Tailoring Natural Abenquines to Inhibit the Photosynthetic Electron Transport through Interaction with the D1 Protein in Photosystem II

Article abstract of DOI:10.1021/acs.jafc.7b04624

Abenquines are natural N-acetylaminobenzoquinones bearing amino acid residues, which act as weak inhibitors of the photosynthetic electron transport chain. Aiming to exploit the abenquine scaffold as a model for the synthesis of new herbicides targeting photosynthesis, 14 new analogues were prepared by replacing the amino acid residue with benzylamines and the acetyl with different acyl groups. The synthesis was accomplished in three steps with a 68-95% overall yield from readily available 2,5-dimethoxyaniline, acyl chlorides, and benzyl amines. Key steps include (i) acylation of the aniline, (ii) oxidation, and (iii) oxidative addition of the benzylamino moiety. The compounds were assayed for their activity as Hill inhibitors, under basal, uncoupled, or phosphorylating conditions, or excluding photosystem I. Four analogues showed high effectiveness (IC₅₀ = 0.1-0.4 μM), comparable with the commercial herbicide diuron (IC₅₀ = 0.3 μM). The data suggest that this class of compounds interfere at the reducing side of photosystem II, having protein D1 as the most probable target. Molecular docking studies with the plastoquinone binding site of Spinacia oleracea further strengthened this proposal.

Full text of DOI:10.1021/acs.jafc.7b04624

Subscriber access provided by RMIT University Library
Article
Tailoring natural abenquines to inhibit the photosynthetic electron
transport through interaction with the D1 protein in photosystem II
Amalyn Nain-Perez, Luiz C. A. Barbosa, Celia RA Maltha, Samuele Gilberti, and Giuseppe Forlani
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04624 • Publication Date (Web): 01 Dec 2017
Downloaded from http://pubs.acs.org on December 2, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 30
Journal of Agricultural and Food Chemistry
1
Tailoring Natural Abenquines to Inhibit the Photosynthetic Electron
Transport through Interaction with the D1 Protein in Photosystem II
2
3
†
∗,†,‡
‡
§
4
Amalyn NainꢀPerez, Luiz C. A. Barbosa,
Celia R. A. Maltha, Samuele Giberti and
∗,§
5
6
Giuseppe Forlani
†
7
8
Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres. Antônio
Carlos, 6627, Campus Pampulha, CEP 31270ꢀ901, Belo Horizonte, MG, Brazil.
‡
9
Department of Chemistry, Universidade Federal de Viçosa, Viçosa, Av. P. H. Rolfs s/n,
10
CEP 36570ꢀ000, Viçosa, MG, Brazil.
§
11
12
Department of Life Science and Biotechnology, University of Ferrara, via L. Borsari 46,
Iꢀ44121 Ferrara, Italy.
13
14
15
16
17
18
19
20
21
*Corresponding author:
Phone: +55 31 38993068. Fax: +55 31 38993065. Eꢀmail: lcab@ufmg.br.
Phone: +39 0532 455311. Fax: +39 0532 249761. Eꢀmail: flg@unife.it.
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 30
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
ABSTRACT
Abenquines are natural Nꢀacetylaminobenzoquinones bearing amino acids residues,
which act as weak inhibitors of the photosynthetic electron transport chain. Aiming to
exploit the abenquine scaffold as a model for the synthesis of new herbicides targeting
photosynthesis, fourteen new analogues were prepared by replacing the amino acid residue
with benzylamines and the acetyl with different acyl groups. The synthesis was
accomplished in three steps with a 68ꢀ95% overall yield from readily available 2,5ꢀ
dimethoxyaniline, acyl chlorides and benzyl amines. Key steps include (i) acylation of the
aniline, (ii) oxidation, and (iii) oxidative addition of the benzylamino moiety. The
compounds were assayed for their activity as Hill inhibitors, under basal, uncoupled or
phosphorylating conditions, or excluding photosystem I. Four analogues showed high
effectiveness (IC50 = 0.1ꢀ0.4 µM), comparable with the commercial herbicide diuron (IC50
= 0.3 µM). The data suggest that this class of compounds interfere at the reducing side of
photosystem II, having protein D1 as the most probable target. Molecular docking studies
with the plastoquinone binding site of Spinacia oleracea further strengthened this proposal.
KEYWORDS
Abenquine analogues; Aminoquinones; Photosynthesis inhibitors; Photosynthetic electron
transport; Herbicides
2
ACS Paragon Plus Environment

Page 3 of 30
Journal of Agricultural and Food Chemistry
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
INTRODUCTION
Presently, agricultural pests1 and weeds2 are largely controlled by the use of
agrochemicals. Since the introduction of the synthetic auxin 2,4ꢀdichlorophenoxyacetic
acid (2,4ꢀD) in 1946, herbicides have become the primary tool for weed management.3,4
Hence, over the years herbicides contributed considerably to increased crop yields,
providing a highly effective, economical and relatively simple means for weed control.3,5
They act through different mechanisms, such as inhibition of amino acids or fatty acids
biosynthesis, or interference with microtubule formation, or the photosynthetic electron
transport light reactions.2 However, a continuous use of the same herbicide or herbicides
sharing the same target leads to the rapid selection of weed biotypes resistant to such
chemicals.3 The identification and characterization of novel herbicides is therefore highly
desirable to manage resistant weeds.5–7 In the last decade some analogues to natural
products have been developed and used as herbicides, but they contribute to only
approximately 8% of chemicals used for crop protection.8 Natural compounds are often
environmentally friendly, as their halfꢀlife in the soil is usually shorter compared with that
of synthetic herbicides.9,10 Moreover, their wide structural diversity offers opportunities to
discover new sites of herbicide action, beyond the targets described to date.11,12 Several of
currently used active principles target the photosynthetic process and these can be
classified as 1) electron transport inhibitors, 2) uncouplers, 3) energy transfer inhibitors, 4)
inhibitory uncouplers or 5) electron acceptors.13
During recent years several natural compounds have been investigated for their
allelopathic activities.14–19 Among this large array of natural substances is sorgoleone, a
lipophilic quinone produced by Sorghum bicolor. The phytotoxic properties of this quinone
have been investigated for many years,20 and its potent herbicidal activity on smallꢀseeded
weeds seems due to the interference with the electron transfer process at the photosystem II
(PSII) level.21 On this basis, several sorgoleone analogues were synthesized that showed
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 30
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
herbicidal activity against weeds as Desmodium tortuosum, Hyptis suaveolens, Hyptis
lophanta, Brachiaria decumbens, and Euphorbia heterophylla.22,23
Abenquines are natural products isolated from Streptomyces sp. strain DB634, a
bacterium found in the Atacama Desert in Chile.24 Abenquines AꢀD (Figure 1) are Nꢀ
acetylaminobenzoquinones bearing different amino acids residues. We recently described
an efficient procedure for their synthesis and of some analogues.25 Several of them showed
a remarkable algicidal activity, inhibiting cyanobacterial growth by 50% at 1.0 µM.26
However, their mode of action has not been elucidated.
