Synthesis of the ligand (z)-2-(3-methoxyphenylamino)-4-oxo-4-phenylbut-2- enoic acid and its antifungal activity against the wood stain fungi mucor plumbeus

Article abstract of DOI:10.4067/S0717-97072013000200015

The ligand (z)-2-(3-methoxyphenylamino)-4-oxo-4-phenylbut-2-enoic acid and its Ni (II) complex were synthesized and their antifungal activity against the fungi wood stain Mucor plumbeus was evaluated. The ligand displayed fungostatic activity while the Ni

Full text of DOI:10.4067/S0717-97072013000200015

J. Chil. Chem. Soc., 58, Nº 2 (2013)
SYNTHESIS OF THE LIGAND (Z)-2-(3-METHOXYPHENYLAMINO)-4-OXO-4-PHENYLBUT-2-ENOIC ACID AND
ITS ANTIFUNGAL ACTIVITY AGAINST THE WOOD STAIN FUNGI MUCOR PLUMBEUS
C. PAZ1*, D. CAJAS-MADRIAGA1, C. TORRES3, Y. MORENO2, M. J. FERNÁNDEZ1,
J. BECERRA1 AND M. SILVA1
1Laboratorio de Química de Productos Naturales, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Chile
2 Fac. Cs. Químicas-Centro de Biotecnología, Universidad de Concepción, Chile
3Laboratorio de Genómica y Biodiversidad (LGB), Departamento de Ciencias Naturales, Universidad del Biobío, Chillán, Chile
(Received: August 6, 2012 - Accepted: January 30, 2013)
ABSTRACT
The ligand (z)-2-(3-methoxyphenylamino)-4-oxo-4-phenylbut-2-enoic acid and its Ni (II) complex were synthesized and their antifungal activity against the
fungi wood stain Mucor plumbeus was evaluated. The ligand displayed fungostatic activity while the Ni (II) complex exhibited antifungal activity with a MIC of
50 μg/mL, moreover the copper complex did not showed biocide activity.
Keywords: wood stain fungi, Mucor plumbeus, Ni-complex.
INTRODUCTION
In the last three decades there has been an increase in sustainable forest
plantations in the central-southern zone of Chile. More than 40% of the arable
land is apt for forest soils, this has allowed the development of a dynamic
forestry sector and industrial basis through monospecific plantations of forest1,
which corresponds to 2.4 million hectares of plantations of exotic species of
Scheme 1
fast-growing eucalyptus and Pinus genera. The forest industry consequently
represents 3.1% of the gross domestic product and is the second most important
economic activity after mining2. For this reason, the damages caused by the
fungi stainers of processed wood become one of the risk factors most relevant
to the forestry sector3. In this regard, the fungi strainers have been a problem
without a solution, by considerably diminishing the commercial value of
the sawn timber due to the appearance of unusual colours and the high risk
of contamination4. It is common to find stacks of sawn timber with serious
damage caused by fungi of the Ceratocystis genera5, however, in recent years
new strainers have been detected, among them we find representatives of
Mucor genera, of which Mucor plumbeus Bon. is one of the most common6.
This fungus is characterized by forming clear light to dark olive grey colonies
sized from 2 to 20 mm occurring on the surface of sawn timber7. Contamination
affects not only the processed wood, can also affect humans, putting at risk the
health of the population8. The most used products for the pathogenic control
of wood are chemical synthetic-based copper salts or chlorinated phenolic
compounds9.
Searching for new compounds with antifungic activity we studied the
acid 4-phenyl-2,4-dioxobutanoic acid, which reported important biological
activities like enzyme inhibitor of HIV-1 integrase10, in bacteria inhibits
KDPG aldolase11 and derivates have shown analgesic activity12. From the
2,4-dioxobutanoic acid, we synthesized the ligand keto-enamine (z)-2-(3-
methoxyphenylamino)-4-oxo-4-phenylbut-2-enoic acid (L) and their Ni (II)
and Cu (II) complexes and studied their fungicide activity in the wood stain
fungi Mucor plumbeus.
