Synthesis and Biological Evaluations of a Series of Thaxtomin Analogues

Article abstract of DOI:10.1021/jf506153t

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 struc

Full text of DOI:10.1021/jf506153t

Article
pubs.acs.org/JAFC
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
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.
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 commer-
cialized 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 ana-
logues, including thaxtomin A (TA) and thaxtomin C (TC)
(Figure 1), have been isolated from natural materials.10−12
MATERIALS AND METHODS
■
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,
Received: December 18, 2014
Revised: March 24, 2015
Accepted: March 25, 2015
Published: March 25, 2015
© 2015 American Chemical Society
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Echinochloa crus-galli and Digitaria sanguinalis, were evaluated
according to a previously reported procedure.21−23
Table 1. Structural Formula of the Title Compounds
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 con-
centrations 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
inhibition (%) = (w − w)/w × 100%
(1)
c
t
c
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) con-
taining 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 proto-
porphyrinogen 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
1
was performed on silica gel (200−300 mesh). H 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.
max − min
y = min +
1 + 10x−log IC
50
(2)
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
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
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1
Table 2. H NMR Data of Title Compounds 1−4
compound
1H NMR
1a
1b
1c
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)
1d
1e
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, indole-
CH2), 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)
2a
2b
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)
2c
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)
2d
2e
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)
2f
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)
3a
3b
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)
4a
4b
4c
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)
4d
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)
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.
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 chromatog-
raphy 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
IC50
S/Km + 1
Ki =
(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
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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 diphos-
phate (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)
HRMS
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
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
(M + H)+ 379.1396
(M + H)+ 395.1356
(M + H)+ 393.1563
(M + H)+ 289.0937
(M + H)+ 345.1564
(M + H)+ 393.1551
(M + H)+ 409.1500
(M + H)+ 407.1715
(M + H)+ 303.1087
(M + H)+ 359.1719
(M + H)+ 409.1504
(M + H)+ 334.1551
(M + H)+ 348.1709
(M + H)+ 435.2020
(M + H)+ 331.1405
(M + H)+ 387.2023
(M + H)+ 376.2021
3a
3b
4a
4b
4c
4d
νi = ν∞ + (ν0 − ν∞)/(1 + [I]/Ki)
(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
a
Table 4. Herbicidal Activity of Compounds (Percent Inhibition) (Rate = 1500 g ha−1)
BC
AR
EC
DS
compound
pre
post
pre
post
pre
0.0
post
0.0
pre
post
1a
1b
1c
85.0 1.2
0.0
30.0 0.4
15.0 2.1
10.0 1.1
0.0
95.0 1.3
0.0
5.0 1.4
15.0 0.8
20.0 1.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.0 0.5
0.0
0.0
0.0
0.0
0.0
1d
1e
2a
2b
2c
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.0 1.7
100.0
0.0
0.0
0.0
0.0
0.0
0.0
100.0
100.0
0.0
61.3 1.6
0.0
100.0
0.0
55.6 2.1
100.0
5.0 0.6
0.0
50.0 1.3
5.0 1.3
5.0 0.9
0.0
0.0
0.0
0.0
0.0
10.0 2.2
10.0 0.6
0.0
0.0
0.0
0.0
0.0
2d
2e
2f
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.0 0.1
100.0
0.0
0.0
0.0
