Article pubs.acs.org/JAFC
Design, Synthesis, Antifungal, and Antioxidant Activities of (E)‑6-((2Phenylhydrazono)methyl)quinoxaline Derivatives Mao Zhang,†,‡,⊥ Zhi-Cheng Dai,†,⊥ Shao-Song Qian,∥ Jun-Yan Liu,§ Yu Xiao,† Ai-Min Lu,† Hai-Liang Zhu,∥ Jian-Xin Wang,† and Yong-Hao Ye*,† †
College of Plant Protection, State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China ‡ Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China § Center for Nephrology and Clinical Metabolomics, Division of Nephrology and Rheumatology, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai 200072, People’s Republic of China ∥ School of Life Sciences, Shandong University of Technology, Zibo 255049, People’s Republic of China S Supporting Information *
ABSTRACT: Different substituted phenylhydrazone groups were linked to the quinoxaline scaffold to provide 26 compounds (6a−6z). Their structures were confirmed by 1H and 13C NMR, MS, elemental analysis, and X-ray single-crystal diffraction. The antifungal activities of these compounds against Rhizoctonia solani were evaluated in vitro. Compound 6p is the most promising one among all the tested compounds with an EC50 of 0.16 μg·mL−1, more potent than the coassayed positive control fungicide carbendazim (EC50: 1.42 μg·mL−1). In addition, these compounds were subjected to antioxidant assay by employing diphenylpicrylhydrazyl (DPPH) and mice microsome lipid peroxidation (LPO) methods. Most of these compounds are potent antioxidants. The strongest compounds are 6e (EC50: 7.60 μg·mL−1, DPPH) and 6a (EC50: 0.96 μg·mL−1, LPO), comparative to or more potent than the positive control Trolox [EC50: 5.90 μg·mL−1 (DPPH) and 18.23 μg·mL−1 (LPO)]. The structure and activity relationships were also discussed. KEYWORDS: quinoxaline, phenylhydrazone, fungicide, antioxidant
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INTRODUCTION Fungal pathogens are the primary causes of both plant diseases and postharvest losses,1,2 and thus blamed for the severe damage of global crop production.3 To guard against fungal pathogens, one traditional approach is to employ synthetic fungicides, which are both economical and efficient, and have played an indispensable role in nourishing more people throughout human history. Unfortunately, drug resistances, environmental hazards, and many other drawbacks emerged along with the fungicide utilization,4,5 which require novel antifungal agents to be discovered. Oxygen-derived free radicals and reactive oxygen species (ROS) such as superoxide anions (O2−•) and hydroxyl radicals (HO•) are generated during the normal bioorganic redox process. They play key roles in regulating cellular functions, since they are intermediate metabolites in several enzymatic reactions involved in post-translational protein turnover and the controlling of signal transductions. There are innate defense mechanisms in human bodies to scavenge free radicals in order to sustain the homeostasis. However, the unbalance of such mechanisms leads to the progression of oxidative stress and numerous diseases, especially various chronic and age-related ones.6−8 As important supplements to the scavenging mechanisms, antioxidants (both naturally occurring and synthetic) have been widely applied in pharmaceuticals, foods, and cosmetics. Quinoxaline is also known as benzo[b][1,4]diazine. Because of the presence of the para-posed double nitrogens, the © 2014 American Chemical Society
condensed ring shows distinctive physicochemical and biological properties.9,10 Quinoxaline derivatives cover nearly all ranges of activities, such as antibacterial,11 antifungal,12 pesticidal,13 antiviral,14 antineoplastic,15,16 antitubercular,17 antimalarial,18 antidepressant,19 and immunosuppressive20 activities. Meanwhile, owing to the unique R1R2NNCR3R4 structure, hydrazones show a variety of activities,21 such as anticonvulsant,22 herbicide,23 antimicrobial,24 antioxidant,25 miticidal,26 and anticancer27 activities. The structure−antioxidant activity relationships study of phenylhydrazones28 disclosed that the NH proton is necessary for the radical scavenging activity (Figure 1). A large number of compounds commercialized or under clinical trials belongs to the quinoxaline or hydrazone family, which has long been offering new pharmaceutical or agrochemical solutions (Figure 2). Carbadox (Pfizer) and Cyadox (Spofa/Chemapol), both used as antimicrobial and growthpromoting agents, are the early successful models of combining these two groups together. Here, to discover novel antifungal and antioxidant candidates, we propose to report the design and syntheses as well as antifungal and antioxidant activity evaluations of (E)-6-((2-phenylhydrazono)methyl)quinoxaline derivatives. Received: January 20, 2014 Accepted: September 17, 2014 Published: September 17, 2014 9637