(Z)-Methyl 2-hydroxy-4-oxo-4-phenylbut-2-enoate (I-A):
A mixture
of acetophenone (1.0 g, 8.3 mmol) and dimethyloxalate (1.2 g, 10 mmol)
were stirred in 150 mL of anhydrous dimethoxyethane, then sodium hydride
(240 mg, 10 mmol) was added. The mixture was stirred under reflux for 4
h. The reaction was cooled and quenched with water 100mL and HCl until
pH 2. The mixture was extracted 3 times with EtOAc, and the organic layer
was evaporated in vacuo. The compound precipitated as a white solid (1.49
g, 84%). 1H-NMR (CDCl3, 300 MHz) ppm: 3.94 (3H, s); 7.10 (1H, s); 7.50
(2H, m); 7.61 (1H, m); 8.00 (2H, m), 15.83 (1H, s, interchangeable). 13C-NMR
(CDCl , 75 MHz) ppm: 55.4; 99.4; 127.5; 129.2; 130.2; 135.2; 162.6; 169.1;
190.8. 3EI-MS: 206 g/mol
(Z)-2-Hydroxy-4-oxo-4-phenylbut-2-enoic acid (II-B): Hydrolysis of I-A
(1.0 g, 4.9 mmol) was carried out with NaOH (194 mg, 4.9 mmol) in 20mL of
water and stirred at 50ºC for 15 min. The reaction was filtered and the reaction
mixture was extracted three times with 10 mL of ethyl acetate. The water layer
was acidified with HCl until pH 2. The product precipitated as a white solid,
then filtered off and dried (752 mg, 80%), mp= 160ºC. 1H-NMR (CDCl3, 300
MHz) ppm: 6.75 (1H, br); 7.52 (2H, m); 7.53 (1H, d, J= 4.2 Hz); 7.87 (2H, d,
J= 4.5 Hz). 13C-NMR (CDCl , 75 MHz) ppm: 98.0; 127.5; 128.0; 128.4; 128.9;
133.0; 135.4; 163.8. EI-MS:3192 g/mol
EXPERIMENTAL
The 1H-NMR spectra were determined using a Bruker ARX 300
instrument, operating at 300.1 MHz (1H) and 75.5 MHz (13C). EI MS spectra
were measured on Trace DSQII GC/MS-system (Axel Semrau GmbH & Co).
FT-IR-spectrometer Nicolet 6700 from Thermo Electron Corporation with the
ATR-unit Smart Performer. Melting points were determined on a Melting Point
SMP10 (Stuart) uncorrected. Column Chromatography was performed using
Merck silica gel 60 (0.063–0.200 mm). TLC was carried out on a Merck silica
gel 60 PF254. Solvents used in this study were distilled prior to use and dried
over appropriate drying agents.
Scheme 2
(z)-2-(3-methoxyphenylamino)-4-oxo-4-phenylbut-2-enoic acid (L):
Condensation of II-B (700 mg, 3.6 mmol) with 3-methoxy aniline (493 mg,
4.0 mmol) was carried out in methanol 50 mL at 0 ºC by 4 h. The product
precipitated as a yellow solid, 962 mg, 90% yield, mp = 168-169°C. 1H-NMR
(DMSO-d6, 300 MHz) ppm: 3.73 (3H, s); 6.12 (1H, s); 6.70 (2H, m); 6.79 (1H,
d, J= 2Hz); 7.22 (1H, t, J= 8.1 Hz); 7.42 (1H, m); 7.48 (2H, m), 7.55 (1H, d, J=
7.2 Hz); 7.92 (2H, d, J= 7.2 Hz); 12.22 (1H, s). 13C-NMR (DMSO-d6, 75 MHz)
ppm: 55.1; 9.44; 106.3; 110.2; 112.9; 127.1; 128.7; 129.9; 131.8; 138.7; 140.5;
154.9; 159.9; 165.7; 1889.5. IR-KBr (cm-1): 3219 NH; 1604 C=O; 1575 N=O.
e-mail: cpaz@udec.cl
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J. Chil. Chem. Soc., 58, Nº 2 (2013)
Complex synthesis
The complexes of Ni(II) and Cu(II) were obtained by reflux of 300 mg
(1.0 mmol) of the ligand, dissolved in 30 ml of methanol, with a solution of 0.5
equivalents of the corresponding metal salt, NiCl2·6H2O (119 mg, 0.5 mmol)
or CuSO4·5H O (125 mg, 0.5 mmol) in 15 ml water was added dropwise with
continuous sti2rring. The mixture was stirred under reflux for 6 h. The solvent
was removed in vacuum obtaining a green solid that was washed with ethyl
acetate three times, followed by cold methanol-water ratio 1:1. The complex
was then dried in vacuum desiccators.