Here we report that abenquine A and some synthetic analogues inhibit the Hill
reaction in vitro, and therefore are potentially endowed with phytotoxic and herbicidal
activity. Some new synthetic analogues showing increased effectiveness were synthesized.
In addition, molecular docking studies were performed aiming to locate their potential site
of action in the photosynthetic system. Results indicate that abenquines and some amide
analogues interact at the D1 protein level of PSII.
MATERIALS AND METHODS
Synthesis. All reagents were purchased from SigmaꢀAldrich and were used without
any purification; solvents were procured from Fluka. All compounds were fully
1
characterized by IR, EIꢀMS, H NMR and 13C NMR spectroscopy. Infrared spectra were
recorded on a Paragon 1000 FTIR spectrophotometer (PerkinꢀElmer, Waltham, MA,
USA), preparing the samples as potassium bromide disks (1% w/w). 1H NMR spectra were
recorded at 400 MHz and 13C NMR spectra at 100 MHz with an Advance 400 (Bruker,
Billerica, MA, U.S.A), using CDCl3 or DMSOꢀd6 as solvent and TMS as internal reference.
Data are reported as follows: chemical shift (δ), multiplicity (s= singlet, d= doublet, t=
triplet, q= quartet, br= broad, m= multiplet), coupling constants (J = Hz) and integration.
The mass spectra were recorded on MSꢀAPI2000 (Applied Biosystem, Halle, Saale,
4
ACS Paragon Plus Environment

Page 5 of 30
Journal of Agricultural and Food Chemistry
96
97
Germany) under electro spray ionization (ESI). Elemental analyses were measured on a
Vario EL unit (FossꢀHeraeus, Halle, Saale, Germany). The melting points were measured
on an MQAPFꢀ301 apparatus (Microquimica Ldta, Belo Horizonte, MG, Brazil) and are
uncorrected. The experimental details for the synthesis of abenquines AꢀD (1ꢀ4) and
compounds 5ꢀ8 and 23 are the same as reported previously.25
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
Synthesis of analogues 9ꢀ22
2ꢀAcetamidoꢀ5ꢀ(2ꢀfluorobenzylamino)ꢀ1,4ꢀbenzoquinone (9). Acylation step. To
a roundꢀbottomed flask (50 mL) were added 2,5ꢀdimethoxyaniline (1.0 g, 6.6 mmol) and
trimethylamine (1.1 mL, 7.6 mmol) dissolved in dichloromethane (25 mL) at 0 ºC. Then, to
the stirred solution was added acetyl chloride (0.6 mL, 7.6 mmol). The resulting solution
was warmed to room temperature over 30 min. When TLC analysis revealed the
consumption of the starting material, the mixture was washed with water (3 x 25 mL),
saturated aqueous sodium hydrogen carbonate solution (25 mL) and brine (25 mL), and
then dried over MgSO4. The mixture was filtered and the solvent removed under reduced
pressure in a rotary evaporator to give a brown solid. This crude product was used in the
next step without further purification.
Oxidation step. To a roundꢀbottomed flask (50 mL) was added the 2,5ꢀ
dimethoxyacetanilide previously obtained (1.0 g, 5.1 mmol) in water (25 mL) and MeOH
(0.64 mL). The mixture was homogenized by magnetic stirring before a slow addition of
phenyl iodine diacetate (2.5 g, 7.7 mmol). The resulting suspension was stirred for 1.5 h,
diluted with water (50 mL) and extracted with dichloromethane (3 x 50 mL). The
combined extracts were washed with water (50 mL) and saturated aqueous sodium
hydrogen carbonate solution (50 mL), dried (Na2SO4), filtered and the solvent was
concentrated under reduced pressure to afford the required quinone as a brown solid. Since
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 30
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
TLC analysis showed only one spot, this crude product was used for the next reaction
without purification.
AzaꢀMichael addition step. To a roundꢀbottomed flask (25 mL) was added 2ꢀ
acetamidoꢀ1,4ꢀbenzoquinone previously obtained (0.12 g, 0.73 mmol) in dichloromethane
(5 mL), followed by the addition of 2ꢀfluorobenzylamine (0.10 g, 0.80 mmol). The reaction
mixture was stirred at room temperature. Initially, the reaction mixture turned from yellow
to red dark. The reaction was quenched after 2 h, before removal of the solvent under
reduced pressure to afford the crude product as a dark red residue. This residue was
purified by silica gel column chromatography eluting with hexane/EtOAc (7:1 v/v) to
afford the required product as red crystals. Overall yield over three steps: 89% (187 mg,
0.65 mmol). M.p 231ꢀ233 °C. IR (KBr) ꢀmax 3290, 2934, 2856, 1725, 1664, 1590, 1500,
̅
1
1470, 1355, 1056, 872 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.57 (s, 1H), 8.15 (t, J =
6.4 Hz, 1H), 7.30ꢀ7.36 (m, 2H), 7.15ꢀ7.21 (m, 3H), 5.35 (s, 1H), 4.43 (d, J = 6.4 Hz, 2H),
2.18 (s, 3H,). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.4, 178.3, 171.1, 147.9, 141.9, 129.3,
124.5, 115.5, 115.1, 109.1, 94.2, 24.5. EIMS m/z 286.9 [MꢀH]ꢀ; 311.1 [M+Na]+; analysis
for C15H13FN2O3, C 62.50, H 4.55, N 9.72%; found: C 62.31, H 4.72, N 9.56%.
The other compounds (10ꢀ22) were prepared following the same procedure
described previously for the synthesis of 9. The overall yield over three steps are reported.
2ꢀAcetamidoꢀ5ꢀ(4ꢀchlorobenzylamino)ꢀ1,4ꢀbenzoquinone (10). Red solid. Yield:
72% (160 mg, 0.53 mmol). M.p 227ꢀ229 °C. IR (KBr) ꢀ̅max 3294, 2924, 2854, 1712, 1654,
1
1588, 1522, 1478, 1342, 1026, 862 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.57 (s, 1H),
8.30 (s, 1H), 7.39 (d, J = 6.5 Hz, 2H), 7.34 (d, J = 6.5 Hz, 2H), 7.22 (s, 1H), 5.35 (s, 1H),
4.39 (brs, 2H), 2.19 (s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.6, 178.3, 171.1, 147.9,
141.9, 136.2, 131.7, 129.1, 128.4, 109.2, 94.5, 44.3, 24.5. EIMS m/z 302.9 [MꢀH]ꢀ; 327.1
6
ACS Paragon Plus Environment

Page 7 of 30
Journal of Agricultural and Food Chemistry
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
[M+Na]+; analysis for C15H13ClN2O3, C 59.12, H 4.30, N 9.19%; found: C 58.92, H 4.41,
N 9.02%.
