Synthesis and Biological Evaluations of a Series of Thaxtomin

Mar 25, 2015 - Molesworth , P. P.; Gardiner , M. G.; Jones , R. C.; Smith , J. A.; Tegg , R. S.; Wilson , C. Aust. J. Chem. 2010, 63, 813– 820 DOI: ...
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Synthesis and Biological Evaluations of a Series of Thaxtomin Analogues Hongbo Zhang,†,‡,§ Qingpeng Wang,†,‡,§,∥ Xin Ning,‡ Hang Hang,‡ Jing Ma,‡,§,∥ Xiande Yang,‡,⊥ Xiaolin Lu,‡,⊥ Jiabao Zhang,‡,⊥ Yonghong Li,§ Congwei Niu,§ Haoran Song,§ Xin Wang,*,‡,§,∥ and Peng George Wang*,‡,§,∥ ‡

College of Pharmacy, §State Key Laboratory of Elemento-organic Chemistry, and ∥Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, People’s Republic of China S Supporting Information *

ABSTRACT: Thaxtomins are a unique family of phytotoxins with unique 4-nitroindole and diketopiperazine fragments possessing potential herbicidal activities. This work presents the total synthesis of natural product thaxtomin C and its analogues. The extensive structure−activity relationship study screens four effective compounds, including thaxtomin A and thaxtomin C. It is indicated that 4-nitro indole fragment is essential for phytotoxicity, while benzyl and m-hydroxybenzyl substituents on the diketopiperazine ring are favorable for the efficacy. The N-methylations on indole and diketopiperazine show weak influence on the herbicidal activities. The four selected compounds show effective herbicidal activities against Brassica campestris, Amaranthus retroflexus, and Abutilon theophrasti, which are comparable or better than dichlobenil, even at a dosage of 187.5 g ha−1. Moreover, these four compounds show good crop-selective properties to different crops and exhibit moderate protoporphyrinogen oxidase (PPO) enzyme inhibition. The antifungal results indicate that thaxtomin C displays inhibition to a wide range of fungi. KEYWORDS: thaxtomin, herbicide, structure−activity relationship, weed control, antifungal

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INTRODUCTION The exploitation of new pesticides, especially the pesticides with new scaffolds and novel action modes, has become an increasing focus in agricultural chemistry. Herbicides, as one of the most important kinds of pesticides, have accounted for approximately 40% of total pesticide expenditures and play a more and more important role in agricultural production all over the world. Nevertheless, resistance of weeds to herbicides has become more and more serious because of the wide application of various herbicides. Moreover, to the best of our knowledge, no herbicide with new target sites was commercialized in the recent 20 years.1−5 Therefore, it is an urgent task to develop new herbicides, especially the herbicides with new target sites and modes of action. Thaxtomins are a unique family of phytotoxins with unique 4-nitroindole and diketopiperazine fragments possessing potential herbicidal activity.6−9 A total of 11 thaxtomin analogues, including thaxtomin A (TA) and thaxtomin C (TC) (Figure 1), have been isolated from natural materials.10−12

the modes of action of these compounds were reported to be different with the known cellulose biosynthesis inhibitors (CBIs) dichlobenil and isoxaben.13−19 Recently, we first reached the chemical total synthesis of TA and its three stereoisomers, and the bioactive results indicated that this unique structural backbone represented a rich resource for developing new herbicides.20 As a continuation of the development of new herbicides, it is of great interest for us to synthesize compounds with the unique thaxtomin scaffold. Herein, a series of thaxtomin analogues (Table 1) are prepared. The influence of N-methylations of R2, R3, and R4 on indole and diketopiperazine were tested by the design of compounds 1, 2, and 4. Moreover, different groups of R5 on the diketopiperazine ring were also investigated. Then, with the aim of exploring the effects of nitro on the indole ring at R1 to bioactivities, compounds 3a and 3b with no nitro as controls were also prepared. The herbicidal properties and antifungal activities of target compounds were evaluated, and the structure−activity relationship and modes of action of these thaxtomin analogues were discussed.

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General. All reactions were carried out under an atmosphere of nitrogen in flame-dried glassware with magnetic stirring, unless otherwise indicated. Reagents were obtained from Alfa Aesar, Aldrich, J&K, and GL Biochem, Ltd. Tetrahydrofuran (THF) was dried by distillation over Na/benzophenone. Dichloromethane (DCM) was

Figure 1. Structures of TA and TC.

Particularly TA, the predominant member of such family, shows effective weed control ability and exerts no toxicity to rice. Biochemically, thaxtomins have been proven to induce weed growth inhibition by inhibiting cellulose synthesis. However, © 2015 American Chemical Society

MATERIALS AND METHODS

Received: Revised: Accepted: Published: 3734

December March 24, March 25, March 25,

18, 2014 2015 2015 2015 DOI: 10.1021/jf506153t J. Agric. Food Chem. 2015, 63, 3734−3741

Article

Journal of Agricultural and Food Chemistry

Echinochloa crus-galli and Digitaria sanguinalis, were evaluated according to a previously reported procedure.21−23 All title compounds were screened at a adosage of 1500 g ha−1. Then, compounds TA, 1a, 2a, and 2f with higher herbicidal activities were further assayed at lower dosages of 750, 375, and 187.5 g ha−1. Dichlobenil was selected as a positive control. All treatments were carried out in triplicate (Tables 4 and 5). Treatment. The emulsions of tested compounds were prepared by dissolving them in 100 μL of N,N-dimethylformamide (DMF) with the addition of a little Tween 20 and diluting with proper water to the desired concentrations. Then, these emulsions were sprayed using a microsprayer. As negative control experiments, the mixture of the same amount of water, DMF, and Tween 20 with no compounds was also tested. Herbicidal activities of dichlobenil at the same concentrations as tested compounds were carried out as positive control experiments. Pre-emergence. Sandy clay (100 g) in a plastic box (11 × 7.5 × 6 cm) was wetted with water. A total of 15 sprouting seeds of the weed under test were planted in fine earth (0.6 cm depth) in the glasshouse and sprayed with the test compound solution. Post-emergence. Seedlings (one leaf and one stem) of the weed were sprayed with the tested compounds at the same rate as used for the pre-emergence test. Data Collection. For both methods, the fresh weights of three parallel plants were determined 20 days later, and the percentage inhibition relative to the controls was calculated by eq 1

