Mg/m3, mꢀ = 0.810 mmꢀ1, F(000) = 1120. All compounds
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were irradiated with Cu Kα radiation (λ =1.54178Å) on the Bruker D8 Venture κꢀgeometry
diffractometer with microꢀfocus Xꢀray source, and Helios multilayer mirror as
monochromator, using an APEX 2 program35 at 296(2) K. Data reduction was achieved
using the SAINT program.35 7291, 2756 and 29262 reflections were collected, from which
1707 (Rint = 0.0582), 1351 (Rint = 0.0784), and 8252 (Rint = 0.0855) reflections were
independent. Structures were solved using direct methods and then refined with the
SHELXS36 and SHELXL36 programs with fullꢀmatrix leastꢀsquares on F2 respectively.
ORTEPꢀ3 software was used for the figures.37 Four crystallographic independent molecules
were found in the asymmetric unit in compound 6.
The final values S=1.091, R1=0.0324, wR2=0.0928 were based on 1668 reflections
observed, 126 parameters for 4. S=1.071, R1=0.0595, wR2=0.1707 based on 1049
reflections observed, 117 parameters for 5. S=1.032, R1 = 0.0446, wR2 = 0.1222, based on
7713 reflections observed, 729 parameters for 6. The largest different peak and hole for 4,
5, and 6 were 0.243, ꢀ0.198 eÅꢀ3, 0.250, ꢀ0.245 eÅꢀ3 and 0.221, and ꢀ0.223 e.Åꢀ3
respectively.
RESULTS AND DISCUSSION
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The tree S. macrocarpum was randomly selected at the “REBIOSH” reserve;12
following the ecological criteria, with symptomless healthy leaves and without herbivore
damage.2,38 This selection was mainly based on the recommendations of Strobel and Daisy
cited in the introduction. S. macrocarpum is a common tree from humid and dry tropical
forests of central North Pacific, and from the region spanning from southern Mexico to
Costa Rica. It is commonly called “Amate prieto”, “Hincha huevos”, "Palo lechón”,
“Amantillo”, “Chileamate”, “Chonte”, “Hierba de la flecha”, “Higuerillo bravo”, “Lechón”,
“Mataise” or “Venenillo”.39 The recommended uses are for making paper, in constructions,
as poles, boxes, and for medicinal use.39,40
Fermentation of X. feejeensis Strain SM3eꢀ1 and Phytogrowth Inhibitory
Activity of the Organic Extracts. The endophytic fungus X. feejeensis strain SM3eꢀ1
isolated from S. macrocarpum was grown under static conditions in liquid substrate
fermentation on PDB. The culture medium and the mycelia were extracted with CH2Cl2 and
EtOAc. TLC analysis of the organic extracts exhibited a similar profile and the presence of
the same major secondary metabolites. The initial phytotoxic activity of the combined
extracts of the mycelia and culture medium was evaluated on the germination of the seed,
root growth, and oxygen uptake of the seedling of four weeds: T. pratense, M. sativa, P.
miliaceum, and A. hypochondriacus. The extract showed potential phytotoxicity with
significant inhibitions in a concentrationꢀdependent way; and, in general was more potent
as germination and seedling respiration inhibitor than as root elongation inhibitor (Figures
3AꢀC). Table 1 compiles the IC50 values of the phytotoxic effect of the organic extract on
the three physiological processes on the four different seeds. The results show that A.
hypochondriacus seeds were the most affected, with IC50 values <400 µg/mL (highest
concentration tested) for the three processes. Root growth of P. miliaceum and seedling
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respiration of M. sativa also showed the IC50 values <400 µg/mL (highest concentration
tested).
Isolation of Phytotoxic Compounds produced by X. feejeensis strain SM3eꢀ1.
Bioassayꢀguided fractionation from the culture medium and mycelium extracts from X.
feejeensis strain SM3eꢀ1 using chromatographic procedures led to the isolation of three
known natural products: one epoxycyclohexenone derivative, (4S,5S,6S)ꢀ4ꢀhydroxyꢀ3ꢀ
methoxyꢀ5ꢀmethylꢀ5,6ꢀepoxycyclohexꢀ2ꢀenone, or coriloxine, 1, and two quinone
derivatives; 2ꢀhydroxyꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione, 2, and 2,6ꢀ
dihydroxyꢀ5ꢀmethoxyꢀ3ꢀmethylcyclohexaꢀ2,5ꢀdieneꢀ1,4ꢀdione or fumiquinone B, 3 (Figure
1). The spectroscopic characteristics of compounds 1, 2, and 3, including IR, 1H NMR, and
13C NMR, are identical to those previously reported for these known compounds.41–43
Compound 3 is new to this species.
Semisynthetic Derivatives of Coriloxine, 1.