dx.doi.org/10.1021/jf504359p | J. Agric. Food Chem. 2014, 62, 9637−9643
Journal of Agricultural and Food Chemistry
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respectively. The substituents introduced here include −Me, −OMe, −F, −Cl, −Br, −NO2, −CF3, −OCF3, and −CN. Hydrazone crystals precipitated from methanol in the last step were filtered and washed gently with petroleum ether. Crystallographic Study. The crystal of 6x was obtained directly from mother methanol liquor without recrystallization. X-ray singlecrystal diffraction data for compound 6x were collected on a Bruker SMART AEPEX CCD diffractometer at 273(2) K using Mo Kα radiation (λ = 0.71073 Å) by the π and ω scan mode. The program SAINT was used for integration of the diffraction profiles. The structure was solved by direct methods using the SHELXS program of the SHELXTL package and refined by full-matrix least-squares methods with SHELXL.31 All non-hydrogen atoms of compound 6x were refined with anisotropic thermal parameters. All hydrogen atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms. Antifungal Activity Assay. The antifungal activity was evaluated as reported protocols.32,33 Preliminary determination of the inhibitory activity of compounds 6a−6z against four plant-pathogenic fungi (Rhizoctonia solani, Fusarium graminearum, Sclerotinia sclerotiorum, and Phytophthora capsici) was conducted in vitro by using the mycelial growth test on PDA (Potato Dextrose Agar) medium. The compounds were dissolved in DMSO and then mixed with sterile molten PDA to obtain final concentrations of 10 μg·mL−1 (containing 4‰ DMSO). PDA with different compounds was poured into 90 mm Petri dishes (15 mL·dish−1), on which 5 mm mycelial disks of the four fungi were planted in the center. The disks were obtained from a pure PDA culture plate by punching at the edge of the actively growing mycelia colony. Three replicates were conducted for each treatment. The widely used commercial fungicides validamycin A and
Figure 1. Reaction pattern of phenylhydrazones with DPPH radical proposed by predecessors,28 in which the NH proton is necessary for the radical scavenging activity.
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METHODS
Materials and spectral data are presented in the Supporting Information. Synthesis and Purification. The reaction route is outlined in Scheme 1. The previously reported protocol29 has been adopted for preparing 2 and 3. The authors changed “heating” to a milder “sun light at room temperature” condition in the bromination step. In the purification step, because of their similar polarities, single and double brominated substances along with nonbrominated compound 2 were collected together from a column using petroleum ether−EtOAc (2:1). Then, the mixture was oxidized by DMSO to give aldehyde 3 which was later purified by column (petroleum ether−EtOAc = 5:1). The preparations of 5a−5z and 6a−6z were performed according to the methods described by Harden et al.30 and Belkheiri et al.,25
Figure 2. Design of the target structure: Above are examples of quinoxaline and hydrazone derivatives commercialized or under clinical trials as agrochemicals or pharmaceuticals. There are earlier successful attempts as Carbadox and Cyadox of combining the two groups together. Herein, we designed the structure below to discover novel biologically active candidates. 9638
dx.doi.org/10.1021/jf504359p | J. Agric. Food Chem. 2014, 62, 9637−9643
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Scheme 1. Synthesis Route and Structures of the Phenylhydrazone Analoguesa
(A) oxalaldehyde (40% aqueous), Na2CO3, 100 °C, 2 h. (B) (i) NBS, benzoyl peroxide, CCl4, sun light, r.t., 3−4 h; (ii) NaHCO3, DMSO, 120 °C, 2 h. (C) (i) HCl, NaNO2, 0 °C, 1 h; (ii) SnCl2, 0 °C, 30 min, r.t., 1 h; (iii) NaOH (40%), pH 7. (D) MeOH, r.t.