Table 1. Structural Formula of the Title Compounds

inhibition (%) = (wc − wt)/wc × 100%

(1)

where wc is the wet weight of the negative control and wt is the wet weight of the tested group. Crop Selectivity. Compounds TA, 1a, 2a, and 2f were selected for crop selectivity evaluation. Conventional wheat, corn, cotton, and peanut were respectively planted in plots (diameter = 12 cm) containing test soil and grown in a greenhouse at 20−25 °C. After the plants had reached the four-leaf stage, the spraying treatment was conducted at different dosages by diluting the formulations of tested compounds with water. The visual injury and growth state of the individual plant were observed at regular intervals. The fresh weights were determined 30 days later, and the percentage inhibition relative to the controls was calculated. Dichlobenil was selected as a positive control (Table 6). Protoporphyrinogen Oxidase (PPO) and Acetohydroxyacid Synthase (AHAS) Inhibition Activity. The recombinant tobacco PPO enzyme (mtPPO) was expressed according to the established protocol.24−27 Oxadiazon, fluazolate, and sulfentrazone were selected as positive controls. The product of the enzymatic reaction has a maximum excitation wavelength at 410 nm and a maximum emission wavelength at 630 nm. PPO activity was assayed by measuring the formation of protoporphyrin IX at room temperature using a fluorescence detector with the excitation and emission wavelengths set at 410 and 631 nm, respectively. The concentration of protoporphyrinogen IX was determined according to the absorption of protoporphyrin IX, and the concentration of protoporphyrin IX was calculated from the calibration graph. In each assay, the stock dimethyl sulfoxide (DMSO) solution (1% total volume) of inhibitor was added to the reaction system. The final inhibitor concentration ranged from 0.005 to 500 μM. The enzymatic reaction rate was measured in a reagent buffer of 100 mM potassium phosphate (pH 7.5), 5 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), Tween 80 (0.03%, v/v), 200 mM imidazole, 5 μM flavin adenine dinucleotide (FAD), and approximately 0−40 μg of protein. The IC50 value was determined by measuring PPO activity over a range of inhibitor concentrations at a fixed substrate concentration. IC50 values were calculated by fitting v versus [I] data to a single binding site model described by eq 2

dried by distillation over CaH2. Thin-layer chromatography (TLC) was performed on silica gel GF254 plates. Column chromatography was performed on silica gel (200−300 mesh). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE AV400 (400 and 100 MHz). All NMR chemical shifts were referenced to residual solvent peaks or to tetramethylsilane (TMS) as an internal standard. All coupling constants J were quoted in hertz. High-resolution mass spectra (HRMS) were obtained on an IonSpec QFT mass spectrometer with electrospray ionization (ESI). Optical rotations were recorded on a PerkinElmer 341 polarimeter. Melting points were measured on an X4 apparatus. Herbicidal Activities. The initial herbicidal activities of compounds 1−4 and TA against dicotyledon weeds, including Brassica campestris and Amaranthus retroflexus, and monocotyledon weeds, including

y = min +

max − min 1 + 10 x − log IC50

(2)

where y is the percentage of maximal rate, with max and min being the y values at which the curve levels off, x is the logarithm of the inhibitor 3735

DOI: 10.1021/jf506153t J. Agric. Food Chem. 2015, 63, 3734−3741

Article

Journal of Agricultural and Food Chemistry Table 2. 1H NMR Data of Title Compounds 1−4 1