2ꢀAcetamidoꢀ5ꢀ(3ꢀchlorobenzylamino)ꢀ1,4ꢀbenzoquinone (11). Red solid. Yield:
68% (151 mg, 0.49 mmol). M.p 231ꢀ232 °C. IR (KBr) ꢀ̅max 3316, 2336, 2930, 1718, 1660,
1
1582, 1488, 1176, 722 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.57 (s, 1H), 8.30 (s, 1H),
7.42 (s, 1H), 7.31ꢀ7.38 (m, 3H), 7.23 (s, 1H), 5.39 (s, 1H), 4.41 (d, J = 5.0 Hz, 2H), 2.20
(s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.6, 178.4, 171.2, 148.0, 141.9, 139.9, 133.2,
130.3, 127.2, 127.1125.9, 109.2, 94.5, 44.4, 24.6. EIMS m/z 302.9 [MꢀH]ꢀ; 326.9 [M+Na]+;
analysis for C15H13ClN2O3, C 59.12, H 4.30, N 9.19%; found: C 58.85, H 4.55, N 8.97%.
2ꢀAcetamidoꢀ5ꢀ(4ꢀnitrobenzylamino)ꢀ1,4ꢀbenzoquinone (12). Red solid. Yield:
76% (175 mg, 0.55 mmol). M.p 250ꢀ252 °C. IR (KBr) ꢀ̅max 3292, 2956, 2924, 2856, 1714,
1654, 1474, 1260, 1026, 804 cmꢀ1. 1H NMR (DMSOꢀd6, 400 MHz) δ: 9.61 (s, 1H), 8.65 (s,
1H), 7.23 (d, J = 6.0 Hz, 2H), 7.61 (d, J = 6.0 Hz, 2H), 7.26 (s, 1H), 5.37 (s, 1H), 4.57 (brs,
2H), 2.21 (s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.5, 178.5, 171.2, 148.0, 146.7,
145.3, 141.9, 128.3, 123.6, 109.3, 94.7, 44.5, 24.5. EIMS m/z 314.4 [MꢀH]ꢀ; 338.4
[M+Na]+; analysis for C15H13N3O5, C 57.14, H 4.16, N 13.33%; found: C 59.94, H 4.30, N
13.12%.
2ꢀAcetamidoꢀ5ꢀ(2ꢀbromobenzylamino)ꢀ1,4ꢀbenzoquinone (13). Red solid. Yield:
85% (217 mg, 0.62 mmol). M.p 225ꢀ227 °C. IR (KBr) ꢀ̅max 3280, 2924, 2854, 1708, 1654,
1
1606, 1566, 1490, 1312, 1174, 1024, 750 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.55 (s,
1H), 7.61ꢀ7.66 (m, 1H), 7.33ꢀ7.39 (m, 2H), 7.21ꢀ7.26 (m, 1H), 7.13 (s, 1H), 5.58 (s, 1H),
4.82 (brs, 2H), 2.18 (s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 185.1, 178.6, 171.1, 151.1,
140.1, 132.7, 132.3, 129.1, 128.0, 127.8, 121.9, 111.6, 100.5, 57.7, 24.5. EIMS m/z 346.8
[MꢀH]ꢀ; 3.71.0 [M+Na]+; analysis for C15H13BrN2O3, C 51.59, H 3.75, N 8.02%; found: C
51.42, H 3.84, N 7.75%.
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 30
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
2ꢀAcetamidoꢀ5ꢀ(3ꢀbromobenzylamino)ꢀ1,4ꢀbenzoquinone (14). Red solid. Yield:
92% (235 mg, 0.67 mmol). M.p 213ꢀ215 °C. IR (KBr) ꢀ̅max 3316, 3242, 2942, 1716, 1658,
1
1580, 1488, 1374, 1332, 1176, 718 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.57 (s, 1H),
8.30 (s, 1H), 7.56 (s, 1H), 7.44ꢀ7.48 (m, 1H), 7.28ꢀ7.36 (m, 2H), 7.24 (s, 1H), 5.39 (s, 1H),
4.41 (d, J = 6.0 Hz, 2H), 2.21 (s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.6, 178.4,
171.1, 147.9, 141.9, 140.1, 130.6, 130.1, 129.9, 126.3, 121.8, 109.2, 94.5, 44.4, 24.5.
EIMS m/z 347.1 [MꢀH]ꢀ; 371.0 [M+Na]+; analysis for C15H13BrN2O3, C 62.50, H 4.55, N
9.72%; found: C 62.37, H 4.61, N 9.51%.
2ꢀAcetamidoꢀ5ꢀ(4ꢀbromobenzylamino)ꢀ1,4ꢀbenzoquinone (15). Red solid. Yield:
78% (199 mg, 0.57 mmol). M.p 222ꢀ225 °C.IR (KBr) ꢀmax 3294, 2958, 2924, 2854, 1712,
̅
1
1654, 1588, 1522, 1342, 1176, 1100, 862 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.57 (s,
1H), 8.29 (s, 1H), 7.52 (d, J = 7.7 Hz, 2H), 7.28 (d, J = 7.7 Hz, 2H), 7.21 (s, 1H), 5.33 (s,
1H), 4.36 (d, J = 5.6 Hz, 2H), 2.18 (s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.6,
178.3, 171.1, 147.9, 141.9, 136.7, 131.1, 129.4, 120.2, 109.2, 94.5, 44.4, 24.5. EIMS m/z
347.0 [MꢀH]ꢀ; 371.3 [M+Na]+; analysis for C15H13BrN2O3, C 51.59, H 3.75, N 8.02%;
found: C 51.41, H 3.93, N 7.94%.
2ꢀAcetamidoꢀ5ꢀ(2ꢀmethylbenzylamino)ꢀ1,4ꢀbenzoquinone (16). Red solid. Yield:
70% (145 mg, 0.51 mmol). M.p 232ꢀ235 °C. IR (KBr) ꢀ̅max 3288, 2922, 2854, 1710, 1654,
1574, 1482, 1348, 1176, 758 cmꢀ1. 1H NMR (DMSOꢀd6, 400 MHz) δ: 9.57 (s, 1H), 8.10 (s,
1H), 7.23 (s, 1H), 7.09ꢀ7ꢀ18 (m, 4H), 5.27 (s, 1H), 4.36 (d, J = 5.7 Hz, 2H), 2.28 (s, 3H),
2.19 (s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.6, 178.2, 171.2, 148.3, 142.1, 135.6,
134.4, 130.2126.4, 125.8, 109.2, 94.4, 43.5, 24.6, 18.7. EIMS m/z 307.1 [M+Na]+; analysis
for C16H16N2O3, C 67.59, H 5.67, N 9.85%; found: C 67.30, H 5.84, N 9.61%.