Ni-Complex: green solid, descomposition over 210ºC. IR-KBr (cm-1):
1591 C=O; 1575 N=O.
Scheme 3
1
The H NMR spectrum of this compound in DMSO-d6 at 20ºC exhibits
sharp signals characteristic of a single conformer (Z). Therefore L presents an
intramolecularly H-bonded Z configuration23.
Fungus isolation
Mucor plumbeus was isolated from wood stain collected in a sawmill
near Concepción, VIII Region, Chile (36º48’S - 72º56’O). Infected wood
samples were suspended in steril water, and dilutions of this suspention were
streaked out onto YMG agar augmented with 200 mg l-1 of streptomycin,
at pH 5.5 and incubated at 22º C by 7 days, procedure repeated 3 times till
pure colonies grow up in the plate as gray mycelium which was verified by
microscopic examination13. This strain has been deposited at the collection of
the Laboratory of Natural Products Chemistry of University of Concepción,
Chile (accession number LQMP-001).
Fungus Identification
In order to validate the morphological identification of Mucor plumbeus
strain, it was amplified the 28S nuclear ribosomal large subunit rRNA (LSU)
using Lr0R/Lr6 primer combination that covers D1, 28S Ribosomal RNA
gene, D2, and D3 regions14. DNA was extracted from fruit bodies using
the E.Z.N.A. fungal DNA MiniKit (Omega-Biotek). PCR reactions were
performed using LR0R (5’-gtacccgctgaacttaagc-3’) as forward primer and
LR06 (5’-cgccagttctgcttacc-3’) as reverse primer. Each reaction was conducted
in a 15 µL volume containing 30-50 ng of DNA, 1X of PCR buffer, 2 mM
MgCl2, 0.1 µM of each dNTP, 0.5 µM of forward and reverse primers, and
1 U of Taq DNA polymerase. PCR amplification was carried out with an
initial denaturation of 4 min at 94ºC, and then 35 cycles of 30 s at 94ºC, 60
s at 50ºC and 60 s at 72ºC s, followed by a final step of 5 min at 72ºC. After
PCR purification and sequencing (both directions) by Macrogen sequencing
service (Seoul, Korea), analyses of sequences were edited using Genious v5.4
software15. The sequence of M. plumbeus 28S Ribosomal RNA gene was
analyzed with BLAST (Basic Local Alignment Search Tool) (http://blast.ncbi.
nlm.nih.gov/Blast.cgi) to determine the percentage of maximal identity with
the sequences of the global database. Finally, the sequence obtained from the
present study was deposited in GenBank with the code BankIt1532924 Seq1
JX123134.
Scheme 4
With this in mind, for the reaction between the ligand and nickel (II), it has
been postulated the following complex (Scheme 5).
Scheme 5
Antifungal activity determination
Agar diffusion test
The infrared spectrum does not have a normal N-H band, because the
system has been deprotonated. Instead the CO stretching mode has been shifted
to 1591 cm-1 due to the Ni-O=C coordination. The apical position could be
occupied by a molecule of oxygen from the carboxylate group of a neighboring
ligand; if possible then it will generate an infinite chain (Scheme 6).
Antifungal activity of L and their Ni(II) and Cu(II) complexes was
qualitatively evaluated by diffusion test in agar YMG. Plates were inoculated
with a spore concentration of 106 spores/mL of M. plumbeus. Paper disks (6
mm) were impregnated with 200 μg of ligand L, Ni(II) complex and Cu(II)
complex. As positive control was used antiblue375 and DMSO as negative
control (solvent). Plates were incubated at 22ºC by 21 days16.
