Synthesis and evaluation of aromatic methoxime derivatives against five postharvest phytopathogenic fungi of fruits. Main structure–activity relationships

Article abstract of DOI:10.1016/j.foodchem.2020.126701

The antifungal activity of a library of twenty-four aromatic methoximes was examined against five representative postharvest phytopathogenic fungi. The panel included Penicillium digitatum, Penicillium italicum, Rhizopus stolonifer, Botrytis cinerea and Monilinia fructicola, all of which cause relevant economic losses worldwide as a result of affecting harvested fruits. The minimum inhibitory concentrations and minimum fungicidal concentrations of each compound were defined and the main structure–activity relationships were determined. Although other congeners were more potent, drug likeliness considerations pointed to the methoxime derived from 2,4-dihydroxypropiophenone as the compound with the most suitable profile. The morphology of the colonies of the fungal strains treated with the methoxime was examined microscopically and the compound was also tested in freshly harvested peaches and oranges, exhibiting promising control profiles in both fruits, similar to those of the commercial agents Imazalil and Carbendazim.

Full text of DOI:10.1016/j.foodchem.2020.126701

Journal Pre-proofs
Synthesis and evaluation of aromatic methoxime derivatives against five post‐
harvest phytopathogenic fungi of fruits. Main structure-activity relationships
Iván Cortés, Melina G. di Liberto, Teodoro S. Kaufman, Marcos G. Derita,
Andrea BJ. Bracca
PII:
S0308-8146(20)30563-X
DOI:
Reference:
https://doi.org/10.1016/j.foodchem.2020.126701
FOCH 126701
To appear in:
Food Chemistry
Received Date:
Revised Date:
Accepted Date:
8 January 2020
24 March 2020
25 March 2020
Please cite this article as: Cortés, I., di Liberto, M.G., Kaufman, T.S., Derita, M.G., Bracca, A.B., Synthesis and
evaluation of aromatic methoxime derivatives against five postharvest phytopathogenic fungi of fruits. Main
structure-activity relationships, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.126701
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Synthesis and evaluation of aromatic methoxime derivatives against
five postharvest phytopathogenic fungi of fruits. Main structure-
activity relationships
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Iván Cortés,a Melina G. di Liberto,b Teodoro S. Kaufman,a,*
Marcos G. Deritab,c,* and Andrea B. J. Braccaa,*
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aInstituto de Química Rosario (IQUIR, CONICET-UNR) and Facultad de Ciencias Bioquímicas y
Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (2000) Rosario, Argentina. E-
mail: kaufman@iquir-conicet.gov.ar; bracca@iquir-conicet.gov.ar
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bFarmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de
Rosario, Suipacha 531, S2002LRK, Rosario, Argentina
cInstituto de Ciencias Agropecuarias del Litoral (ICiAgro Litoral, Universidad Nacional del
Litoral-CONICET), Kreder 2805, (3080) Esperanza, Argentina. E-mail: mgderita@hotmail.com
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Abstract
The antifungal activity of a library of twenty-four aromatic methoximes was examined against
five representative postharvest phytopathogenic fungi. The panel included Penicillium digitatum,
Penicillium italicum, Rhizopus stolonifer, Botrytis cinerea and Monilinia fructicola, all of which
cause relevant economic losses worldwide as a result of affecting harvested fruits. The minimum
inhibitory concentrations and minimum fungicidal concentrations of each compound were defined
and the main structure-activity relationships were determined. Although other congeners were more
potent, drug likeliness considerations pointed to the methoxime derived from 2,4-
dihydroxypropiophenone as the compound with the most suitable profile. The morphology of the
colonies of the fungal strains treated with the methoxime was examined microscopically and the
compound was also tested in freshly harvested peaches and oranges, exhibiting promising control
profiles in both fruits, similar to those of the commercial agents Imazalil and Carbendazim.
Keywords: Bioactive methoximes; Postharvest fruit phytopathogens; Antifungal activity; Synthetic
antifungal compounds
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1. Introduction
Food safety and environmental health are matters of growing public concern. Pesticides are
extensively used in agriculture and undoubtedly play a pivotal role in retaining current high
production and quality of the crops (Felsot & Racke, 2007). Nevertheless, with the escalating
demand of environmental protection and food safety, the economic, health, and environmental costs
of pesticides have to be considered (Li et al., 2014). This requires that pesticides must be more
selective, effective and safe, also leaving less residue.
In addition, despite their different modes of action, most pesticides could lose efficacy due to
resistance after long-term usage. Thus, continuous efforts are needed to develop new control agents
with broad spectrum, low dosage and cost, long efficacy duration, less environmental pollution, and
with increased safety to humans (Liu et al., 2016).
The functional groups embedded in organic compounds play a critical role in eliciting or
potentiating their biological activities. For example, the transformation of the 5,7-dimethoxyflavone
carbonyl into oxime derivatives was found to enhance its antifungal activity against Candida
albicans (Yenjai, Wanich, Pitchuanchom & Sripanidkulchai, 2009). Notwithstanding, oximes
became valuable synthetic targets because of their low toxicity to non-target organisms and their
unique mode of action (Sun, Li, Lü, Xiong & Wang, 2010).
Currently, the oxime motif is present in a number of compounds with antibacterial (Brain,
Forrest, Hunt, Shillingford & Wilson, 1989), antifungal (Zhu & Shi, 2009) and antiprotozoan (Doré,
Lacroix, Lacroix & Viel, 1987) properties, as well as with anticonvulsant (Karakurt, Dalkara,
Özalp, Özbey, Kendi & Stables, 2001), anti-HIV (Emami, Falahati, Banifatemi, Amanlou &
Shafiee, 2004), antienteroviral (Chern et al., 2004), antineoplastic (Jindal, Chattopadhaya, Guleria
& Gupta 2003) and herbicidal (Chen et al., 2007) activity.
Oxime esters have been used in the molecular design of pesticides, such as the commercial
agents aldicarb (insecticide) and trifopsome (herbicide), as well as in investigational antifungal
drugs (see Figure S1). On the other hand, oxime ethers have shown anticholinergic, insecticidal
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(Ling, Liu, Chen, Wei, Wang & Yin, 2006) and acaricidal properties (Prabhu, Brown, & Delgado,
1981). Furthermore, several methoximes, such as picoxystrobin, dimoxystrobin, trifloxystrobin and
pyrifenox are listed in the Pesticide Manual and display herbicidal and fungicidal activity (Tomlin,
2003).