2ꢀAcetamidoꢀ5ꢀ(3ꢀmethylbenzylamino)ꢀ1,4ꢀbenzoquinone (17). Red solid. Yield:
68% (141 mg, 0.49 mmol). M.p 215ꢀ217 °C. IR (KBr) ꢀ̅max 3300, 3286, 3022, 2926, 2858,
1
1712, 1654, 1572, 1482, 1344, 1176, 768 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.55 (s,
8
ACS Paragon Plus Environment

Page 9 of 30
Journal of Agricultural and Food Chemistry
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
1H), 8.24 (s, 1H), 7.19ꢀ7.22 (m, 2H, Hꢀ3), 7.05ꢀ7.11 (m, 3H), 5.32 (s, 1H), 4.34 (d, J = 5.6
Hz, 2H), 2.28 (s, 3H), 2.18 (s, 3H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.7, 178.2, 171.1,
148.1, 142.0, 137.6, 137.1, 128.4, 127.8, 127.1, 109.1, 94.4, 45.1, 24.5, 20.9. EIMS m/z
282.9 [MꢀH]ꢀ; 307.0 [M+Na]+; analysis for C16H16N2O3, C 62.67.59, H 5.67, N 9.85%;
found: C 67.42, H 5.81, N 9.57%.
2ꢀPentanamidoꢀ5ꢀ(benzylamino)ꢀ1,4ꢀbenzoquinone (18). Red crystals. Yield:
90% (204 mg, 0.5065mmol). M.p 209ꢀ211 °C. IR (KBr) ꢀmax 3314, 2970, 2870, 1696,
̅
1
1654, 1600, 1508, 1328, 1178, 870 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ: 9.44 (s, 1H),
8.28 (s, 1H), 7.22ꢀ7.31 (m, 6H, Hꢀ3), 5.33 (s, 1H), 4.39 (s, 2H), 2.41ꢀ2.59 (m, 2H), 1.44ꢀ1ꢀ
53 (m, 2H), 1.19ꢀ1.31 (m, 2H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (DMSOꢀd6, 100 MHz)
δ: 183.6, 178.2, 173.9, 148.1, 141.9, 137.1, 128.5, 127.2, 109.0, 94.4, 45.1, 36.2, 26.7,
21.6, 13.6. EIMS m/z 311.5 [MꢀH]ꢀ; 335.3 [M+Na]+; analysis for C18H20N2O3, C 69.00, H
6.60, N 9.85%; found: C 69.21, H 6.45, N 9.97%.
3ꢀMethylbutamidoꢀ5ꢀ(benzylamino)ꢀ1,4ꢀbenzoquinone (19). Red solid. Yield:
82% (186 mg, 0.59 mmol). M.p 198ꢀ200 °C. IR (KBr) ꢀ̅max 3318, 2975, 2872, 1698, 1652,
1
1605, 1502, 1324, 1179, 880 cmꢀ1. H NMR (CDCl3, 400 MHz) δ: 8.53 (brs, 1H), 7.39 (s,
1H), 7.25ꢀ7.37 (m, 5H), 6.44 (s, br, 1H), 5.47 (s, 1H), 4.33 (d, J = 5.8 Hz, 2H), 2.29 (d, J =
13
7.0 Hz, 2H), 2.12ꢀ2.22 (m, 1H), 0.99 (d, J = 6.6 Hz, 6H). C NMR (CDCl3, 100 MHz) δ:
183.4, 179.3, 172.1, 148.0, 141.2, 135.4, 129.2, 128.4, 127.7, 109.7, 94.9, 47.2, 46.9, 26.1,
22.5. EIMS m/z 311.3 [MꢀH]ꢀ; 335.7 [M+Na]+; analysis for C18H20N2O3, C 69.08, H 6.70,
N 9.80%; found: C 69.00, H 6.45, N 9.36%.
2ꢀPivalamidoꢀ5ꢀ(benzylamino)ꢀ1,4ꢀbenzoquinone (20). Red solid. Yield: 95%
(216 mg, 0.69 mmol). M.p 206ꢀ208 °C. IR (KBr) ꢀmax 3324, 3090, 3066, 3030, 2970,
̅
1
2932, 2870, 1696, 1654, 1600, 1456, 1256, 992 cmꢀ1. H NMR (DMSOꢀd6, 400 MHz) δ:
8.93 (brs, 1H), 8.44 (brs, 1H), 7.25ꢀ7.31 (m, 5H), 7.13 (s, 1H), 5.39 (s, 1H), 4.42 (s, 2H),
1.20 (s, 12H). 13C NMR (DMSOꢀd6, 100 MHz) δ: 183.2, 177.6, 177.4, 148.6, 141.1, 136.9,
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 30
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
128.4, 127.1, 108.4, 93.9, 45.1, 40.2, 26.6. EIMS m/z 311.4 [MꢀH]ꢀ; 335.1 [M+Na]+;
analysis for C18H20N2O3, C 69.13, H 6.62, N 9.73%; found: C 69.21, H 6.45, N 9.97%.
2ꢀIsobutyramidoꢀ5ꢀ(benzylamino)ꢀ1,4ꢀbenzoquinone (21). Red solid. Yield: 75%
(163 mg, 0.54 mmol). M.p 225ꢀ228 °C. IR (KBr) ꢀmax 3298, 3256, 2972, 2932, 1706,
̅
1654, 1592, 1494, 1330, 1178, 734 cmꢀ1. 1H NMR (DMSOꢀd6, 400 MHz) δ: 9.42 (brs, 1H),
8.32 (brs, 1H), 7.23ꢀ7.33 (m, 6H), 5.36 (s, 1H), 4.40 (s, 2H), 2.97 (brs, 1H), 1.06 (brs, 6H).
13C NMR (DMSOꢀd6, 100 MHz) δ: 178.1, 177.8, 155.3, 148.2, 142.1, 137.1, 128.5, 127.2,
109.2, 94.4, 45.1, 34.9, 19.1. EIMS m/z 297.0 [MꢀH]ꢀ; 299.4 [M+Na]+; analysis for
C17H18N2O3, C 68.21, H 6.23, N 9.27%; found: C 68.44, H 6.08, N 9.39%.