Four coriloxine derivatives were prepared according to the synthetic approaches
depicted in Figure 2, also described on the Materials and Methods section. Compounds 4ꢀ7
1
were identified from their IR and H NMR spectroscopic data. Single crystal diffraction
confirmed the structure of compounds 4, 5 and 6. Figure 4 shows an ORTEP drawing of
these structures. In addition, anomalous dispersion effects in diffraction measurements on
the crystal of 4 and 6 allowed to assess the absolute configuration. It is important to notice
that these coriloxine derivatives have not been reported before.
Treatment of coriloxine with an aqueous solution of methylamine in presence of a
catalytic amount of LiClO4 produced compound 5.30 For compounds 6 and 7 a catalytic
amount of SiO2 was used to promote the opening of the epoxide ring44; in both cases the
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nucleophilic attack of the aromatic amine (aniline or pꢀbutylaniline) took place at the less
sterically hindered carbon atom.
When designing semisynthetic derivatives with herbicidal activity, it is important
that they satisfy the requirements established by Lipinski’s and Tice’s rules,45,46 thus,
before preparing the derivatives depicted in Figure 2, it was confirmed that they posses
adequate physicochemical properties for their application as herbicides. Besides, since
lipophilicity is a key parameter that rules the pharmacokinetic behavior of agrochemicals,
by enhancing their in vivo transport and passive diffusion across membranes, we designed
derivatives with different degrees of lipohilicity, using methylamine, aniline and
butylaniline to open the epoxide, derivatives 5 – 7.
In our attempts to open the epoxide ring of coriloxine, an electrochemical reduction
of compound 1 was carried out in anhydrous acetonitrile under N2 atmosphere.
Surprisingly, these conditions led to an oxidized coriloxine derivative, compound 2. The
formation of 2 can be rationalized as follows: the electrochemical reduction of the epoxy
moiety proceeds through a twoꢀelectron process that promotes the ring opening, yielding
the corresponding hydroxy derivative, 1ʹ (Figure 5);47 then, by a ketoꢀenol equilibrium, the
hydroquinone of 2 is formed, 2ʹ, which is easily oxidized by the O2 in the air during
workup. Compound 2 was also obtained when coriloxine was reacted with a strong organic
base, such as sodium methoxide. Although, NaOMe is both a strong base and a good
nucleophile, no nucleophilic addition of the methoxy group was observed under our
experimental conditions. In another attempt to open the epoxide by the introduction of a
methoxyl group, coriloxine was reacted with methanol using a catalytic amount of
indium(III)chloride and SiO2;29 again, methoxy addition was not achieved; instead, we
obtained derivative 4. Knowing that about 65% of all the known agrochemicals have
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halogens in their structure,48 we considered important to evaluate the activity of the
chlorine derivative 4.
PhytogrowthꢀInhibitory Activity of the Secondary Metabolites and
Semisynthetic Derivatives of Coriloxine, 1. The inhibitory effect of the natural
compounds 1−3, and the semisynthetic derivatives of coriloxine, compounds 4−7, was
evaluated in the same manner than the extracts for the three physiological processes on the
four weeds. Figure 3 shows the phytotoxic effect, expressed as inhibition percentage, for
the different compounds tested at 200 ꢁg/mL. As it is observed in the Figure, all the
compounds exhibited a significant phytotoxic effect on the growth of the four target species
and their root elongation inhibition activity is more effective than their ability to inhibit
germination and seedling respiration (Figure 3AꢀC).
For the root elongation process of the four weeds, the seven evaluated compounds
had significant inhibition percentages between 19 and 100% at 200 μg/mL. The greatest
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effect was on root elongation of A. hypochondriacus (Figure 3B) with inhibitions ranging
from 45 to 100%; compounds 1 and 6 caused 100% inhibition, and 3 and 4 caused 80 and
96% inhibition respectively; compound 5 had no significant effect. Positive controls,
Rival® (31.8%) and ExterPro® (56.2%), are less effective than compounds 1, 3, 4, and 6.
Additionally, IC50 values of these compounds were found to be between 0.1 and 0.4 mM,
whereas the positive controls Rival® and ExterPro® values were >1.2 and >0.8 mM
respectively (Table 1).
On the other hand, the evaluated compounds reduced the germination of the four
weeds; however, the percentages of inhibition were low. Again, A. hypochondriacus was
the most affected weed with percentages of inhibition from 19 to 100% (Figure 3A).
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Compounds 1 and 6 caused 100% of inhibition; IC50 values were found to be 0.7 mM and
0.2 mM respectively, while positive control Rival® presents an IC50 value >1.2 mM (Table
1).
Finally, with exception of the seeds of M. sativa, the compounds also inhibited the
seedling respiration significantly at 200 ꢁg/mL, though percentages of inhibition were low.