Postharvest fungal phytopathogens like Penicillium digitatum, P. italicum, Rhizopus
stolonifer, Botrytis cinerea and Monilinia fructicola are recognized as serious threats to many
valuable fruits. This demands the development of novel and improved crop-protection agents and
the chemical control approach is a convenient alternative in pest management. Plant fungicides
based on synthetic chemicals are extensively used in agriculture, and more than 110 active
ingredients have been registered worldwide as fungicides (Knight et al., 1997).
The synthesis and evaluation of oxime ether derivatives have attracted considerable interest in
recent years. We have developed a facile, eco-friendly and efficient catalytic method for the
synthesis of methoxime derivatives of aromatic aldehydes and ketones (Cortés, Kaufman & Bracca,
2018) and also became interested in contributing to combat postharvest pathogens (Pergomet, Di
Liberto, Derita, Bracca & Kaufman, 2018; Di Liberto, Stegmayer, Svetaz & Derita, 2019).
Taking into account all the above precedents, we hypothesized that the methoxime moiety
may be a pharmacophore, so that methoximes may be active against postharvest fruit pathogenic
fungi and that knowledge of basic structure-activity relationships may aid in the development of
more powerful agents. Therefore, and in continuation of our work, herein we present our results on
the synthesis and biological evaluation of diversely substituted methoxime derivatives of aromatic
aldehydes and ketones as potential antifungal agents to prevent the damage caused by five
representative postharvest fruit pathogens and an analysis of the main structure-activity
relationships among the set of synthetic methoximes.
2. Materials and methods
2.1 General information
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All the reactions were executed under anhydrous argon atmospheres, employing oven-dried
glassware and freshly distilled anhydrous solvents. Anhydrous chlorobenzene was prepared by
refluxing the solvents over P2O5 for 4 h, followed by distillation. Anhydrous EtOH was prepared by
treatment of the PA reagent with clean magnesium turnings and a crystal of iodine, followed by
reflux until complete consumption of the metal was achieved. The solvent was distilled from the so
formed magnesium methoxide. All the anhydrous solvents were transferred via cannula and stored
in dry Young ampoules containing molecular sieves. All other commercial reagents were used as
received, without further purification.
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The reactions were monitored by TLC, run on plates of Kieselgel 60 GF254, employing
different solvent systems of different polarity (hexane:EtOAc). The chromatographic spots were
detected by irradiation of the plates with UV light (254 nm), followed by exposure to iodine
vapours or by spraying with ethanolic p-anisaldehyde/sulfuric acid reagent and careful heating for
improving selectivity. All new compounds gave single spots. The flash column chromatographies
were run with slurry-packed silica gel 60 H (particle size 63–200 μm), eluting with hexane-EtOAc
mixtures, under positive pressure and employing gradient of solvent polarity techniques.
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2.2. Equipment
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The melting points were measured on an Ernst Leitz Wetzlar model 350 hot stage apparatus.
The structures of the products were determined employing a combination of spectroscopic methods,
including IR, 1D and 2D NMR (DEPT, COSY, HSQC and HMBC) and HRMS.
The IR spectra were recorded with a Shimadzu Prestige 21 spectrophotometer, as thin films
held between NaCl cells, or as solid dispersions in KBr disks.
The NMR spectra were acquired in a Bruker Avance 300 MHz spectrometer in CDCl3 (300
MHz for 1H and 75 MHz for 13C). Chemical shift data are informed in ppm with TMS (1H, 0 ppm)
and CDCl3 (13C, 76.9 ppm) as internal standards. Signal splitting patterns are reported as follows: s
(singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), dq
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(doublet of quartets), ddd (doublet of doublet of doublets), and m (multiplet). Coupling constant
values (J) are informed in Hertz. The signs asterisk (*) and hashtag (#) designate signals which
attribution can be exchanged.
The high-resolution mass spectra were obtained from UMyMFOR (Buenos Aires, Argentina)
with a Bruker MicroTOF-Q II instrument. Detection of the ions was performed in electrospray
ionization, positive ion mode.
The optical microscopy studies were developed employing a Correct optical microscope
(Seiwa Optical Co., Tokyo, Japan), fitted with 10×, 40× and 100× objectives and equipped with a
5.0 Megapixels Beion CMOS digital camera (Beion Medical Techonology, Shanghai, China) at a
resolution (H × V) = 2592 × 1944.
2.3. Synthetic procedures
2.3.1. General method for the synthesis of the precursor ketones
A mixture of the appropriate carboxylic acid (5.44 mmol) and resorcinol (1.36 mmol) in
C6H5Cl (0.6 mL) was treated with BF3.Et2O (5.44 mmol). The reaction was stirred at 80 °C until
complete consumption of resorcinol was assessed by TLC. The reaction mixture was treated with
brine (10 mL) and the products were extracted with EtOAc (3×10 mL). The organic layers were
combined, dried over MgSO4 and concentrated under reduced pressure. The residues were purified
by column chromatography, eluting with mixtures of hexanes and EtOAc.
2.3.2. General method for the synthesis of the methoximes
A stirred mixture of the aldehyde or ketone (1.0 equiv.), MeONH2.HCl (1.5 equiv.) and
anhydrous NaOAc (1.5 equiv.) in absolute EtOH (2.5 mL) was treated with CeCl3.7H2O (5 mol%).
The reaction was heated to 50-80 °C without special protection against atmospheric oxygen and its
progress was monitored by TLC. After consumption of the starting material, brine (10 mL) was
added and the products were extracted with EtOAc (3 × 10 mL). The organic layers were combined,
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dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column
chromatography, eluting with mixtures of hexanes and EtOAc.
2.3.3. Characterization of representative synthetic methoximes
2.3.3.1. (E)- and (Z)-1-(4-Hydroxy-2-methoxyphenyl)propan-1-one O-methyl oxime (E-1B and
Z-1B)
Yellowish oil (E/Z = 75:25); yield: 36.1 mg (91%); IR (film, ῡ): 3258, 2938, 1612, 1508, 1462,
1429, 1300, 1200, 1163, 1047, 957, 874 and 833 cm–1.