0.0
0.0
100.0
95.0 0.7
0.0
80.0 2.4
0.0
100.0
0.0
20.0 1.1
50.0 0.8
0.0
65.0 0.7
3a
3b
4a
4b
4c
10.0 0.4
0.0
10.0 1.1
15.0 0.7
5.0 0.5
0.0
0.0
0.0
0.0
0.0
0.0
10.0 0.7
0.0
0.0
0.0
10.0 1.2
5.0 1.0
10.0 0.8
10.0 1.1
100.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
15.0 1.6
5.0 0.8
80.0 1.5
20.0 1.2
0.0
0.0
0.0
0.0
0.0
0.0
4d
TA
Dicb
0.0
0.0
0.0
0.0
0.0
0.0
100.0
100.0
70.0 1.1
5.0 1.5
40.0 0.9
95.4 1.7
30.0 0.6
0.0
45.0 1.4
64.9 2.1
10.0 1.3
0.0
100.0
a
BC, B. campestris; AR, A. retroflexus; EC, E. crus-galli; DS, D. sanguinalis; post, post-emergence; pre, pre-emergence; and Dicb, dichlobenil.
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than zero, the data were reanalyzed with ν∞ = 0. Kiapp was calculated
by nonlinear least squares and the simplex method for error min-
imization.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.
1
The detailed H 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 post-
emergence 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-emer-
gence 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
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Table 6. Crop Selectivity of Compounds TA, 1a, 2a, and 2f (Post-emergence; Percent Inhibition)
compound
dosage (g ha−1
)
wheat
corn
cotton
0.0
peanut
1a
750
375
750
375
750
375
750
375
750
375
4.1 0.2
0.0
43.6 0.5
13.4 1.5
31.8 0.8
24.1 0.4
27.1 0.7
9.9 0.4
0.0
7.7 1.0
5.0 0.7
0.0
2a
0.0
0.0
26.2 2.2
14.6 0.8
36.0 0.9
22.4 0.1
36.8 1.7
24.5 0.5
25.0 2.1
7.9 0.6
0.0
0.0
2f
15.9 1.6
13.3 0.3
17.5 1.2
0.0
8.6 0.4
0.0
TA
Dicb
10.7 0.8
0.0
0.0
7.6 0.7
4.7 0.5
36.2 1.2
24.9 0.4
0.0
0.0
Table 7. PPO and AHAS Inhibition Activity of Compounds
TA, 1a, 2a, and 2f (Ki, μM)
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.
a
compound
PPO
AHAS
1a
2a
2f
75.04 7.43
41.51 2.38
39.94 1.83
45.75 0.42
/
−
−
−
−
TA
chlorimuron ethyl
bispyribac sodium
oxadiazon
0.0221 0.0064
/
24.0 15.8
0.024 0.0007
0.0062 0.0009
0.030 0.0006
/
/
/
fluazolate
sulfentrazone
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.
a
“−” = no activity, and “/” = not tested.
Table 8. Antifungal Data as MIC (μg mL−1) for
Compounds
ab
,
fungi
compound
C. albicans
C. utiliz
S. cerevisiae
A. flavus
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
128
32
256
256
256
128
256
64
256
512
256
128
128
64
64
128
128
128
256
64
64
64
256
64
256
128
256
128
256
256
256
128
128
256
64
256
128
128
128
256
256
256
64
256
128
256
128
256
256
256
128
128
128
128
64
256
64
64
128
128
128
128
128
128
256
64
3a
3b
4a
4b
4c
4d
TA
128
256
128
128
16
256
4
128
256
fluconazole
16
a
MICs were determined by the microbroth dilution method for
b
microdilution plates. C. 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 sub-
stituents, 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,
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 com-
pounds also caused substantial wilting on all species following
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post-emergence applications, which was similar to PPO or
AHAS inhibitors. Thus, the PPO and AHAS inhibition experi-
ments 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 inhibi-
tion. 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.
AUTHOR INFORMATION
■
Corresponding Authors
*Telephone: +086-022-23501642. Fax: +86-022-23505369.
E-mail: wangxinnk@nankai.edu.cn.
*E-mail: pwang@nankai.edu.cn.
Author Contributions
†Hongbo Zhang and Qingpeng Wang contributed equally to
this work.
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 con-
trast to compounds 3a and 3b. Especially the natural com-
pound 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 syn-
thesized and evaluated for herbicidal property, PPO and AHAS
inhibition, crop selectivity and antifungal activity. The green-
house 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.
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-methyl-
tryptophan 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.
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