compound 1a

1b

1c 1d 1e 2a 2b 2c

2d 2e 2f

3a 3b

4a 4b 4c

4d

H NMR

1.98−2.11 (m, 1H, CH2), 2.70 (dd, J1 = 13.6 Hz, J2 = 5.3 Hz, 1H, CH2), 2.79 (dd, J1 = 13.5 Hz, J2 = 4.9 Hz, 1H, CH2), 3.02 (dd, J1 = 14.5 Hz, J2 = 4.2 Hz, 1H, CH2), 3.78 [m, 1H, diketopiperazine (DKP) CH], 4.06 (d, J = 5.3 Hz, 1H, DKP CH), 7.09 (d, J = 8.2 Hz, 3H, Ph-H), 7.23 (q, J = 8.0 Hz, 2H, Ph-H), 7.31 (t, J = 7.4 Hz, 2H, indole CH), 7.65 (d, J = 2.8 Hz, 1H, indole CH), 7.76 (t, J = 9.0 Hz, 2H, indole CH, DKP NH), 7.96 (s, 1H, DKP NH), 11.72 (s, 1H, indole NH) 2.03 (dd, J1 = 14.2 Hz, J2 = 8.9 Hz, 1H, CH2), 2.55−2.62 (m, 1H, CH2), 2.69 (dd, J1 = 13.7 Hz, J2 = 4.7 Hz, 1H, CH2), 3.05 (dd, J1 = 14.3 Hz, J2 = 4.2 Hz, 1H, CH2), 3.69 (m, 1H, DKP CH), 3.95 (m, 1H, DKP CH), 6.64−6.77 (m, 2H, Ph-H), 6.82−6.94 (m, 2H, Ph-H), 7.14 (d, J = 1.8 Hz, 1H, indole CH), 7.22 (t, J = 7.9 Hz, 1H, indole CH), 7.56 (d, J = 2.8 Hz, 1H, indole CH), 7.77 (t, J = 8.0 Hz, 2H, DKP NH, indole CH), 7.86 (d, J = 2.5 Hz, 1H, DKP NH), 9.23 (s, 1H, OH), 11.61−11.99 (m, 1H, indole NH) 1.37−1.50 (m, 2H, CH2), 1.61 (m, 1H, CH2), 1.95−2.05 (m, 1H, CH2), 2.12−2.30 (m, 2H, CH2), 3.70 (m, 1H, DKP CH), 4.07 (m, 1H, DKP CH), 7.03 (d, J = 7.5 Hz, 2H, Ph-H), 7.17 (m, 2H, Ph-H, indole CH), 7.26 (t, J = 7.5 Hz, 2H, Ph-H), 7.50 (d, J = 2.3 Hz, 1H, indole CH), 7.71 (m, 2H, DKP NH, indole CH), 7.86 (s, 1H, DKP NH), 8.07−8.17 (m, 1H, indole CH), 11.86 (s, 1H, indole NH) 3.23−3.30 (m, 2H, CH2), 3.43−3.49 (m, 2H, DKP CH2), 3.84−3.95 (m, 1H, DKP CH), 7.24 (t, J = 7.9 Hz, 1H, indole CH), 7.51 (d, J = 2.6 Hz, 1H, indole CH), 7.74−7.86 (m, 3H, DKP NH, indole CH), 7.91 (d, J = 3.1 Hz, 1H, DKP NH), 11.86 (s, 1H, indole NH) 0.61−0.91 (m, 8H, i-butyl CH2, CH3), 1.01 (m, 1H, i-butyl CH), 1.54 (m, 1H, DKP CH), 3.27 (d, J = 8.2 Hz, 1H, indole-CH2), 3.57 (m, 1H, indoleCH2), 4.05 (m, 1H, DKP CH), 7.21 (t, J = 7.9 Hz, 1H, indole CH), 7.48 (s, 1H, indole CH), 7.63−8.15 (m, 4H, DKP NH, indole CH), 11.81 (s, 1H, indole NH) 2.12 (m, 1H, CH2), 2.59−2.69 (m, 2H, CH2), 2.73 (s, 3H, DKP-CH3), 3.02 (m, 1H, CH2), 3.84 (m, 1H, DKP CH), 3.94 (m, 1H, DKP CH), 6.87−6.93 (m, 2H, Ph-H), 7.18−7.32 (m, 5H, indole CH, Ph-H), 7.72−7.92 (m, 3H, DKP NH, indole CH), 11.75−11.94 (m, 1H, indole NH) 2.05 (m, 1H, CH2), 2.57 (m, 1H, CH2), 2.65−2.68(m, 1H, CH2) 2.70 (s, 3H, DKP-CH3), 3.00 (m, 1H, CH2), 3.78 (t, J = 6.2 Hz, 1H, DKP CH), 3.84−3.88 (m, 1H, DKP CH), 6.63−6.74 (m, 4H, Ph-H), 7.17−7.28 (m, 2H, indole CH), 7.72−7.77 (m, 1H, indole CH), 7.77−7.83 (m, 2H, DKP NH, indole CH), 9.22 (s, 1H, OH), 11.87 (s, 1H, indole NH) 0.84 (m, 1H, CH2), 1.44 (m, 1H, CH2), 2.24 (m, 1H, CH2), 2.34 (m, 1H, CH2), 2.83 (s, 3H, DKP-CH3), 3.36 (d, J = 5.7 Hz, 1H, CH2), 3.49−3.57 (m, 2H, DKP CH, CH2), 4.04 (t, J = 5.2 Hz, 1H, DKP CH), 6.97−7.04 (m, 2H, Ph-H), 7.14−7.22 (m, 2H, Ph-H, indole CH), 7.26 (t, J = 7.5 Hz, 2H, Ph-H), 7.43 (d, J = 2.5 Hz, 1H, indole CH), 7.66 (d, J = 8.0 Hz, 1H, indole CH), 7.74 (d, J = 7.8 Hz, 1H, indole CH), 8.22 (d, J = 3.1 Hz, 1H, DKP NH), 11.90 (m, 1H, indole NH) 2.72 (s, 3H, DKP-CH3), 2.93 (m, 1H, CH2), 3.31 (m, 1H, CH2), 3.43 (m, 2H, DKP CH2), 3.93 (t, J = 5.8 Hz, 1H, DKP CH), 7.25 (t, J = 7.9 Hz, 1H, indole CH), 7.46 (d, J = 2.5 Hz, 1H, indole CH), 7.82 (d, J = 7.8 Hz, 3H, DKP NH, indole CH), 11.93 (s, 1H, indole NH) 0.65 (t, J = 6.7 Hz, 6H, i-butyl CH3), 0.99−1.13 (m, 2H, i-butyl CH2), 1.16−1.19 (m, 1H, i-butyl CH), 2.76 (s, 3H, DKP-CH3), 3.35−3.36 (m, 2H, indole-CH2), 3.44−3.49 (m, 1H, DKP CH), 3.97−4.06 (m, 1H, DKP CH), 7.22 (t, J = 7.9 Hz, 1H, indole CH), 7.44 (d, J = 2.5 Hz, 1H, indole CH), 7.76 (dd, J1 = 10.2 Hz, J2 = 7.9 Hz, 2H, indole CH), 8.09 (d, J = 3.3 Hz, 1H, DKP NH), 11.70−12.04 (m, 1H, indole NH) 2.33 (dd, J1 = 13.4 Hz, J2 = 6.3 Hz, 1H, CH2), 2.44−2.49 (m, 1H, CH2), 2.58−2.71 (m, 4H, DKP-CH3, CH2), 2.85 (m, 1H, CH2), 3.74 (t, J = 6.5 Hz, 1H, DKP CH), 3.97 (m, 1H, DKP CH), 6.38 (d, J1 = 7.6 Hz, 1H, Ph-H), 6.47−6.55 (m, 1H, Ph-H), 6.63 (dd, J1 = 8.0 Hz, J2 = 1.6, 1H, Ph-H), 7.09 (t, J = 7.8 Hz, 1H, Ph-H), 7.17 (d, J = 2.5 Hz, 1H, indole CH), 7.24 (t, J = 7.9 Hz, 1H, indole CH), 7.79 (t, J = 7.6 Hz, 2H, indole CH), 7.88 (d, J = 3.2 Hz, 1H, DKP NH), 9.39 (s, 1H, OH), 11.88 (d, J = 2.5 Hz, 1H, indole NH) 1.86 (m, 1H, CH2), 2.45 (d, J = 3.0 Hz, 1H, CH2), 2.55 (d, J = 5.7 Hz, 1H, CH2), 2.77−2.88 (m, 1H, CH2), 3.86 (s, 1H, DKP CH), 3.98 (s, 1H, DKP CH), 6.71 (d, J = 7.2 Hz, 2H, Ph-H), 6.94−7.02 (m, 2H, Ph-H), 7.07 (t, J = 7.5 Hz, 1H, Ph-H), 7.16 (m, 3H, indole CH), 7.31 (t, J = 10.4 Hz, 1H, indole CH), 7.49 (d, J = 7.8 Hz, 1H, indole CH), 7.70 (s, 1H, DKP NH), 7.90 (s, 1H, DKP NH), 10.88 (s, 1H, indole NH) 0.88−0.99 (m, 1H, CH2), 1.22−1.28 (m, 1H, CH2), 1.80−1.88 (m, 2H, CH2), 3.02 (dd, J1 = 14.4 Hz, J2 = 4.7 Hz, 1H, CH2), 3.29 (d, J = 4.0 Hz, 1H, CH2), 3.58−3.63 (m, 1H, DKP CH), 4.16 (m, 1H, DKP CH), 6.75−6.81 (m, 2H, Ph-H), 6.92−6.97 (m, 1H, Ph-H), 6.99−7.04 (m, 1H, Ph-H), 7.06 (d, J = 2.3 Hz, 1H, indole CH), 7.08−7.13 (m, 1H, Ph-H), 7.15−7.24 (m, 3H, indole CH), 7.62 (d, J = 7.8 Hz, 1H, indole CH), 8.06 (d, J = 2.3 Hz, 1H, DKP NH), 8.14 (d, J = 2.2 Hz, 1H, DKP NH), 10.89 (s, 1H, indole NH) 1.12−1.31 (m, 1H, CH2), 1.47−1.65 (m, 1H, CH2), 2.45 (m, 2H, CH2), 2.67 (s, 3H, DKP-CH3), 2.82 (s, 3H, DKP-CH3), 3.43 (m, 2H, CH2), 3.71 (s, 3H, indole-CH3), 3.71 (s, 1H, DKP CH), 4.03 (t, J = 4.8, 1H, DKP CH), 7.07 (d, J = 8.0 Hz, 2H, Ph-H), 7.20−7.32 (m, 4H, indole CH, Ph-H), 7.48−7.55 (m, 1H, indole CH), 7.67−7.73 (m, 1H, indole CH), 7.77−7.86 (m, 1H, indole CH) 2.42 (d, J = 1.3 Hz, 3H, DKP-CH3), 2.54 (d, J = 2.0 Hz, 1H, DKP CH2), 2.81 (s, 3H, DKP-CH3), 3.27−3.31 (m, 1H, DKP CH2), 3.39 (d, J = 5.4 Hz, 1H, indole-CH2), 3.43 (d, J = 8.0 Hz, 1H, indole-CH2), 3.86 (m, 3H, indole-CH3), 4.04 (t, J = 5.0 Hz, 1H, DKP CH), 7.32 (t, J = 8.0 Hz, 1H, indole CH), 7.41 (s, 1H, indole CH), 7.84 (d, J = 7.8 Hz, 1H, indole CH), 7.92 (d, J = 8.2 Hz, 1H, indole CH) 0.81−0.91 (m, 6H, i-butyl CH3), 1.29 (m, 1H, i-butyl CH), 1.53−1.68 (m, 2H, i-butyl CH2), 2.71 (s, 3H, DKP-CH3), 2.79 (s, 3H, DKP-CH3), 3.34−3.41 (m, 2H, indole-CH2), 3.74 (d, J = 5.7 Hz, 1H, DKP CH), 3.86 (s, 3H, indole-CH3), 3.95 (t, J = 6.0 Hz, 1H, DKP CH), 7.31 (t, J = 8.0 Hz, 1H, indole CH), 7.56 (s, 1H, indole CH), 7.81 (dd, J1 = 8.0 Hz, J2 = 1.0 Hz, 1H, indole CH), 7.89 (dd, J1 = 8.1 Hz, J2 = 1.0 Hz, 1H, indole CH) 1.48 (m, 1H, CH2), 2.15−2.29 (m, 1H, CH2), 2.44 (d, J = 3.8 Hz, 3H, DKP-CH3), 2.77 (dd, J1 = 14.9 Hz, J2 = 3.9 Hz, 1H, CH2), 2.85 (d, J = 3.7 Hz, 3H, DKP-CH3), 2.94 (dd, J1 = 14.8 Hz, J2 = 4.1 Hz, 1H, CH2), 3.73 (d, J = 3.7 Hz, 3H, indole-CH3), 3.97 (m, 1H, DKP CH), 4.17 (m, 1H, DKP CH), 6.54−6.64 (m, 2H, Ph-H), 7.03−7.24 (m, 6H, indole CH, Ph-H), 7.39 (d, J = 8.0 Hz, 1H, indole CH), 7.48 (d, J = 7.6 Hz, 1H, indole CH)