E-1B: ¹H NMR (300 MHz, CDCl3): δ = 6.99 (1H, d, J = 8.2, H-6’), 6.26 (1H, d, J = 2.3, H-3’), 6.17
(1H, dd, J = 2.2, 8.2, H-5’), 3.93 (3H, s, NOCH3), 3.66 (3H, s, OCH3), 2.67 (2H, q, J = 7.5, CH2)
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and 0.99 (3H, t, J = 7.5, CH3). C NMR (75 MHz, CDCl3): δ = 163.2 (C-1), 158.6 (C-2’),* 158.4
(C-4’),* 130.8 (C-6’), 117.0 (C-1’), 107.4 (C-5’), 99.3 (C-3’), 61.3 (NOCH3), 55.2 (OCH3), 23.0
(C-2) and 10.2 (C-3).
Z-1B: ¹H NMR (300 MHz CDCl3): δ = 6.87 (1H, d, J = 8.5, H-6’), 6.37–6.33 (2H, m, H-3’ and H-
5’) 3.80 (3H, s, NOCH3), 3.67 (3H, s, OCH3), 2.48 (3H, q, J = 7.7, CH2) and 1.00 (3H, t, J = 7.6,
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CH3); C NMR (75 MHz, CDCl3): δ = 159.5 (C-1), 157.7 (C-2’),* 157.0 (C-4’),* 129.4 (C-6’),
115.2 (C-1’), 107.3 (C-5’), 99.5 (C-3’), 61.3 (NOCH3), 55.5 (OCH3), 28.5 (C-2) and 11.3 (C-3);
HRMS m/z calcd. For C11H16NO3 210.1125 ([M+H]+); found: 210.1129.
2.3.3.2. (E)-1-(2-Hydroxy-4-methoxyphenyl)propan-1-one O-methyl oxime (2B)
White solid; yield: 67.5 mg (92%); mp 48-50 °C; IR (KBr, ῡ): 3566, 3466, 3447, 3412, 1628, 1616,
1597, 1558, 1522, 1508, 1458, 1290, 1167, 1150, 1051, 1022, 851 and 800 cm–1; ¹H NMR (300
MHz, CDCl3): δ = 11.58 (1H, s, 2’-OH), 7.30 (1H, d, J = 8.8, H-6’), 6.50 (1H, d, J = 2.7, H-3’),
6.46 (1H, dd, J = 2.7, 8.8, H-5’), 3.96 (3H, s, NOCH3), 3.80 (3H, s, 4’-OCH3), 2.78 (2H, q, J = 7.6,
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H-2) and 1.17 (3H, t, J = 7.6, H-3); C NMR (75 MHz, CDCl3): δ = 163.2 (C-1), 161.6 (C-2’),*
160.2 (C-4’),* 128.4 (C-6’), 110.4 (C-1’), 106.2 (C-5’), 101.8 (C-3’), 62.2 (NOCH3), 55.3 (OCH3),
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18.8 (C-2) and 11.4 (C-3), in agreement with the literature (Saha, Perveen, Nihesh & Sekar, 2019).
2.3.3.3. (E)- and (Z)-1-(2,4-Dimethoxyphenyl)propan-1-one O-methyl oxime (E-3B and Z-3B)
Colorless oil (E/Z = 73:27); yield: 34.4 mg (95%); IR (film, ῡ): 2936, 2837, 1611, 1504, 1462,
1306, 1281, 1209, 1159, 1047 and 1034 cm–1.
E-3B: ¹H NMR (300 MHz, CDCl3): δ = 7.17 (1H, d, J = 8.1, H-6’), 6.53–6.44 (2H, m, H-3’ and H-
5’), 3.93 (3H, s, NOCH3), 3.81 (3H, s, 2’-OMe),* 3.79 (3H, s, 4’-OMe),* 2.67 (2H, q, J = 7.5, H-2)
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and 0.99 (3H, t, J = 7.5, H-3); C NMR (75 MHz, CDCl3): δ = 161.6 (C-1), 161.5 (C-2’),# 158.6
(C-4’),# 130.9 (C-6’), 118.8 (C-1’), 104.2 (C-5’), 98.7 (C-3’), 61.5 (NOCH3), 55.4 (2’-OCH3),*
55.4 (4’-OCH3),* 22.6 (C-2) and 10.3 (C-3).
Z-3B: ¹H NMR (300 MHz, CDCl3): δ = 7.00 (1H, d, J = 9.1), 6.53–6.44 (2H, m), 3.81 (3H, s), 3.80
(3H, s), 3.79 (3H, s), 2.49 (2H, q, J = 7.6) and 1.02 (3H, t, J = 7.6); 13C NMR (75 MHz, CDCl3): δ
= 160.9 (C-1), 158.1 (C-2’),# 157.0 (C-4’),# 129.5, 116.4, 104.3 (C-5’), 98.9 (C-3’), 61.5 (NOCH3),
55.6 (2’-OCH3),* 55.3 (4’-OCH3),* 28.5 (C-2) and 11.3 (C-3); HRMS m/z calcd. For C12H18NO3
224.1281 ([M+H]+); found: 224.1286.
2.3.3.4. (E)-1-(2,5-Dihydroxyphenyl)propan-1-one O-methyl oxime (4B)
Yellow oil; yield: 18.0 mg (96%); IR (film, ῡ): 3395, 2968, 2938, 1504, 1495, 1487, 1215, 1051
and 781 cm–1; ¹H NMR (300 MHz, CDCl3): δ = 10.94 (1H, s, 2’-OH), 6.91 (1H, d, J = 2.9, H-6’),
6.86 (1H, d, J = 8.8, H-3’), 6.77 (1H, dd, J = 2.9, 8.8, H-4’), 4.60 (1H, s), 3.99 (3H, s), 2.77 (2H, q,
J = 7.6) and 1.18 (3H, t, J = 7.6); 13C NMR (75 MHz, CDCl3): δ = 162.8 (C-1), 152.3 (C-2’), 147.9
(C-5’), 118.1 (C-3’ and C-4’), 117.4, 113.6 (C-6’), 62.5 (NOCH3), 18.8 (C-2) and 11.3 (C-3);
HRMS m/z calcd. For C10H14NO3 196.0968 ([M+H]+); found: 196.0969.