Microtitre plate test
The minimal inhibitory concentration (MIC) was determined following
the method described by Sarker et al 200717,18. Microplates were cultivated in
liquid media YMG, pH 5.5 and inoculated with 25 μL of solution 105 FCU/
mL per well. Solutions of L and Ni(II) complex were added in decreasing
concentrations from 400 μg/mL to 3,2 μg/mL. Plates were incubated at 22ºC
by 21 days. The activity was evaluated by spectroscopic methods at 450 nm
in EpochTM (BIOTEK®) spectrophotometer recording the absorbance of
each well after incubation and correcting the blank control. Each sample was
analyzed in triplicate.
RESULTS AND DISCUSSION
Scheme 6
The synthesized compound (z)-2-(3-methoxyphenylamino)-4-oxo-4-
phenylbut-2-enoic acid (L), displays a conjugate system, enaminoketone, give
rise to intramolecular hydrogen bonding. The IR spectrum of L exhibits bands
at 3220 and 1604 cm-1 for the NH and CO stretching modes, respectively. The
ligand conjugate system undergoes a tautomeric equilibrium (keto-enol//imine-
enamine) as is reported in other similar systems19-22, showed in the scheme 3
and 4.
The interpretation of infrared to the complex is difficult because the metal
ion coordination causes displacement of the bands; and it is possible that one
of the tautomeric forms predominates over the other24. In spite of this, an
assignation for the bands is proposed, Table 1.
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J. Chil. Chem. Soc., 58, Nº 2 (2013)
Table 1: Infra-red analysis for Ni(II) complex.
Further Quantitative assays against the native fungus M. plumbeus LQMP-
001 by microtitre plate test, n=3, determinate than the Ni complex has a minimal
inhibitory concentration MIC, of 50 μg/mL. The Cu-complex has a MIC higher
than 200 μg/mL. The control antiblue375, showed a MIC of 10 μg/mL, n=3.
Molecular vibration
Ligand (cm-1)
Complex (cm-1)
N-H
C=O
C=N
3219
1604
1575
-
1591
1575
CONCLUSIONS
We have synthesized and evaluated in vitro the antifungal activity of the
ligand (z)-2-(3-methoxyphenylamino)-4-oxo-4-phenylbut-2-enoic acid and its
metal complex’s of nickel and cupper against the wood stain fungus Mucor
plumbeus LQMP-001. According to the in vitro results, the ligand displays
fungostatic activity. The incorporation of Ni to the ligand molecule, formed a
Ni(II) complex with antifungal activity, MIC of 50 μg/mL. Furthermore, the
Cu (II) complex did not have bioactivity at concentration of 200 μg/mL or
lower. The Ni complex could be a promising candidate as new antifungal agent
for control of wood stain fungi.
Fungal strain
The microscopic analysis of the strain is coincident with the description
for Mucor species presented by Schipper7 in 1976; colony varying from 2-20
mm in height, Mouse Gray, Deep Mouse Gray or Light Olive Gray colour;
sporangiophores branching in a sympodial and in a monopodial fashion
(Fig. 1A), up to 21 µm in diam., constricted and infrequently recurved below
sporangia, with slightly incrusted walls. Columellae pyriform, obovoid on a
truncate base, ellipsoidal to cylindrical-ellipsoidal, with incrusted walls that
rupture at maturity (Fig. 1B). The analysis of the RNA sequence fragment 972
pb, performed in BLAST, confirmed the identity of the fungus as M. plumbeus
(BankIt1532924 Seq1 JX123134) with a one hundred percent match to M.
plumbeus strain available from Genbank database (JN938896)25.
The mechanism resistance to organometallic compounds of these metals is
still unknown26. Furthermore, several studies reported that the organometallic
compounds of the divalent cations are more toxic than their metallic forms,
particularly when compared to their own inorganic equivalents27, 28
.
ACKNOWLEDGEMENTS
Authors would like to thank the financial support from the Project Fondecyt
3130378, Project Basal PFB-27 (PCS-009), Universidad de Concepción and
CONICYT for of its Doctoral Scholarship Program.
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Figure 2. Antifungal activity against Mucor plumbeus at 200 μg/disk
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