a 2 L round-bottomed flask. Then, the culture was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) when A600 reached 0.7− 0.8. Cells were grown for an additional 3 h at 37 °C and harvested by centrifugation at 6000 rpm for 10 min. The cell pellet was suspended in 10 mL of solution (10 mM imidazole, 0.5 M KCl, 50 mM Tris−HCl at pH 8.0, and 20 μM FAD) for every gram. The solution was cooled in an ice bath and subjected to disruption in a SONICS VCX500 model ultrasonic processor with a 1.3 cm flat-tip probe for 60 cycles (1 s each with 9 s pauses). The disrupted cells were centrifuged at 27000g for 1 h at 4 °C to remove cellular debris. The purification of 6× His tag AHAS II was carried out to near homogeneity in a single step by nickel−nitrilotriacetic acid (Ni−NTA) affinity chromatography as described in QIAexpressionist (Qiagen).28 The enzyme was stored in aliquots at −80 °C. One aliquot was analyzed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE), with protein concentrations determined by the method of Bradford.29−32 The inhibitory activity of the title compounds against AHAS enzyme

concentration, and IC50 is the concentration of the inhibitor that caused 50% of the total inhibition. Because the inhibitors belong to the class of competitive inhibitors, the calculated Ki value can be obtained by applying the following relationship among Ki, Km, and IC50 at any saturating substrate concentration (S) by the following eq 3. Ki =