2.3.3.5. (E)-1-(2-Hydroxy-5-methoxyphenyl)propan-1-one O-methyl oxime (5B)
Yellow solid; yield: 18.1 mg (98%); mp 38-40 °C; IR (KBr, ῡ): 3545, 3526, 3474, 3414, 2980,
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2943, 1636, 1618, 1570, 1491, 1398, 1285, 1211, 1184 1042, 970, 930, 862, 822, 762 and 685 cm–1;
¹H NMR (300 MHz, CDCl3): δ = 10.89 (1H, s, 2’-OH), 6.95 (1H, d, J = 2.9, H-6’), 6.92 (1H, d, J =
8.8, H-3’), 6.86 (1H, dd, J = 2.9, 8.8, H-4’), 4.00 (3H, s, NOCH3), 3.79 (3H, s, OCH3), 2.80 (2H, q,
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J = 7.6, CH2) and 1.19 (3H, t, J = 7.6, CH3); C NMR (75 MHz, CDCl3): δ = 163.0 (C-1), 152.4
(C-2’), 152.3 (C-5’), 117.9 (C-3’), 117.3 (C-1’), 116.2 (C-4’), 112.9 (C-6’), 62.5 (NOCH3), 56.0
(OCH3), 18.8 (CH2) and 11.3 (CH3); HRMS m/z calcd. For C11H16NO3 210.1125 ([M+H]+); found:
210.1125.
2.3.3.6. (E)-1-(2,4-Dihydroxyphenyl)butan-1-one O-methyl oxime (6B)
Yellow solid; yield: 46.7 mg (81%); mp 56–58 °C; IR (KBr, ῡ): 3412, 2930, 2371, 1653, 1636,
1616, 1558, 1541, 1522, 1508, 1458, 1180, 1057, 976 and 829 cm–1; ¹H NMR (300 MHz, CDCl3): δ
= 7.25 (1H, d, J = 8.8, H-6’), 6.47 (1H, d, J = 2.4, H-3’), 6.41 (1H, dd, J = 2.6, 8.7, H-5’), 3.94 (3H,
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s, NOCH3), 2.72 (2H, t, J = 7.9, H-2), 1.67–1.52 (2H, m, H-3) and 1.00 (3H, t, J = 7.4, CH3); C
NMR (75 MHz, CDCl3): δ = 162.10 (C-1), 159.7 (C-2’),* 158.0 (C-4’),* 129.0 (6’), 111.0 (C-1’),
107.2 (C-5’), 103.9 (C-3’), 62.2 (NOCH3), 27.2 (C-2’), 20.5 (C-3’) and 14.2 (CH3); HRMS m/z
calcd. For C11H16NO3 210.1125 ([M+H]+); found: 210.1118.
2.3.3.7. (E)-1-(2,4-Dihydroxyphenyl)propan-1-one O-methyl oxime (14 B)
Yellowish solid; yield: 43.4 mg (90%); mp 60–62 °C; IR (KBr, ῡ): 3482, 3246, 1634, 1605, 1524,
1458, 1439, 1327, 1298, 1252, 1180, 1142, 1053, 968 and 804 cm-1; ¹H NMR (300 MHz, CDCl3): δ
= 11.73 (1H, s, 2’-OH), 7.27 (1H, d, J = 8.6, H-6’), 6.46 (1H, d, J = 2.7, H-3’), 6.41 (1H, dd, J =
2.7, 8.6, H-5’), 5.67 (1H, s, 4’-OH), 3.96 (3H, s, NOCH3), 2.77 (2H, q, J = 7.6, H-2) and 1.17 (3H,
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t, J = 7.9, H-3); C NMR (75 MHz, CDCl3): δ = 163.2 (C-1), 160.0 (C-2’),* 157.9 (C-4’),* 128.8
(C-6’), 110.7 (C-1’), 107.1 (C-5’), 103.9 (C-3’), 62.3 (NOCH3), 18.8 (C-2) and 11.4 (C-3), in
agreement with the literature (Cortés, Kaufman & Bracca, 2018).
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2.4. Compound-stability testing
2.4.1. Hydrolytic stability
The short-term chemical stability test was conducted with the immersion solution containing
compound 14B. A volume of solution equivalent to 5 mg of 14B was extracted with EtOAc (3  20
mL). The combined organic extracts were dried over MgSO4, concentrated under reduced pressure,
dissolved in CDCl3 and examined by 1H NMR spectroscopy.
2.4.2. Photostability
A sample of the methoxime 14B (~ 12 mg) was transferred to a NMR tube and dissolved in
CDCl3 (~ 0.6 mL). The tube was tightly closed, placed at 7 cm of a 1000 W tungsten lamp (exposed
sample area = 1.5 cm2) and periodically checked by H NMR spectroscopy for decomposition
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products.
2.5. Antifungal studies
2.5.1. Microorganisms and media
Monosporic strains of each fungus were obtained from fruits that exhibited the corresponding
disease symptom and were morphologically characterized by the Mycology Reference Center
(CEREMIC, Rosario, Argentina), the National Institute of Agricultural Technology (INTA, San
Pedro, Argentina) and the Microbiology Department of the Chemical Engineering Faculty (FIQ-
UNL, Santa Fe, Argentina). The strains of P. digitatum CCC-102, P. italicum CCC-101, B. cinerea
CCC-100, M. fructicola INTA-SP345 and R. stolonifer LMFIQ-317, were grown on Potato-
Dextrose-Agar (PDA) medium using Petri dishes for 48 h to 6 days at 15-20 °C (as needed for the
growth of each one), and sub-cultured every 15 days to prevent pleomorphic transformations. The
inoculum of spore suspensions were obtained according to the CLSI reported procedures and
adjusted to 1 × 104 Colony Forming Units (CFU)/mL (CLSI, 2008).
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2.5.2. In vitro susceptibility tests
2.5.2.1. Determination of MIC
The Minimum Inhibition Concentration (MIC) values were determined by using broth
microdilution techniques according to the CLSI guidelines (CLSI, 2008) for filamentous fungi
(document M38-A2). MIC values were determined in RPMI-1640 medium (Sigma, St. Louis, MO,
USA) buffered to pH 7.0 with MOPS (Remel, Lenexa, KS, USA). Microtiter trays were incubated
at 15-20 °C in a moist and dark chamber, and MIC values were visually recorded at 48 h for R.
stolonifer and at a time according to the control growth, for the remaining fungi.