2ꢀ((Methoxycarbonyl)amino)ꢀ5ꢀ(benzylamino)ꢀ1,4ꢀbenzoquinone (22). Red
solid. Yield: 86% (179 mg, 0.62 mmol). M.p 210ꢀ212 °C. IR (KBr) ꢀ̅max 3318, 3230, 2946,
1742, 1654, 1578, 1370, 1342, 1186, 1050, 866 746 cmꢀ1. 1H NMR (DMSOꢀd6, 400 MHz)
δ: 8.69 (s, 1H), 8.35 (s, 1H), 7.25ꢀ7.31 (m, 5H), 6.83 (s, 1H), 5.34 (s, 1H), 4.39 (s, 2H),
13
3.70 (s, 3H). C NMR (DMSOꢀd6, 100 MHz) δ: 182.7, 177.2, 152.8, 148.4, 142.7, 137.1,
128.5, 127.2, 107.4, 94.2, 52.9, 45.2. EIMS m/z 284.9 [MꢀH]ꢀ; 287.1 [MꢀH]+; 309.3
[M+Na]+; analysis for C15H13BrN2O3, C 62.77, H 5.11, N 9.57%; found: C 62.93, H 4.93,
N 9.79%.
Biological Tests
Measurement of the Photosynthetic Electron Transport. Photosynthetically
active thylakoid membranes were isolated from market spinach (Spinacia oleracea L.)
leaves. Briefly, 20 g of deveined plant material were resuspended in 100 mL of iceꢀcold 20
mM Tricine/NaOH buffer (pH 8.0) containing 10 mM NaCl, 5 mM MgCl2 and 0.4 M
sucrose, and homogenized for 30 s in a blender at maximal speed. The homogenate was
filtered through surgical gauze, and the filtrate was centrifuged at 4 °C for 1 min at 500 g;
the supernatant was further centrifuged for 10 min at 1500 g. Pelleted chloroplasts were
10
ACS Paragon Plus Environment

Page 11 of 30
Journal of Agricultural and Food Chemistry
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
osmotically disrupted by resuspension in sucroseꢀlacking buffer, immediately diluted 1:1
with sucroseꢀcontaining buffer, and kept on ice in the dark until used. Following dilution
with 80% (v/v) acetone, the chlorophyll content was calculated using the Arnon’s formula.
The basal rate of photosynthetic electron transport was measured following the lightꢀdriven
reduction of ferricyanide. Aliquots of membrane preparations corresponding to 15 ꢁg of
chlorophyll were incubated at 24 °C in 1 mL cuvettes containing 20 mM tricine/NaOH
buffer (pH 8.0), 10 mM NaCl, 5 mM MgCl2, 0.2 M sucrose and 1 mM K3[Fe(CN)6]. The
assay was initiated by exposure to saturating light (800 ꢁmol/m2/s), and the rate of
ferricyanide reduction was measured at 1 min intervals for 20 min in a Novaspec Plus
spectrophotometer (GE Healthcare, Milan, Italy) at 420 nm against an exact blank.
Activity was calculated over the linear portion of the curve from a molar extinction
coefficient of 1000/M/cm. Compounds 6ꢀ10 and 17ꢀ20 were dissolved in DMSO and then
diluted with water, as required. Their effect on the photosynthetic electron transport chain
was measured by adding concentrations in the range from 0.1 to 100 ꢁM to the above
reaction mixture, and comparing the results with untreated parallel controls. Each assay
was repeated in triplicate, and results were expressed as percentage of untreated controls.
Phosphorylating photosynthetic electron rate was determined under the same conditions,
but in the presence of 0.5 mM ADP and 2 mM K2HPO4. Uncoupled photosynthetic
electron rate was measured following the addition of 1 mM NH4Cl to the basal reaction
mixture. In these last two cases, ferricyanide reduction was determined at intervals of 30 s
for 10 min. Reported values are mean ± SE over replicates. The concentrations causing
50% inhibition (IC50) and their confidence limits were estimated by nonlinear regression
analysis using Prism 6 for Windows, version 6.03 (GraphPad Software, La Jolla, CA).
Molecular docking. Molecular docking studies for compounds 5, 10, 14, 15, 21
and lenacil with the active site of spinach photosystem II (PDB: 3JCU) were performed by
using AutoDockTools 4.2 program suite.27,28 Each compound was generated using
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 30
276
277
278
279
280
281
282
283
284
285
ChemDraw 14.0 followed by MM2 energy minimization. The target enzyme was prepared
for molecular docking simulation using the chain A by removing water, and all hydrogens
were added, Gasteiger charges were calculated and nonꢀpolar hydrogens were merged to
carbon atoms. A grid box size of 40 x 40 x 40 point (x, y, z) with a spacing of 0.486 Å was
centered on the X, Y, and Z at ꢀ41.788, 3.115, and ꢀ18.728, respectively. Then, to evaluate
the binding free energy of the inhibitor–macromolecule, automated docking studies were
carried out. The genetic algorithm with local search (GALS) was used to find the best
conformers. The Lamarckian genetic algorithm with default settings was used as well.
Each docking experiment was performed 50 times, yielding 50 docked conformations.
286
287
288
RESULTS AND DISCUSSION
Abenquine A is a mild inhibitor of the photosynthetic electron transport, but
some of its synthetic analogues show a remarkable activity. Natural abenquines and
some synthetic analogues were recently found to inhibit the photoautotrophic growth of a
model cyanobacterial strain.25,26 In photosynthetic organisms, plastoquinone plays a central
role carrying electrons from PSII to the cytochrome b6f complex.29 Therefore, we
investigated the ability of abenquines to interfere with the chloroplast electron transport
chain by measuring the Hill reaction using thylakoid membranes isolated from spinach
leaves.30,31 For this initial investigation, natural abenquines AꢀD and their synthetic
analogues 5ꢀ8 were prepared in high yields using the methodology previously developed.25
The results from such bioassays (Table 1) showed that they may indeed exert a significant
inhibition of ferricyanide reduction. Natural abenquines were inactive or scarcely active.
Only abenquine A reduced electron transport rate at submillimolar concentrations.
Remarkably, however, some synthetic analogues were much more effective, with IC50
289
290
291
292
293
294
295
296
297
298
299
300
12
ACS Paragon Plus Environment

Page 13 of 30
Journal of Agricultural and Food Chemistry
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
values lower than 100 µM. Moreover one of them, namely compound 5 (IC50= 1.1 µM),
was almost as active as diuron,32 a commercial herbicide targeting PSII (IC50 0.3 µM).