Natural compounds 1−3 inhibited seedling respiration of A. hypochondriacus by ≥60% at
200 ꢁg/mL, showing greater efficiency than the positive controls Rival® (6.0%) and
ExterPro® (12.0%) at the same concentration (Figure 3C). IC50 values of compounds 1ꢀ3
were found to be 1.1, 1.1, and 0.8 mM respectively, whereas positive controls Rival® and
ExterPro® were >1.2 mM and >0.8 mM (Table 1). Products 4 and 6 did not have a
significant effect on the oxygen consumption of A.hypochondriacus, P. miliaceum and T.
pretense.
Table 1 summarizes the phytotoxic effect, expressed as IC50 values, of the three
isolated compounds, and the four coriloxine derivatives. The results show that all the tested
compounds have a high phytotoxic activity, inhibiting the three physiological processes on
the four seeds in a concentrationꢀdependent way. The IC50 values confirmed that, in
general, the evaluated compounds strongly inhibit the root growth, and have a weaker
effect on the germination and seedling respiration processes.
In conclusion, this work demonstrates that the combined culture medium and
mycelium extract, the three secondary metabolites from X. feejeensis strain SM3eꢀ1 (1−3),
and the four semisynthetic derivatives of coriloxine (4−7), possess phytotoxic properties
against the evaluated physiological processes of three dicotyledon species, T. pratense, M.
sativa, and A. hypochondriacus; and one monocotyledon species, P. miliaceum. Based on
the results from this investigation, additional studies will be performed in order to have a
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better understanding of the action mechanisms associated with the phytotoxic effects of the
phytotoxins, semisynthetic derivatives and organic extracts produced by endophytic fungus
Xylaria feejeensis strain SM3eꢀ1b isolated from S. macrocarpum.
ASSOCIATED CONTENT
Supporting Information
A comparative Nucleotide BLAST analysis of the SM3eꢀ1b sequence is available free of
charge via the Internet at http://pubs.acs.org. Crystallographic data for the semisynthetic
derivatives of coriloxine, compounds 4, 5 and 6 have been deposited at the Cambridge
Crystallographic Data Centre (CCDC) as supplementary publication # CCDC 1450883,
CCDC 1450884 and CCDC 1450885 respectively. Copies of the data can be obtained, free
of charge, from http://www.ccdc.cam.ac.uk/data_request/cif.
AUTHOR INFORMATION
Corresponding Author
*(M.L.M.R) Phone: + 52 (55) 56 22 44 32. Fax: + 52 (55) 56 16 22 17.
Eꢀmail address: mmacias@iquimica.unam.mx; mamaciasr@gmail.com
Author Contributions
∇Taken in part from the Ph.D. thesis of Marbella C. García Méndez
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Funding
This work was funded by Consejo Nacional de Ciencia y Tecnología, México (CONACyT,
grant 179194).
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We wish to thank Baldomero Esquivel Rodríguez, Nuria Esturau Escofet, Elizabeth Huerta,
Héctor Ríos, Ma. de los Ángeles Peña, Beatriz Quiroz, Isabel Chávez, Rocío Patiño, Javier
Pérez Flores, Luis VelascoꢀIbarra, and Ma. del Carmen García González, Instituto de
Química, UNAM for recording NMR, IR, UV, and MS spectra. To MSc Rafael Ibarra
Contreras, Facultad de Química, UNAM for the language revision. Marbella García
acknowledges the fellowship awarded by CONACyT during her graduate studies.
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FIGURE CAPTIONS
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Figure 1. Structures of phytotoxic compounds isolated from the endophytic fungus Xylaria
feejeensis strain SM3eꢀ1b.
Figure 2. Schematic diagram of the synthetic pathway of compounds 4, 5, 6, and 7.
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Figure 3. Phytotoxic effect of organic extract, secondary metabolites and semisynthetic
compounds from endophytic fungus Xylaria feejeensis strain SM3eꢀ1b (200 ꢁg/mL) on A.
hypochondriacus, T. pratense, M. sativa, and P. miliaceum: A. seed germination, B. root
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Figure 4. Xꢀray crystal structures of (4R,5S,6R)ꢀ6ꢀchloroꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀ
methylcyclohexꢀ2ꢀenone,
4,
(4R,5R,6R)ꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylꢀ6ꢀ
(metylamino)cyclohexꢀ2ꢀenone, 5, and (4R,5R,6R)ꢀ4,5ꢀdihydroxyꢀ3ꢀmethoxyꢀ5ꢀmethylꢀ6ꢀ
(phenylamino)cyclohexꢀ2ꢀenone, 6.
Figure 5. Mechanism for the electrochemical formation of compound 2.
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