For the assay, solutions of each compound (12.5 mg/mL) were prepared in DMSO. Aliquots
(40 μL) of these were diluted in RPMI media (960 μL) to obtain stock solutions (500 μg/mL) that
were serially diluted from 500 to 7.9 μg/mL (final volume = 100 μL) in the corresponding wells of a
microtiter plate. A volume of fungal suspension (100 μL) was added to each well, except for the
sterility control, where sterile water was used instead. The commercial antifungal agents Imazalil,
Carbendazim and Captan were used as positive controls.
The MIC endpoints were defined as the lowest concentration of compound visually resulting
in total inhibition of fungal growth compared to the growth in the control wells containing no
antifungal agent.
2.5.2.2. Determination of MFC
The Minimum Fungicidal Concentration (MFC) was defined as the lowest concentration of
compound that fully killed the fungi. The MFC values were determined after assessing the
corresponding MIC data, by transferring sample aliquots (5 μL) from each clear well of the
microtiter tray onto a 150-mm RPMI-1640 agar plate buffered with MOPS. The inoculated plates
were incubated at 15-20 °C; the MFC values were recorded after 48 h for R. stolonifer and at later
times, according to the corresponding control growth, for the rest of the fungi.
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2.5.2.3. Mycelial growth inhibition test
The antifungal activity of the methoximes 6B and 14B was demonstrated in vitro by
examining the inhibition of the mycelial radial growth of the five representative phytopathogenic
fungi (P. digitatum, P. italicum R. stolonifer, B. cinerea and M. fructicola).
A stock solution of each test compound (10 mg/mL) was prepared by dissolution of the
compound in DMSO. This was further diluted to the testing concentrations (31.25, 62.5 and 125
μg/mL), according to the results of the in vitro susceptibility tests, with Potato-Dextrose-Agar
(PDA) medium at 50°C and poured into sterilized Petri dishes (6 cm in diameter). After
solidification, the conidial suspensions (10 µL) adjusted to 1 × 104 CFU/mL of the active fungi,
were placed at the center of the Petri dishes.
The dishes were sealed and incubated at 15-20°C for 2-4 days as appropriate for each of the
fungi. The corresponding growth controls were prepared by admixing volumes of DMSO with PDA
to the equivalent final concentrations. The growth of the fungal mycelia for the treated plates was
measured as the average of three orthogonal diameters when the control plates reached full growth.
The percentage of reduction in colony diameter [antifungal index (%)] was calculated by the
formula of Eq. 1,
Antifungal index (%) = 100 × (C-T)/C
[Eq. 1]
where T and C are the mean diameters of the mycelia in the Test and corresponding Control
samples.
2.5.3. Ex vivo antifungal assay in wounded fruits
2.5.3.1. General
The fruits, namely Salustiana sweet oranges (Citrus sinensis (L.) Osbeck, Rutaceae) and Red
Globe peaches (Prunus persica) were harvested from the Campo Experimental de Cultivos
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Intensivos y Forestales (Facultad de Ciencias Agrarias, Universidad Nacional de Litoral, Esperanza,
Argentina) at the mature stage and sorted based on size and absence of physical injuries or disease
infection.
The fruits were collected before any commercial post-harvest treatment had been applied and
were surfaced-disinfected with 2% (w/v) sodium hypochlorite for 3 min, rinsed with tap water and
then air-dried. The treated fruits were put in 300 mm  500 mm  100 mm plastic boxes covered in
the bottom with filter papers embedded with 25 mL of sterile water in order to maintain a high
relative humidity (90-95%).
2.5.3.2. Test on oranges
The test was carried out according to the literature (Badawy & Rabea, 2009). The fruits were
distributed at random into 3 groups (test, control and comparator), with 10 units each. Each orange
was wounded (3 mm deep and 3 mm wide) with a sterile nail in the upper zone and 10 µL of a
conidial suspension (105 CFU/mL) of P. digitatum was placed into each wound. After 2 h, the fruits
were individually exposed to the different treatments, by immersion during 3 seconds of each
inoculated fruit into a beaker containing the corresponding solutions.
One group was submitted to treatment with compound 14B (125 μg/mL) according to the
results of the in vitro antifungal assay. A second group was used as a negative control and treated
with sterile water, whereas the third group was a positive control where commercial Imazalil (7.8
μg/mL) was used as comparator, according to the results of the in vitro assay.
2.5.3.3. Test on peaches
The test was carried out according to the literature (Egüen, Melgarejo & De Cal, 2015). The
fruits were distributed at random into 3 groups (test, control and comparator), with 21 units each.
Each peach was wounded with a sterile tip in the upper zone and 10 µL of a conidial suspension
(105 CFU/mL) of M. fructicola was placed into each wound. After 2 h, the inoculated fruits were
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individually exposed to the different treatments, by immersion during 3 seconds in a beaker
containing the corresponding solutions. One group was submitted to treatment with compound 14B
(62 μg/mL) according to the results of the in vitro antifungal assay. A second group was used as a
negative control and submerged in sterile water, while the third group was a positive control where
commercial Carbendazim (0.97 μg/mL) was used as comparator, according to the results of the in
vitro assay.
2.5.3.4. Test development and evaluation
The fruits exposed to the different treatments were stored at 20 ºC for 14 days (oranges) or 10
days (peaches). Then, the degree of sporulation (sporulation index) of P. digitatum (for oranges) or
M. fructicola (for peaches) on the surface of the decayed fruits was evaluated on a 0 to 4 scale, in
which the value 0 was assigned to negligible sporulation and 4 referred to a dense fungal
sporulation over the entire fruit. The index value for each fruit was treated as a biological replicate,
and the treatment means were subjected to statistical analysis.
2.5.3.5. Statistical analysis
The experimental data were analyzed statistically by a one-way ANOVA followed by
Tukey’s multiple comparison test (α = 0.05) using the GraphPad Prism v.7.0 software.
3. Results and discussion
3.1. Chemistry. Synthesis of the methoximes
This investigation began with the evaluation of two groups of selected compounds from the
set of methoximes synthesized in our previous work (Cortés, Kaufman & Bracca, 2018), namely
those derived from natural products and simple aromatic derivatives of aldehydes and ketones.
Their chemical structures are shown in Table 2, along with the outcome of their evaluation.