Tailoring the abenquine scaffold by replacing the amino acid residue for
substituted benzylamine groups, or changing the acetyl group by other acyl groups,
afforded new analogues with increased effectiveness. Aiming to obtain more active
molecules that could be potentially developed as new active principles for weed control,
new abenquine analogues were prepared by employing a synthetic methodology that
involves a threeꢀstep protocol.25 An initial acylation of 2,5ꢀdimethoxyaniline with the
corresponding acyl chloride afforded the required amides in quantitative yields. These
amides were immediately oxidized with phenyl iodine diacetate affording the
corresponding quinones. Final treatment of crude quinones with a diversity of amines
resulted in the isolation of required products (9ꢀ22) in overall yields ranging from 68 to
95% (Figure 2). Based on the fact that analogue 5 bearing an acetyl and a Nꢀbenzyl moiety
was the most active, one set of compounds was prepared keeping the acetyl unit and
modifying the substituents on the Nꢀbenzyl group in order to evaluate the stereoꢀelectronic
effect on this portion of the molecule. For another set of compounds, the Nꢀbenzyl group
was kept unchanged and the acyl portion was modified in order to evaluate its size effect.
Standard spectroscopic analysis was carried out for structural characterization of
these new compounds. In short, mass spectra obtained under electrospray conditions (EIꢀ
MS) showed the characteristic molecular ions as expected for each compound. The IR
spectra showed absorption bands characteristics of NH, C=O and C=C bonds.33 On the
1
other hand, the H NMR spectra showed a similar pattern for all compounds, with signals
at δ 2.18ꢀ2.22 corresponding to the methyl group (CH3ꢀCO), as well the methylene of
benzylamine fragment (ꢀCH2ꢀNH) as doublet at δ 4.36ꢀ4.81. The aromatic portion of the
molecules showed signals in the range of δ 7.04ꢀ8.23. The signals corresponding to the Nꢀ
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 30
327
328
329
330
H amine were observed at δ 8.09ꢀ8.40 due to the NꢀH amide were at δ 9.55ꢀ9.60. In the 13C
NMR spectra were observed signals corresponded to the methyl group (CH3ꢀCO) at δ 24.5ꢀ
24.6 as well the methylene of benzylamine fragment (ꢀCH2ꢀNH) at δ 43.5ꢀ57.7. The signals
due to carbons of the amide carbonyl groups appeared around 171 ppm.
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
All analogues (9ꢀ22) were tested for their ability to interfere with the lightꢀdriven
reduction of ferricyanide by spinach chloroplasts, and the results are summarized in Table
2. Among compounds 9ꢀ17, which share the acetamide moiety attached to the quinone
core, compounds 9 (oꢀfluoroꢀsubstitution) and 12 (pꢀnitroꢀsubstitution) bearing a strong
electron withdrawing group showed IC50 values in the 10 to 100 µM range, whereas
compounds with Cl or Br atom in either meta or paraꢀposition (10 and 11; 14 and 15)
exhibited IC50 values as low as 0.18ꢀ0.37 µM, comparable to that found for the herbicide
diuron (IC50 0.3 µM).32 Compound 13, with Br atom in orthoꢀposition, was significantly
less effective (IC50 = 4.7 µM). A similarity tendency was observed also with a methyl
substituent, which in the orthoꢀposition (16) resulted in an IC50 value of 3.6 µM, whereas
in the metaꢀposition (17) exhibited an IC50 value of 0.38 µM. Even though no clear
structureꢀactivity relationship can be deduced at this stage due to the limited number of
analogues evaluated, we observed that the analogues bearing substituent in metaꢀposition
are better inhibitors of the photosynthetic electron transport. From such preliminary data it
is clear that the electronic nature and position of the substituent on the benzyl ring is
associated with the activity.
The second set of compounds has a benzylamine moiety in the quinone core but
bear a different aliphatic chain on the amide group (18ꢀ22). For comparison purpose
compound 23 is included in Table 2. This compound bears a benzoyl group in place of the
acetyl group. From the data presented (Table 2), compound 20 with the tertꢀbutyl moiety
was inactive, and 19 with isobutyl group was scarcely effective. Compound 23 having the
14
ACS Paragon Plus Environment

Page 15 of 30
Journal of Agricultural and Food Chemistry
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
large benzene ring was also virtually inactive, causing only 24% inhibition at 100 µM.
However, compound 18 with nꢀbutyl exerted a moderate inhibition (IC50 = 2.6 µM). On the
other hand, compounds 21 (bearing an isopropyl group) and 22 (a carbamate) showed IC50
values as low as 0.27 and 0.95 µM, respectively. If compared with the IC50 value for 5 (1.1
µM), this result clearly suggests that the acyl group volume influences the inhibitory
potential, with the activity reducing as the steric hindrance increases. However, it seems
that the best size for the acyl group is isopropyl, since derivative 21 is approximately 4
times more effective than 5. Also, the only carbamate derivative 22 was as active as 5,
indicating that the target site can also accommodate this pharmacophore. Considering that
the previously reported 2,5ꢀbis(alkylamino)ꢀ1,4ꢀbenzoquinones did not inhibit the Hill
reaction at 100 ꢀM and caused only 10% inhibition at 200 ꢀM, being less active than its
benzoyl analogue 23 (Table 2),31 we suggest that the carbonyl group attached to one of the
nitrogen atoms is essential for activity, most likely for some hydrogen bond formation in
the active site, while a group attached to it should also have an optimal size to fit into the
cavity of the reactive center.
Both the evaluation of the inhibitory effect of abenquine analogues on the
photosynthetic electron transport chain under different conditions and docking
studies suggest the PSII protein D1 as their likely target. The activity of the most potent
abenquine analogues (10, 14, 15, 21) was further characterized. The comparison of the
inhibitory patterns under basal, uncoupling or phosphorylating conditions (Figure 3)
allowed us to rule out the possibility that these compounds may act as energy coupling
inhibitors or as uncouplers, thereby impeding ATP synthesis.34
Being true Hill inhibitors, other experiments were performed to identify their
molecular target inside the so called Z scheme, i.e. if they interact with the photosynthetic
machinery at the photosystem II, cytochrome b6f complex or photosystem I level.4,29 When
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 30
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
the electron flow was suppressed by addition of the cytochrome b6f inhibitor 2,5ꢀdibromoꢀ
3ꢀmethylꢀ6ꢀisopropylꢀpꢀbenzoquinone (2 µM), and then an electron flow excluding
photosystem I was established by the addition of 0.1 mM phenylenediamine, compounds
10, 14, 15, 21 were still inhibitory (Figure 4). Consequently, the compounds studied here
are most likely interacting with the PSII.