IC50 S/K m + 1

(3)

The expression vector pQE-GMwt, containing the wild-type Escherichia coli AHAS II gene, was provided by professor D. M. Chipman (Ben-Gurion University). The wild-type E. coli AHAS II was expressed from the plasmid pQE-GMwt. A single colony of the E. coli strain XL1-Blue, transformed with the plasmid pQE-GMwt, was inoculated in 20 mL of Luria−Bertani (LB) medium containing 100 μg mL−1 ampicillin. The culture was incubated overnight at 37 °C. The overnight medium was transferred into 500 mL of LB medium in 3736

DOI: 10.1021/jf506153t J. Agric. Food Chem. 2015, 63, 3734−3741

Article

Journal of Agricultural and Food Chemistry

in vitro was measured according to Westerfeld33−35 and Singh36,37 methods. Chlorimuron ethyl and bispyribac sodium were selected as a positive control. The reaction mixture contained a 50 mM potassium phosphate buffer (pH 7.5), 10 mM MgCl2, 1 mM thiamine diphosphate (ThDP), 10 μM FAD, 100 mM pyruvate, and the enzyme in the absence or presence of various concentrations of inhibitors. The mixture was preincubated at room temperature for 5 min before initiation of the reaction. Then, the reaction was started by the addition of AHAS enzyme and allowed to incubate at 37 °C for 1 h, before being stopped by the addition of 3 M H2SO4 (1/10 final volume). Acetolactate was converted to acetoin by incubation at 60 °C for 15 min. Acetoin was then quantified by measuring the absorbance at 525 nm after incubation at 60 °C for 15 min with 0.5% creatine and 5% (w/v) α-naphthol. The apparent inhibition constant (Ki) values were determined by fitting the data to eq 4. The Ki values represented the concentration of the inhibitor at 50% inhibition.

Table 3. Melting Point and HRMS Data of Title Compounds 1−4 compound

mp (°C)

1a 1b 1c 1d 1e 2a 2b 2c 2d 2e 2f 3a 3b 4a 4b 4c 4d

255.0−256.0 256.5−258.0 276.5−278.2 294.0−296.2 277.4−279.4 252.0−254.0 165.0−167.0 236.5−238.0 225.0−226.5 267.5−269.5 163.0−165.0 254.5−256.5 270.5−272.5 255.0−257.0 215.0−217.0 250.0−252.5 266.4−268.2

HRMS (M (M (M (M (M (M (M (M (M (M (M (M (M (M (M (M (M

+ + + + + + + + + + + + + + + + +

H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+ H)+

379.1396 395.1356 393.1563 289.0937 345.1564 393.1551 409.1500 407.1715 303.1087 359.1719 409.1504 334.1551 348.1709 435.2020 331.1405 387.2023 376.2021

νi = ν∞ + (ν0 − ν∞)/(1 + [I ]/K i)

(4)

In eq 4, νi and ν0 represented the rates in the presence or absence of inhibitor, respectively, and [I] was the concentration of the inhibitor. If the initial analysis indicated that the residual activity (ν∞) at a saturating inhibitor concentration was not significantly greater

Scheme 1. Synthesis of Title Compounds

Table 4. Herbicidal Activity of Compounds (Percent Inhibition) (Rate = 1500 g ha−1)a BC compound 1a 1b 1c 1d 1e 2a 2b 2c 2d 2e 2f 3a 3b 4a 4b 4c 4d TA Dicb a

pre 85.0 0.0 10.0 0.0 0.0 100.0 5.0 5.0 0.0 0.0 100.0 10.0 0.0 10.0 5.0 10.0 10.0 100.0 100.0

± 1.2 ± 0.5

± 1.3 ± 0.9

± 0.4 ± ± ± ±

AR post

1.2 1.0 0.8 1.1

30.0 15.0 10.0 0.0 10.0 100.0 0.0 0.0 0.0 5.0 100.0 10.0 15.0 5.0 0.0 15.0 5.0 80.0 20.0

pre

± 0.4 ± 2.1 ± 1.1 ± 1.7

± 0.1 ± 1.1 ± 0.7 ± 0.5 ± ± ± ±

1.6 0.8 1.5 1.2

95.0 0.0 0.0 0.0 0.0 100.0 0.0 10.0 10.0 0.0 95.0 0.0 0.0 0.0 0.0 0.0 0.0 100.0 100.0

± 1.3

± 2.2 ± 0.6 ± 0.7

EC post 5.0 0.0 0.0 0.0 0.0 61.3 0.0 0.0 0.0 0.0 80.0 0.0 10.0 0.0 0.0 0.0 0.0 70.0 5.0

± 1.4

± 1.6

± 2.4 ± 0.7

± 1.1 ± 1.5

DS

pre

post

0.0 0.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 40.0 ± 0.9 95.4 ± 1.7

0.0 0.0 0.0 0.0 0.0 55.6 ± 2.1 0.0 0.0 0.0 0.0 20.0 ± 1.1 0.0 0.0 0.0 0.0 0.0 0.0 30.0 ± 0.6 0.0

pre 15.0 0.0 0.0 0.0 0.0 100.0 5.0 0.0 0.0 0.0 50.0 0.0 0.0 0.0 0.0 0.0 0.0 45.0 64.9

± 0.8

± 0.6

± 0.8

± 1.4 ± 2.1

post 20.0 0.0 0.0 0.0 0.0 50.0 0.0 0.0 0.0 0.0 65.0 0.0 0.0 0.0 0.0 0.0 0.0 10.0 0.0

± 1.2

± 1.3

± 0.7

± 1.3

BC, B. campestris; AR, A. retroflexus; EC, E. crus-galli; DS, D. sanguinalis; post, post-emergence; pre, pre-emergence; and Dicb, dichlobenil. 3737