The need of additional compounds to find out and confirm more precisely some structure-
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activity relationships was satisfied mainly by means of a two-step general strategy. This one was
initiated with the synthesis of the precursor aryl ketones (A) by acylation of properly substituted
phenol derivatives (C) with carboxylic acids, under Friedel Crafts conditions, employing BF3.Et2O
as catalyst (Table 1). Then, the resulting ketones were further submitted to methoximation with
MeONH2.HCl employing our previously developed CeCl3.7H2O-based catalytic approach (Cortés,
Kaufman & Bracca, 2018).
Place Table 1 here
The first stage afforded the ketones 4A (Martínez-Cifuentes, Monroy-Cárdenas, Millas-
Vargas, Weiss-López & Araya-Maturana, 2019), the homologous series 6A-9A (Nandinsuren, Shi,
Zhang, Bai & Gao, 2016), and 10A-13A (Liu et al., 2014) in good to excellent yields (52-99%),
whereas in the second step, the methoximes of general structure B (1B-13B) were obtained in good
to excellent yields (56-98%).
In specific cases, such as those of compounds 1A-3A (Janser et al., 2010) and 5A (Tauber,
Imbri, Colligs & Opatz, 2014), additional synthetic steps were executed, taking advantage of the
presence of a rather strong hydrogen bond between the ketone carbonyl and the hydrogen atom of
the neighboring phenol moiety, which enabled to differentiate the phenol group ortho to the
carbonyl moiety from the other free phenols.
Compounds 2A and 5A were prepared in 80% and 64% yield (see Scheme S1), respectively
by mild selective O-methylation of the respective resorcinol derivatives 4A (Martínez-Cifuentes,
Monroy-Cárdenas, Millas-Vargas, Weiss-López & Araya-Maturana, 2019) and 14A (Saha, Perveen,
Nihesh & Sekar, 2019) whereas ketone 3A was synthesized in 86% yield by exhaustive methylation
of 14A (Kusakabe et al., 2017).
On the other hand, the synthesis of compound 1A was performed from 14A by selective
protection of the 4-OH group with dihydropyran (DHP) under TsOH catalysis to afford the
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tetrahydropyranyl ether (THP) 15A, followed by a Williamson methylation. A final deprotection of
the THP ether was performed by acidification with 2M HCl, providing 16A in 52% overall yield for
the three steps.
The syntheses of ω-bromo derivatives 10A-12A were carried out in 68-87% yield from the
corresponding commercial ω-bromoacids (Kawakita et al., 1999), whereas the carboxylic acid 13A
was prepared following the general method, in the presence of excess glutaric acid (Liu et al.,
2014).
Imine derivatives, including oximes, are known to display syn/anti geometric isomerism as well
as rotamerism due to hindered rotation around a C-C bond (Karabatsos & Hsi, 1967). The anti-
isomer was always obtained as the prevalent compound, as stemmed from results of NOE
experiments, which revealed signal enhancement of the neighboring protons in the aliphatic side
chain when the oxime methoxy moiety was irradiated. On the other hand, it was observed that the
three bromoderivatives 10B-12B exhibited NMR signals which evidenced the existence of
1
rotamers. Surprisingly, the most affected H signals were those of the methylene group attached to
the bromine atom observed as pairs of triplets with different areas, in ratios ranging from 0.37:1 to
13
0.94:1. In their C NMR spectra, the corresponding carbon atoms displayed noticeably different
shieldings, with values of ∆δ = 11.3 ± 0.1 ppm. Their unequivocal attribution was confirmed by
HMBC and HSQC experiments.
3.2. Biological activity
3.2.1. Determination of MIC and MFC. Main structure-activity relationships
The tests in vitro were carried out employing the commercial antifungics Imazalil,
Carbendazim and Captan as comparators or positive controls. In the initial screen, it was observed
that the methoxime of 2,3-dimethoxy benzaldehyde (17B) as well as the methoximes derived from
methyl- and benzyl- (18B-21B) ethers of differently substituted acetophenones, and even the
benzophenone methoxime (22B) proved to be essentially inactive (Table 2).
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Luckily, however, examination of the methoximes of some related phenolic aldehydes (23B
and 24B) resulted in mild increase of activity, suggesting the need of a free phenol group.
It was also observed that the nitrophenol derivative 25B also displayed a convenient
bioactivity profile; however, in view of the known toxicity issues related to 2-nitrophenol
derivatives, the preparation of analogs of the latter was not pursued.
Instead, the effect of extending the side chain of resorcinol derivatives was examined,
observing in the ketone derivatives a further improvement in the general activity profile (14B and
26B vs. 24B). Contrastingly, however, in agreement with previous observations, the monomethyl
ether derivative 27B proved to be inactive.
In order to assess the role of the methoxime moiety in the detected bioactivity, the
corresponding ketone precursors (26A and 14A) were also tested. As expected, and in agreement
with our working hypothesis, they proved to be essentially inactive, confirming the relevance of the
methoxime functionality.
Place Table 2 here
In view of the promising activity profile exhibited by the propiophenone methoxime
derivative 14B, the requirement of free phenols in a 1,3-arrangement (resorcinol-type) was
assessed. As shown in Table 3, blocking either one or both phenolic moieties through their
conversion into the corresponding methyl ethers (Compounds 1B-3B) afforded inactive products.
These results were in agreement with the observations with compounds 20B, 26B and 27B, further
confirming that formation of methyl ethers abolishes the antifungal properties of the methoximes;
they also suggested that both free phenols are required for the observed activity.
On the other hand, while the hydroquinone derivative 4B conserved a weak fungistatic
activity, the related monomethyl ether 5B proved to be inactive against the five tested fungal
strains, confirming not only the effect of the O-methylation of the free phenols but also that an
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ortho phenol and the resorcinol-type substitution pattern are the most suitable for eliciting the
observed bioactivity. Next, the optimum side chain length required for the antifungal activity was
investigated through the preparation of the methoximes of entries 6B-9B (4, 5, 6 and 12 carbon
atoms, respectively).
The assays revealed that not all tested strains were equally sensitive to the methoximes and
that the propiophenone methoxime 14A exhibited the best fungistatic potency against P. digitatum,
whereas its congener, the butyrophenone derivative 6B was the most potent analog against P.
italicum and M. fructicola.
In the same way, the valerophenone (C-5) methoxime 7B was the most effective agent against
B. cinerea and the caprophenone (C-6) methoxime 8B was the most powerful analog against R.
stolonifer, being inactive against both strains of Penicillium. On the other hand, the long-chain
analog, the laurophenone (C-12) methoxime 9B proved to be inactive in all cases.