The great majority of herbicides inhibiting PSII are known to displace the
plastoquinone QB from its binding site and to inhibit the electron transfer from QA to
QB.35,36 In silico analyses of the interaction between some of these herbicides, such as
diuron (3ꢀ(3,4ꢀdichlorophenyl)ꢀ1,1ꢀdimethylurea), atrazine, terbutryn and bromacil, and the
plastoquinone binding site have been carried out using the structure of PSII from either
bacteria, like Rhodopseudomonas viridis,37 Tricondyloides elongatus,36,38 T. vulcanus,39
and Blastochloris viridis,40 or plants, like Phalaris minor41 and Spinacia oleracea.39 For
the natural quinone sorgoleone a docking study using the QBꢀbinding site of the PSII
complex of bacterium Rhodopseudomonas viridis was carried out.42 In the current work a
docking analysis was performed for the most active abenquine analogues (5, 10, 14, 15,
21) and the herbicide lenacil using the program AutoDockTools 4.2.27,28 Molecular
docking was carried out by using the crystallographic structure of the reaction center of
PSII (D1) from spinach (PDB ID: 3JCU). Results suggested that compounds 14 and 15 are
bonded via oxygen and hydrogen. The carbonyl group of the quinone interacts with an OH
of Ser264 in 2.063 Å for 14, or with both Ser264 and the NH of the His215 with a distance
bond of 2.094 and 2.226 Å, for 15 (Figure 5), with docking scores of ꢀ7.83 for 14 and ꢀ7.69
for 15. Moreover, hydrophobic interactions were found with Phe265, Phe274, Phe255, His252
,
Leu271, Leu218 and His215 for 14, and Phe255, Phe274, His252, Leu271, Ala251, Leu218 and
Met214 for 15. Consequently, the compounds 14 and 15 are surrounded by the above
residues. Thus, the interaction of the ligands with these residues might lead to an inhibition
of PSII (D1). In addition, most of the herbicides described above prefer the position near
16
ACS Paragon Plus Environment

Page 17 of 30
Journal of Agricultural and Food Chemistry
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
the loop made by Ser264, and Phe265 in the PSII. The same orientation for abequine
analogues 5, 10, 14, 15, 21 and lenacil herbicide in the binding site was observed.
In summary, based on the initial observation that natural abenquine A inhibits the
electron transport chain in the 10ꢀ4ꢀ10ꢀ3 M range, we carried out the synthesis of new, more
active analogues. The remarkable biological activity of analogue 5 bearing a benzyl group
indicated that such a group is critical for the inhibition of the photosynthetic machinery. In
addition, new analogues bearing several substituents in the benzylic ring (9ꢀ17) or in the
amide moiety (18ꢀ22) were synthesized, with Cl, Br or methyl aromatic substituents
resulting in potent inhibitors of the Hill reaction, with IC50 values (0.1ꢀ0.4 µM) of the same
order of magnitude as some commercial herbicides targeting PSII.
ASSOCIATED CONTENT
Supporting Information
Figure of molecular docking and 1H and 13C NMR spectra for synthetic abenquines
analogues. This material is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding authors
*(L.C.A.B.) Phone: +55 31 38993068. Fax: +55 31 38993065. Eꢀmail: lcab@ufmg.br.
*(G.F.) Phone: +39 0532 455311. Fax: +39 0532 249761. Eꢀmail: flg@unife.it.
ORCID
Luiz Cláudio Almeida Barbosa: 0000ꢀ0002ꢀ5395ꢀ9608
Giuseppe Forlani: 0000ꢀ0003ꢀ2598ꢀ5718
Notes
The authors declare no competing financial interest.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 30
430
431
432
433
434
435
436
437
438
439
440
441
442
443
ACKNOWLEDGEMENTS
We are grateful to the following Brazilian agencies: Conselho Nacional de
Desenvolvimento Cientifíco e Tecnológico (CNPq, grant 400746ꢀ2014) for research
fellowships (L.C.A.B.); Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG,
grant APQ1557ꢀ15) for financial support; and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) for a research fellowship (A.N.P). Support from the
University of Ferrara (Fondo di Ateneo per la Ricerca 2016) is also acknowledged. The
authors thank Dr R. Csuk for mass spectra data and elemental analysis experiments, and Dr
A. Straforini for technical assistance.
REFERENCE
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
(1)
Duke, S. O.; Cantrell, C. L.; Meepagala, K. M.; Wedge, D. E.; Tabanca, N.;
Schrader, K. K. Natural toxins for use in pest management. Toxins (Basel). 2010, 2,
1943–1962.
(2)
(3)
(4)
(5)
(6)
Baucom, R. S. The remarkable repeated evolution of herbicide resistance. Am. J.
Bot. 2016, 103, 1–3.
Shaner, D. L. Lessons learned from the history of herbicide resistance. Weed Sci.
2014, 62, 427–431.
Teixeira, R. R.; Pereira, J. L.; Pereira, W. L. Photosynthetic inhibitors. In Applied
Photosynthesis; Najafpour, M. M., Ed.; InTech, 2012; pp 3–22.
Heap, I. Global perspective of herbicideꢀresistant weeds. Pest Manag. Sci. 2014, 70,
1306–1315.
Burgos, N. R.; Tranel, P. J.; Streibig, J. C.; Davis, V. M.; Shaner, D.; Norsworthy, J.
K.; Ritz, C. Review: Confirmation of resistance to herbicides and evaluation of
resistance levels. Weed Sci. 2012, 61, 4–20.
(7)
Powles, S. B.; Yu, Q. Evolution in action: plants resistant to herbicides. Annu. Rev.
18
ACS Paragon Plus Environment

Page 19 of 30
Journal of Agricultural and Food Chemistry
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
Plant Biol. 2010, 61, 317–347.
(8)
(9)
Cantrell, C. L.; Dayan, F. E.; Duke, S. O. Natural products as sources for new
pesticides. J. Nat. Prod. 2012, 75, 1231–1242.
Duke, S. O.; Dayan, F. E.; Romagni, J. G.; Rimando, A. M. Natural products as
sources of herbicides: current status and future trends. Weed Res. 2000, 40, 99–111.
(10) Soltys, D.; Krasuska, U.; Bogatek, R.; Gniazdowska, A. Allelochemicals as
bioherbicides
517–542.
– pesent and perspectives. Herbic. ꢀ Curr. Res. Case Stud. Use 2013,
(11) Jablonkai, I. Molecular mechanism of action of herbicides. In Herbicides –
Mechanisms and Mode of Action; 2011, pp 3–24.
(12) Dayan, F. E.; Duke, S. O. Natural compounds as next generation herbicides. Plant
Physiol. 2014, 166, 1090–1105.
(13) Fuerst, P. E.; Norman, M. A. Interactions of herbicides with photosynthetic electron
transport. Weed Sci. 1991, 39, 458–464.
(14) Macias, F. A.; Galino, J. C. G.; Molinillo, J. M. G. Allelopathy: chemistry and mode
of action of allelochemicals; CRC Press. Taylor & Francis Group, 2003.
(15) Barbosa, L. C. A.; Demuner, A. J.; de Alvarenga, E. S.; Oliveira, A.; KingꢀDiaz, B.;
LotinaꢀHennsen, B. Phytogrowthꢀ and photosynthesisꢀinhibiting properties of
nostoclide analogues. Pest Manag. Sci. 2006, 62, 214–222.