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1.4

2.4 1.9

0.6 1.2

1.1

0.6 0.4

0.4

0.7

1.3 1.5

1.2 1.1

/ 0.0 2.2 ± / 0.0 0.0 / 22.2 ± 7.7 ± / 4.6 ± 0.0 /

0.0 0.0

post pre

0.0 0.0 / 100.0 100.0 / 65.1 ± 30.7 ± / 3.2 ± 0.0 / 55.6 ± 24.6 ± / ± 1.4 ± 0.6

1.8 1.2 1.0 1.2 0.6 1.5 1.5 0.7 0.7 0.9 ± ± ± ± ± ± ± ± ± ±

post

15.0 10.0 2.1 15.7 10.8 2.8 59.2 36.2 16.6 15.2 0.0 0.0 2.1 1.7 0.0 1.1 1.7 1.3 1.3 1.2 0.9 0.7 0.6 1.3 1.3 1.7 1.1 0.8 1.2 0.8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

pre

60.6 50.3 29.1 56.0 41.8 36.5 46.5 39.4 30.1 33.0 29.4 17.0 73.4 57.1 42.9 1.4 0.5 1.2 0.4 0.1 1.3 1.1 0.7 0.5 0.6 0.3 0.6 0.7 0.9 0.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.1 ± 1.1 ± 0.8

± 1.5

post

17.9 14.4 6.8 14.1 6.5 3.8 3.5 2.6 1.2 3.8 2.4 1.8 11.8 8.5 5.3 0.7 1.2 0.8 0.8 0.7 0.2 ± ± ± ± ± ±

pre

67.6 57.4 52.2 91.9 91.2 83.1 100.0 100.0 100.0 99.3 96.3 94.9 100.0 100.0 96.3 0.3 0.8 0.5 1.5 2.4 1.1 1.8 1.3 1.0 0.5 0.4 1.5 1.5 0.7 0.1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.5

± 0.6

± 1.5 ± 0.4

± 1.3 ± 1.5 ± 0.6

Dicb

TA

2f

2a

post

15.6 10.5 9.2 76.1 26.1 18.1 20.9 13.0 9.7 52.1 44.1 25.1 8.8 5.9 1.0 ± 1.1 ± 0.2

pre

49.5 0.6 0.0 88.1 86.0 68.9 100.0 98.1 86.2 100.0 100.0 98.1 100.0 100.0 87.6 750 375 187.5 750 375 187.5 750 375 187.5 750 375 187.5 750 375 187.5 1a

dosage (g ha−1) compound

BC, B. campestris; AR, A. retroflexus; AT, A. theophrasti; EC, E. crus-galli; DS, D. sanguinalis; SG, S. glauca; SV, S. viridis; CV, Cernuella virigata; post, post-emergence; pre, pre-emergence; and Dicb, dichlobenil. b“/” = not tested.

pre post pre post

29.1 ± 1.6 12.1 ± 1.2 / 93.1 ± 0.3 88.5 ± 1.5 / 67.8 ± 1.8 42.6 ± 1.1 / 70.5 ± 0.7 33.6 ± 1.8 / 19.9 ± 0.4 15.8 ± 1.4 / 30.4 ± 1.7 18.3 ± 0.6 / 90.2 ± 0.1 54.4 ± 1.1 / 38.2 ± 0.7 31.0 ± 1.5 / 51.5 ± 0.8 48.5 ± 0.9 / 61.6 ± 0.8 50.8 ± 0.9 /

pre pre

3.5 ± 0.0 / 3.5 ± 0.9 ± / 20.9 ± 10.4 ± / 6.1 ± 0.0 / 0.0 0.0 /

0.5

14.6 ± 2.2 5.6 ± 1.3 / 40.4 ± 0.7 14.6 ± 1.3 / 38.2 ± 1.3 12.2 ± 1.2 / 0.0 0.0 / 0.0 0.0 /

post

34.9 ± 0.9 7.3 ± 1.4 / 89.7 ± 0.9 46.7 ± 0.3 / 51.3 ± 0.4 10.7 ± 0.9 / 41.8 ± 1.0 33.0 ± 1.2 / 22.4 ± 1.2 10.7 ± 2.1 /

SV SG DS EC AT AR BC

Table 5. Further Herbicidal Activity of Compounds TA, 1a, 2a, and 2f (Percent Inhibition)a,b

than zero, the data were reanalyzed with ν∞ = 0. Kiapp was calculated by nonlinear least squares and the simplex method for error minimization.38−41 The Ki values were summarized in Table 7. Antifungal Activity. Compounds 1−4 and TA were evaluated for their antifungal activities against Candida albicans [American Type Culture Collection (ATCC) 76615], Candida utiliz, Saccharomyces cerevisia, and Aspergillus flavus according to the National Committee for Clinical Laboratory Standards (NCCLS).42 Fluconazole was selected as the control. A spore suspension in sterile distilled water was prepared from a 1-day-old culture of the fungi growing on Sabouraud agar (SA) media. The final spore concentration was 1−5 × 103 spore mL−1. From the stock solutions of the tested compounds and reference antifungal fluconazole, dilutions in sterile RPMI 1640 medium (Neuronbc Laboraton Technology Co., Ltd., Beijing, China) were made resulting in 11 wanted concentrations (0.25−256 μg mL−1) of each tested compound. These dilutions were inoculated and incubated at 35 °C for 24 h. The growth was monitored visually and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of fungi was regarded as the minimum inhibitory concentration (MIC). All of the antifungal activities were tested 3 times. The MICs were summarized in Table 8. Data Collection. The tested compounds and reference drugs were prepared in Mueller−Hinton broth by 2-fold serial dilution to obtain the required concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg/mL. The data were collected only if three of the MIC values were consistent or three of the MICs were ranged in two neighboring concentrations (two MICs were consistent, and this concentration was collected).