Considering the whole set of the homologous 2,4-dihydroxyphenone methoximes (24B, 26B,
14B and 6B-9B), it can be concluded that the chain length impacts on the potency of the
compounds, which also depends on the tested fungal strain. The agents with the best profiles were
those with 3-5 carbon atoms on their side chains (14B 6B and 7B).
To further elucidate the effects of the side chain on the antifungal activity, three ω-
bromoderivatives with chain lengths of 5, 6 and 8 carbon atoms were prepared (10B-12B). The
bioactivity assays revealed that the ω-bromovalerophenone (10B) displayed an interesting
fungistatic profile; however, the fungistatic and fungicide potencies weakened within this series
when the side chains became longer, to the point that the ω-bromocaprylophenone (12B) was
inactive against all the test strains, except for R. stolonifer.
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Place Table 3 here
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It can also be assumed that the terminal bromine atom may also have a positive effect.
Furthermore, in view of the profile of the ω-bromocaprophenone 11B compared to those of its
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congeners 10B and 12B, it can be safely inferred that the chain length has impact on the potency of
the compound, and that neither the latter nor the activity itself are related to the alkylating ability of
the alkyl bromide motif.
The online applet Osiris Property Explorer (Sander, 2019) is a web tool useful to predict
various physicochemical and toxicological molecular properties of small organic molecules. The
application contains a database of traded drugs and commercially available compounds assumable
as non-drug-like molecules, which is used to assess the occurrence frequency of each fragment in
the individual tested structure (sub-structure similarity) in order to estimate its drug likeliness.
The program can also compute several drug-relevant properties and provide a drug-score, as a
measure of the potential of the compound to meet the criteria of a possible drug candidate. This is a
weighted average of several predictions, including cLogP, logS, molecular weight and drug
likeliness, along with the risks of mutagenicity and tumorigenicity, and the probability of
reproductive and irritating effects.
The Osiris Property Explorer predicted that the members of the homologous series of the
resorcinol-derived methoximes were neither mutagenic nor tumorigenic, being also devoid of
irritating effects or reproductive risk (see Supporting Information).
The application also revealed that, among the compounds of the homologous series, the
propiophenone methoxime 14B has the highest values of drug likeliness (1.38; score = 0.80) and
drug score (0.84). In addition, it uncovered that, despite slightly improved potency in the in vitro
test, the values of drug likeliness sharply decay for the higher analogs (6B-9B), with more than
three carbon atoms in the side chain.
Furthermore, the predictions on the brominated derivatives 10B-12B, suggested that they may
possess all of the above undesirable risks; as a result, their drug likeliness was negative (score = 0)
and the drug score dropped below 0.05. Therefore, it was concluded that the propiophenone
methoxime derivative 14B displayed the best parameters.
The hydrolytic cleavage of carbon–nitrogen double bonds is reversible and their hydrolysis is
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catalyzed by acids; in addition, the oximes are hydrolytically labile, even under neutral conditions
(Kalia & Raines, 2008). For example, the half-life of the methoxime of pivaldehyde is 25 days.
Therefore, the short-term hydrolytic stability of compound 14B was tested under neutral conditions,
proving to be stable for 2 weeks, to the 1H NMR sensitivity (no evident signs of the related ketone
14A were observed in the spectrum).
On the other hand, when the photostability of 14B was tested, it was observed that the
compound slowly degrades to the related oxime (probably through iminyl radical formation),
exhibiting the concomitant generation of 5% MeOH (δ 3.50 ppm) after a 6 h exposure to white
light.
3.2.2. Macroscopic examination of agar plates
The antifungal activity of compounds 6B and 14B was also assessed in agar plates against B.
cinerea, M. fructicola, R. stolonifer and P. italicum for the first one and against M. fructicola and P.
digitatum for the second one, by verifying the inhibition of the radial growth of their mycelia
(Figure 1). The solid medium assay was performed by triplicate at a final concentration of 31.25,
62.5 or 125 μg/mL according to the corresponding results of MICs. At these levels, the compounds
inhibited the hyphal growth of all tested phytopathogenic fungi.
As shown in Figure 1A, compound 6B inhibited the mycelial growth of B. cinerea compared
to the control (antifungal index = 86%). Further, the vegetative mycelium in the control plates
resulted lax and expansive, while the treated plates exhibited a compact and reduced core. At the
same time, as depicted in Figure 1B, it was interesting to observe that the oxime was able to
completely inhibit the growth of the pathogen M. fructicola in this assay.
Compound 6B was also tested against P. italicum, where the observed antifungal index was
62% after 96 h incubation at a final concentration of 62.5 µg/mL (Figure 1C). In this case, the
vegetative mycelium in the control plates clearly exhibited an expansive growth, accompanied by
reproductive structures in the central zone as usually observed in this fungus; on the contrary, the
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reproductive mycelium was hardly visualized in the treated plates.
Insert Figure 1 here
Regarding R. stolonifer (Figure 1D), it proved to strongly inhibit the mycelial growth
compared to the control, showing an antifungal index = 83% after 48 h, at a final concentration of
31.25 µg/mL. Notably, the corresponding growth control exhibited its characteristic hairy
appearance and the vegetative mycelium essentially covered the whole plate. In comparison, the
treated sample just developed into expansion, but grew up over the inoculation zone.
The activity of 14B was also examined against M. fructicola. At a final concentration of 62.5
µg/mL, it displayed an unequivocal inhibition of the mycelial growth compared to the control,
showing an antifungal index = 92% after 96 h (Figure 1E). The untreated colonies displayed their
characteristic pattern of greyish concentric rings, which were rather absent in the treated samples.
The vegetative mycelium in the control plates was loose and covered the entire plate, while in the
treated plates it was presented as a very compact and caked mass of hyphae.
Finally, when P. digitatum was exposed to 14B, at a final concentration of 125 µg/mL, the
antifungal index displayed was 50% after a 96 h incubation period (Figure 1F). The vegetative
mycelium in the control plates demonstrated an expansive growth, accompanied by its reproductive
structures in the central zone, as usually seen with this fungus; meanwhile, in the treated plates,
reproductive mycelia are hardly visualized. Interestingly, however, the premature presence of
spores in the treated plates could be an indication that the fungus would try to escape from the
hostile environment caused by the oxime.