a
Table 1. Crystallographic Data of Compound 6x compound
6x
chemical formula formula weight crystal system space group crystal color a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å) Z Dcalc (g·cm−3) θ range (deg) hkl range F(0 0 0) no. collected refl. no. ind. refl. (Rint) data/restraints/parameters absorption coefficient (mm−1) R1; wR2 [I > 2σ(I)] R1; wR2 (all data) GOOF
C16H11F3N4 316.29 monoclinic P21/c light yellow 15.322(4) 8.462(2) 11.824(3) 90.00 106.013(9) 90.00 1473.5(7) 4 1.426 2.77−25.71 −16 ≤ h ≤ 18; −10 ≤ k ≤ 10; −14 ≤ l ≤ 14 648 13602 2806 (0.0584) 2806/0/208 0.115 0.0822; 0.2189 0.1456; 0.2448 0.956
DPPH Radical Scavenging Assay (RSA). The scavenging rate of DPPH radical was carried out by the improved protocol of previous literatures.34,35 Briefly, in 96-well microliter plates, 100 μL of different concentrations (2.5, 5, 10, 20, 40, 80, and 160 μg·mL−1 in EtOH) of compounds 6a−6z were mixed with 100 μL of DPPH solution (0.2 mmol·L−1 in EtOH) to be detected as OD6a−OD6z. 100 μL of different concentrations (as above) of these compounds were mixed with 100 μL of EtOH to be detected as OD6a′−OD6z′. 100 μL of DPPH was mixed with 100 μL of EtOH to be detected as ODblank. 200 μL of EtOH was to be detected as ODE. And 100 μL of different concentrations (as above) of Trolox (positive control) were mixed with 100 μL of DPPH solution to be detected as ODT. The reaction mixtures were shaken vigorously and stored in the dark at 25 °C for 30 min, and the absorbance (OD) was measured at 517 nm using a SpectraMax M5 microplate reader. All of the assays were performed
carbendazim were co-tested as positive controls while mere DMSO (4‰ contained in PDA) as negative control. After a certain incubation period (1.5 d for R. solani, 3 d for F. graminearum, 2.5 d for S. sclerotiorum, and 4 d for P. capsici, according to their different mycelial growth rates) at 25 °C in the dark, mycelial growth diameters were measured. The inhibition percentages were calculated as (B − A)/(B − 5) × 100%, where A is the mycelial diameter (mm) in Petri dishes with compounds and B is the diameter (mm) of the negative control. Furthermore, the fungicidal activity of these compounds against R. solani was assessed in the same method as above. According to the preliminary test, these compounds were dissolved in DMSO to obtain different final concentration grads. Mycelial growth diameters were measured, and the inhibition percentages relative to the control with 4‰ DMSO were calculated. The EC50 values were calculated using linear-regression analysis. 9639
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three times. The radical scavenging rate was measured as a decrease in the OD of DPPH and was calculated using the following equation: scavenging rate % = 100 × [(OD blank − ODE ) − (OD6(a − z) − OD6(a − z)′)]/(ODblank − ODE ) The concentration of a certain compound necessary to decrease the initial DPPH concentration by 50% (EC50 μg·mL−1) was determined by linear regression analysis of data obtained by plotting the scavenging rate % against the concentrations of that compound. Inhibition of Microsomal LPO. Mouse liver microsomes used for the LPO studies were prepared adopting the method in the literature.36 The adult male mice (8−12 weeks old) of Kunming strain (purchased from the Comparative Medicine Center of Yangzhou University) were used for the preparation of liver microsomes. The protein content of microsomes was measured using the method in the literature.37 Briefly, microsomes (0.67 mg of protein/mL) were incubated at 37 °C for 60 min with test compounds at varying concentrations, 10 mM FeSO4 and 0.1 mM ascorbic acid in 1.0 mL of potassium phosphate buffer solution (0.2 M, pH 7.4). Afterward, the reaction was stopped by 20% (w/v) trichloroacetic acid (1.0 mL) and 0.67% (w/v) 2-thiobarbituric acid (1.5 mL) in succession. Then, the solution was heated to 100 °C for 15 min. After centrifugation of precipitated protein, the absorbance (OD) of the color reaction of malondialdehyde (MDA)−TBA complex was detected at 535 nm by a SpectraMax M5 microplate reader. Trolox was the positive control. In the same way as DPPH-RSA, the inhibition rates and EC50 (μg·mL−1) values were determined.
Figure 3. ORTEP view showing the atom-labeling scheme with thermal ellipsoids drawn at 30% probability for compound 6x.
Table 2. Inhibitory Percentage Test against the Mycelial Growtha inhibitory percentage of compounds in 10 μg·mL−1 (%) compound
Rhizoctonia solani
Fusarium graminearum
Sclerotinia sclerotiorum
Phytophthora capsici
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 6p 6q 6r 6s 6t 6u 6v 6w 6x 6y 6z
78.34 73.25 79.62 75.16 81.04 86.43 81.57 77.14 77.71 75.16 67.52 83.14 58.57 76.43 89.17 92.99