■

RESULTS AND DISCUSSION Chemistry. The total chemical synthesis of a series of thaxtomin analogues differs in R1 and R2 on indole, and R3, R4, and R5 on the diketopiperazine ring are also synthesized. The detailed 1H NMR (Table 2), 13C NMR, optical rotation, and high-resolution mass spectra as well as melting point data (Table 3) of all title compounds are reported, and NMR and high-resolution mass spectrometry (HRMS) spectra are presented (see the Supporting Information). The synthetic route of title compounds are given in Scheme 1. Compounds 843 and 920 were prepared according to the reported synthetic routes. Compounds 5 and 6 could be obtained in 90% yields by the deprotection of precursors 8 and 9 in solution of dichloromethane/trifluoroacetic acid (4:1, v/v) at room temperature for 30 min. The tryptophan methyl ester 7 was commercially available. The condensation of compounds 5, 6, and 7 with N-Boc-amino acid respectively in the presence of propylphosphonic acid anhydride (T3P), following by the deprotection of Boc moiety in dichloromethane/trifluoroacetic acid (4:1, v/v), afforded the key intermediates. The cyclization step44 of the key intermediates with morpholine as a base in dichloromethane at room temperature for 48 h produced the target compounds 1a−1e, 2a−2f, 3a, and 3b in yields of 50− 90%. The further methylation of compounds 1c−1e and 3a with MeI and Ag2O afforded compounds 4a−4d in yields of 80−90%. Herbicidal Activities. The pre-emergence and postemergence herbicidal activities in Table 4 manifest that substituents at R1 and R5 significantly affect the activities of title thaxtomin analogues, while the methylations of R2, R3, and R4 show a weak influence on the herbicidal efficiency. It is observed that this series of compounds generally exhibits more effective pre-emergence herbicidal activities than post-emergence herbicidal activities. Compounds TA, 1a, 2a, and 2f with the benzyl group and m-hydroxybenzyl groups at R5 show significant pre-emergence herbicidal activity against dicotyledon

a

/ 71.1 ± 0.2 28.9 ± 0. 5 / 0.0 0.0 / 0.0 0.0 / 55.6 ± 0.7 2.2 ± 1.4 / / 36.3 ± 0.5 21.4 ± 1.2 / 41.0 ± 0.8 11.5 ± 1.5 / 38.2 ± 0.6 0.0 / 36.3 ± 0.6 0.0 /

0.0 0.0 / 0.0 0.0 / 0.0 0.0 / 0.0 0.0 / 0.0 0.0 / 0.0 0.0 0.0 0.0

CV

post

Journal of Agricultural and Food Chemistry

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Article

Journal of Agricultural and Food Chemistry Table 6. Crop Selectivity of Compounds TA, 1a, 2a, and 2f (Post-emergence; Percent Inhibition) dosage (g ha−1)

compound

wheat

750 375 750 375 750 375 750 375 750 375

1a 2a 2f TA Dicb

4.1 0.0 0.0 0.0 15.9 13.3 17.5 0.0 7.6 4.7

corn

± 0.2

43.6 13.4 31.8 24.1 27.1 9.9 0.0 0.0 36.2 24.9

± 1.6 ± 0.3 ± 1.2 ± 0.7 ± 0.5

compound

PPO ± ± ± ± / / 0.024 ± 0.0062 ± 0.030 ± 75.04 41.51 39.94 45.75

a

AHAS − − − − 0.0221 ± 0.0064 24.0 ± 15.8 / / /

7.43 2.38 1.83 0.42

0.0007 0.0009 0.0006

“−” = no activity, and “/” = not tested.

Table 8. Antifungal Data as MIC (μg mL−1) for Compoundsa,b fungi compound

C. albicans

C. utiliz

S. cerevisiae

A. flavus

1a 1b 1c 1d 1e 2a 2b 2c 2d 2e 2f 3a 3b 4a 4b 4c 4d TA fluconazole

128 32 64 64 256 64 256 128 256 128 256 256 256 128 128 256 64 256 4

256 256 256 128 256 64 256 128 128 128 256 256 256 64 128 256 128 128 16

256 512 256 128 128 64 256 128 256 128 256 256 256 128 128 128 128 64 16

64 128 128 128 256 64 256 64 64 128 128 128 128 128 128 256 64 128 256

cotton 0.5 1.5 0.8 0.4 0.7 0.4

± 1.2 ± 0.4

0.0 0.0 0.0 0.0 8.6 ± 0.4 0.0 10.7 ± 0.8 0.0 0.0 0.0

peanut 7.7 5.0 26.2 14.6 36.0 22.4 36.8 24.5 25.0 7.9

± ± ± ± ± ± ± ± ± ±

1.0 0.7 2.2 0.8 0.9 0.1 1.7 0.5 2.1 0.6

compounds 3a and 3b as controls were tested. The results manifest that the herbicidal activities disappear with the removal of the nitro group. Noticeably, compound 3a exhibits rather decreased activities (≤10.0%) in comparison to its precursor 1a with nitro on indole. Then, further methylations of ineffective compounds 1c−1e and 3a at R2, R3, and R4 to afford compounds 4a−4d lead no significant enhancements of their weed control competence. Besides, benzyl and m-hydroxybenzyl at R5 of these thaxtomin analogues are more favorable for herbicidal activities than other substituents. Nitro at R1 is essential for herbicidal efficacy, and the N-methylation of R2, R3, and R4 is not helpful for herbicidal activity. Compounds TA, 1a, 2a, and 2f with effective activities were selected for further herbicidal testing at lower dosages (Table 5). The results indicate that compound 1a shows moderate activity at 750 g ha−1 dosage to weeds but low activity at a lower dosage in most instances. Compounds TA, 2a, and 2f exhibit generally better weed control ability against dicotyledons (B. campestris, A. retroflexus, and Abutilon theophrasti) than monocotyledons, which are comparable or even better than positive control dichlobenil even at low dosage of 187.5 g ha−1. Especially, TA and compound 2f show excellent pre-emergence herbicidal activity against B. campestris and A. retroflexus with an inhibitory percent up to 86.2% at a dosage of 187.5 g ha−1, which are superior to dichlobenil. Meanwhile, compound 2a also exerts effective inhibition to monocotyledons E. crus-galli, Setaria glauca, and Setaria viridis. These results enable these compounds to be of much potential for further investigations as new herbicides. Crop Selectivity. On the basis of the herbicidal activities mentioned above, compounds TA, 1a, 2a, and 2f were selected for crop selectivity evaluation. As depicted in Table 6, four compounds display different crop selectivity to tested crops. Wheat and cotton exhibit absolute tolerance to compounds 1a and 2a with benzyl at R5 by post-emergence application even at the dosage of 750 g ha−1. Meanwhile, compound 2f with m-hydroxybenzyl is safe for cotton at a dosage of 375 g ha−1. Then, TA with C-hydroxyl diketopiperazine ring exerts no injury to corn at a high dosage of 750 g ha−1, while wheat and cotton are also absolutely tolerant to TA at the dosage of 375 g ha−1. However, peanut is susceptible to all four tested compounds. These results indicate that the substituents, especially the benzyl moiety at R5 and the hydroxyl group on diketopiperazine, affect crop selectivity properties of thaxtomin analogues. The tested compounds may have the potential as herbicides in wheat, corn, and cotton fields. PPO and AHAS Inhibition Activity. It was observed that the injury symptoms of thaxtomins in most instances were similar to known CBI dichlobenil. Meanwhile, the tested compounds also caused substantial wilting on all species following