3.2.3. Morphology of the colonies. Microscopic examination
The morphology of the colonies in the Petri plates was examined employing optical
microscopy. Representative photographs exhibiting hyphae malformations of some of the
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phytopathogens in response to their exposure to the oximes and the corresponding controls are
shown in Figure 2.
Figures 2A and 2D show the images of mycelia obtained from the outer part of the colonies of
M. fructicola stained with Lactophenol Blue. In the control plate (Figure 2D) the presence of a
healthy vegetative mycelium and the lemon-shaped spore chains characteristic of this
phytopathogen are clearly visible. In contrast, the hyphae of M. fructicola treated with the test
oxime 6B (Figure 2A) are shorter and show a significantly smaller diameter; in addition, they
exhibit a tip-shaped apex, unlike the rounded edge that is visualized in the control preparation.
These observations may indicate a possible alteration of the fungal cell wall.
On the other hand, Figures 2B and 2E show images corresponding to the mycelia obtained
from the outer part of the colonies of R. stolonifer. In the control sample (Figure 2E), broad and
coenocytic hyphae characteristic of Rhizopus are observed, whereas spores released over a
sporangium structure are notably visible. Contrastingly, however, in the counterpart treated with
14B (Figure 2B) the hyphae are narrower and no reproductive structures are observed.
Insert Figure 2 here
In addition, the Figures 2C and 2F show the images of mycelia obtained from the outer part of
the colonies of P. italicum. The control sample (Figure 2F) displays a typical mycelium, where
hyphae and conidiophores bearing the characteristic conidia of the Penicillium genus are clearly
observed. Instead, the colony exposed to the oxime 14B (Figure 2C) evidenced thinner and less
expanded hyphae.
Summing up, significant differences were observed in the fungi treated with the most active
methoximes 6B and 14B. The data revealed dramatically reduced growth of fungal hyphae in the
treated colonies compared with the controls. Moreover, the hyphae in the specimens exposed to the
methoximes were thinner and apparently exhibited undeveloped structures, compared with the
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corresponding controls.
3.2.4. Effect on freshly collected fruits. Peaches and oranges
As a final test of our hypothesis, the effect of compound 14B, one of the most active
methoximes was examined ex-vivo, in fresh Red Globe peaches infected with M. fructicola. The
results were compared with the application of the commercial product Carbendazim (Figure 3A),
through the determination of the sporulation index of infected fruits (Vilanova, Teixidó, Torres,
Usall, Viñas & Sánchez-Torres, 2016). To that end, 14B was applied to wound-inoculated peaches
by immersion in a 62 μg/mL solution of the methoxime. A control set without antifungal agents was
also prepared.
Insert Figure 3 here
After 10 days, it was observed that the control set exhibited clear signs of fungal infection, to
the extent to require discarding the whole lot of fruits. Additionally, the sets of treated fruits
indicated beyond doubt that both, 14B and Carbendazim, have protected the fruits, reducing
significantly the brown rot sporulation index with respect to the control peaches.
In addition, examination of the set of peaches treated with the commercial agent gratifyingly
revealed that while some fruits had clear signs of fungal infection, less specimens of those exposed
to 14B seemed to be infected. Furthermore, despite no significant difference was observed between
the fruits treated with Carbendazim and those with 14B, the results suggest that the latter could be
even more powerful than the commercial agent under the test conditions.
An analogous test was also carried out on freshly harvested Salustiana sweet oranges, infected
with P. digitatum (Figure 3B), using Imazalil as commercial comparator, where the wound-
inoculated oranges were exposed to 14B by immersion in a 125 μg/mL solution of the compound.
After 14 days, essentially the same profile of results was observed. The control set showed obvious
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signs of attack by the pathogen, displaying 100% of infected fruits; conversely, the sets of treated
oranges revealed signs of infection with noticeable lower intensity.
Both treatments reduced significantly (P < 0.05) green mold sporulation index with respect to
control oranges. Only 40% of the oranges exposed to Imazalil were visibly infected, whereas solely
20% of the fruits treated with 14B exhibited clear infection; furthermore, the infected area seemed
to be smaller in the oranges treated with the methoxime 14B than in those exposed to the
commercial product and the control set.
The experimental results hint that the potencies of the methoxime 14B and the commercial
antifungal agents against the tested phytopathogens are not too different. They also suggest that the
methoxime should be considered as an excellent candidate for further development.
4. Conclusions
Finding potent and economic alternatives to current fungicides for controlling postharvest
phytopathogenic fungi is indispensable. Following our hypothesis, that the methoxime moiety is a
suitable pharmacophore that may provide a solution to this case, a series of methoxime derivatives
of aromatic aldehydes and ketones has been prepared in good to excellent yield, characterized by
spectroscopic means and evaluated in vitro as antifungal agents against a panel of five relevant
strains of postharvest phytopathogenic fungi, namely B. cinerea, M. fructicola, P. digitatum, P.
italicum and R. stolonifer.
Success in the demonstration of the hypothesis was crowned by a structure-activity
relationship analysis of the in vitro results, which revealed that the observed biological activity is
strongly related to the presence of the methoxime moiety, a 2,4-dihydroxyarene pattern and a
suitable side chain of 3-5 carbon atoms. Blocking the phenolic hydroxyls or removing the
methoxime unit to unveil the corresponding ketone abolished the observed bioactivity.
As a final verification of the hypothesis, the activity of one of the compounds (14B) was
tested ex vivo on freshly collected oranges and peaches, demonstrating in both cases that under the
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experimental conditions, the results of applying the methoxime are not statistically different than
those obtained from exposing the fruits to the commercial products Carbendazim and Imazalil. The
overall results are promising and suggest that these compounds, among them 14B may have great
potential to be further developed as usable protective-contact fungicides.
Furthermore, the synthesis of 14B and its congeners is simple and straightforward, requiring
inexpensive and easily available precursors and uncomplicated synthetic methodology. Research is
in progress to design and develop methoxime derivatives with more enhanced biological activity.
Acknowledgments
I.C. and M.G.D.L. thank CONICET for their fellowships.
Funding
This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET, PUE IQUIR 2016 and PIP N° 2015-0524) and Agencia Nacional de Promoción
Científica y Tecnológica (ANPCyT, PICT 2017-0149 and PICT 2015-2259).
Conflict-of-interest
The authors declare no conflict of interest.
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