Table 7. PPO and AHAS Inhibition Activity of Compounds TA, 1a, 2a, and 2f (Ki, μM)a 1a 2a 2f TA chlorimuron ethyl bispyribac sodium oxadiazon fluazolate sulfentrazone

± ± ± ± ± ±

a

MICs were determined by the microbroth dilution method for microdilution plates. bC. albicans, Candida albicans (ATCC 76615); C. utiliz, Candida utiliz; S. cerevisia, Saccharomyces cerevisia; and A. flavus, Aspergillus flavus.

weeds B. campestris (≥85.0%) and A. retroflexus (≥95.0%). However, when the group at R5 is exchanged by other substituents, such as p-hydroxybenzyl, phenylethyl, or alkyl groups or hydrogen (compounds 1b−1e and 2b−2e), the herbicidal properties disappear (percent inhibitions ≤15.0% to all weeds). Then, to investigate the influence of the nitro moiety at R1, 3739

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Journal of Agricultural and Food Chemistry

■

post-emergence applications, which was similar to PPO or AHAS inhibitors. Thus, the PPO and AHAS inhibition experiments of compounds TA, 1a, 2a, and 2f were carried out. Results shown in Table 7 indicate that TA, 1a, 2a, and 2f all exhibit moderate PPO inhibitory activity but no AHAS inhibition. These results might be a reasonable explanation for why thaxtomins cause substantial wilting on weeds beside CBI mode of action and indicate that thaxtomins might act as multi-target herbicides. Antifungal Activities. It is revealed that thaxtomins could inhibit the synthesis of cellulose, which is a major component of the fungal cell wall. This situation inspired us to evaluate the antifungal activity of title thaxtomin analogues. The MIC values in Table 8 manifested that the nitro group at R1 is favorable for antifungal activities, and the introduction of the methyl moiety on indole and diketopiperazine at R2, R3, and R4 has no significant influence on their bioactivities. Compounds 1, 2, and TA with nitro group R1 give relatively better activities in contrast to compounds 3a and 3b. Especially the natural compound TC (2a) exhibits the most broad and effective antifungal efficacy among the tested compounds, with MICs of 64 μg mL−1 to all strains. Additionally, compounds 1b−1d show moderate inhibition to C. albicans (MIC = 32−64 μg mL−1); meanwhile, compounds 1a and 2c−2d could inhibit the growth of A. flavus at a concentration of 64 μg mL−1. Subsequently, the N-methylations of R2, R3, and R4 in compounds 1c−1e and 3a to synthesize compounds 4a−4d do not result in improved antifungal activities. In summary, compound 2a displays the broadest and most effective activities among the tested thaxtomin analogues and might be a potential lead compound for further development of novel antifungal agents. In conclusion, a series of thaxtomin analogues were synthesized and evaluated for herbicidal property, PPO and AHAS inhibition, crop selectivity and antifungal activity. The greenhouse experiments indicate that nitro at R1 and benzyl or m-hydroxybenzyl fragments at R5 significantly affect herbicidal activities of thaxtomin analogues; meanwhile, N-methylations at R2, R3, and R4 lead to a slight influence on the efficacy. Especially compounds TA, TC (2a), 1a, and 2f exhibit effective activities, which are comparable or even superior to positive control dichlobenil at a dosage of 187.5 g ha−1. Moreover, the selected compounds show moderate PPO inhibition and satisfactory crop selectivity results. The above results indicate that the selected compounds TA, TC, 1a, and 2f might be potential lead compounds for future development of novel herbicides. Additionally, antifungal bioassays show that some compounds, especially TC, display effective inhibition to the tested fungi. Eventually, an attractive biological performance of thaxtomins mentioned above could prove to be of prime importance in future endeavors at understanding the mode of action of thaxtomins and developing new herbicides based on this natural resource backbone.

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Article

AUTHOR INFORMATION

Corresponding Authors

*Telephone: +086-022-23501642. Fax: +86-022-23505369. E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

Hongbo Zhang and Qingpeng Wang contributed equally to this work. Author Contributions ⊥

Xiande Yang, Xiaolin Lu, and Jiabao Zhang contributed equally to this work.

Funding

This research was supported by the National Natural Science Foundation of China (Grants 21102076, 91013013, and 31100587) and the Natural Science Foundation of Tianjin (Grant 10JCYBJC04100). Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

* Supporting Information S

General, synthetic procedures for the preparation of 4-nitro tryptophan methyl ester 5, preparation of 4-nitro N-methyltryptophan methyl ester 6, preparation of the title compounds 1a−1e, preparation of the title compounds 2a−2f, preparation of the title compounds 3a and 3b, preparation of the title compounds 4a−4d, and preparation of TA, and NMR and HRMS spectra. This material is available free of charge via the Internet at http://pubs.acs.org. 3740

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