(16) MoralesꢀFlores, F.; Aguilar, M. I.; KingꢀDíaz, B.; LotinaꢀHennsen, B. Derivatives
of diterpen labdaneꢀ8α,15ꢀdiol as photosynthetic inhibitors in spinach chloroplasts
and growth plant inhibitors. J. Photochem. Photobiol. B Biol. 2013, 125, 42–50.
(17) Macías, F. A.; Marín, D.; OliverosꢀBastidas, A.; Molinillo, J. M. G. Optimization of
benzoxazinones as natural herbicide models by lipophilicity enhancement. J. Agric.
Food Chem. 2006, 54, 9357–9365.
(18) MacíasꢀRubalcava, M. L.; RuizꢀVelasco Sobrino, M. E.; MeléndezꢀGonzález, C.;
KingꢀDíaz, B.; LotinaꢀHennsen, B. Selected phytotoxins and organic extracts from
endophytic fungus Edenia gomezpompae as light reaction of photosynthesis
inhibitors. J. Photochem. Photobiol. B Biol. 2014, 138, 17–26.
(19) KingꢀDíaz, B.; GranadosꢀPineda, J.; Bah, M.; RiveroꢀCruz, J. F.; LotinaꢀHennsen,
B. Mexican propolis flavonoids affect photosynthesis and seedling growth. J.
Photochem. Photobiol. B Biol. 2015, 151, 213–220.
(20) Dayan, F. E. Factors modulating the levels of the allelochemical sorgoleone in
Sorghum bicolor. Planta 2006, 224, 339–346.
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 30
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
(21) Rimando, A. M.; Dayan, F. E.; Czarnota, M. A.; Weston, L. A.; Duke, S. O. A new
photosystem II electron transfer inhibitor from Sorghum bicolor. J. Nat. Prod. 1998,
61, 927–930.
(22) Barbosa, L. C. A.; Ferreira, M. lucia; Demuner, A. J. Preparation and phytotoxicity
of sorgoleone analogues. Quim. Nova 2001, 24, 751–755.
(23) Lima, L. S.; Barbosa, L. C. A.; de Alvarenga, E. S.; Demuner, A. J.; da Silva, A. A.
Synthesis and phytotoxicity evaluation of substituted paraꢀbenzoquinones. Aust. J.
Chem. 2003, 56, 625–630.
(24) Schulz, D.; Beese, P.; Ohlendorf, B.; Erhard, A.; Zinecker, H.; Dorador, C.; Imhoff,
J. F. Abenquines AꢀD: aminoquinone derivatives produced by Streptomyces sp.
strain DB634. J. Antibiot. (Tokyo). 2011, 64, 763–768.
(25) NainꢀPerez, A.; Barbosa, L. C. A.; Maltha, C. R. A.; Forlani, G. First total synthesis
and phytotoxic activity of Streptomyces sp. metabolites abenquines. Tetrahedron
Lett. 2016, 57, 1811–1814.
(26) NainꢀPerez, A.; Barbosa, L. C. A.; Maltha, C. R. A.; Forlani, G. Natural abenquines
and their synthetic analogues exert algicidal activity against bloomꢀforming
cyanobacteria. J. Nat. Prod. 2017, 80, 813–818.
(27) Sanner F., M. Python: A programming language for software integration and
development. J. Mol. Graph. 1999, 17, 57–61.
(28) Forli, S.; Huey, R.; Pique, M. E.; Sanner, M. F.; Goodsell, D. S.; Olson, A. J.
Computational protein–ligand docking and virtual drug screening with the
AutoDock suite Stefano. Nat. Protoc. 2016, 11, 905–919.
(29) Witt, H. T. Primary reactions of oxygenic photosynthesis. Berichte der
Bunsengesellschaft für Phys. Chemie 1996, 100, 1923–1942.
(30) LotinaꢀHennsen, B.; Achnine, L.; Ruvalcaba, N. M.; Ortiz, A.; Hernández, J.;
Farfán, N.; AguilarꢀMartínez, M. 2,5ꢀDiaminoꢀpꢀbenzoquinone derivatives as
photosystem
I
electron acceptors:ꢂ synthesis and electrochemical and
physicochemical properties. J. Agric. Food Chem. 1998, 46, 724–730.
(31) NainꢀPerez, A.; Barbosa, L. C. A.; Picanço, M. C.; Giberti, S.; Forlani, G. Aminoꢀ
substituted paraꢀbenzoquinones as potential herbicides. Chem. Biodivers. 2016, 13,
1008–1017.
(32) Pereira, A.; Barbosa, L. C. A.; Demuner, A. J.; Silva, A. A.; Bertazzini, M.; Forlani,
G. Rubrolides as model for the development of new lactones and their aza analogs
as potential photosynthesis inhibitors. Chem. Biodivers. 2015, 12, 987–1006.
20
ACS Paragon Plus Environment

Page 21 of 30
Journal of Agricultural and Food Chemistry
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
(33) Barbosa, L. C. A. Espectroscopia no Infravermelho na caraterização de compostos
orgânicos; Editora UFV, 2011.
(34) Barbosa, L. C. A.; Demuner, A. J.; Alvarenga, E. S.; Oliveira, A.; KingꢀDiaz, B.;
LotinaꢀHennsen, B. Phytogrowthꢀ and photosynthesisꢀinhibiting properties of
nostoclide analogues. Pest Manag. Sci. 2006, 62, 214–222.
(35) Barr, R.; Crane, F. L. Selective thiol inhibition of ferricyanide reduction in
photosystem II of spinach chloroplasts. Biochem. Biophys. Res. Commun. 1981, 67,
1190–1194.
(36) Lambreva, M. D.; Russo, D.; Polticelli, F.; Scognamiglio, V.; Antonacci, A.;
Zobnina, V.; Campi, G.; Rea, G. Structure/function/dynamics of photosystem II
plastoquinone binding sites. Curr. Protein Pept. Sci. 2014, 15, 285–295.
(37) Xiong, J.; Subramaniam, S.; Govindjee. Modeling of the D1/D2 proteins and
cofactors of the photosystem II reaction center: Implications for herbicide and
bicarbonate binding. Protein Sci. 1996, 5, 2054–2073.
(38) Rea, G.; Polticelli, F.; Antonacci, A.; Lambreva, M.; Pastorelli, S.; Scognamiglio,
V.; Zobnina, V.; Giardi, M. T. Computational biology, protein engineering, and
biosensor technology: a close cooperation for herbicides monitoring. In Herbicides,
Theory and Applications; Larramendy, M., Ed.; InTech, 2011.
(39) FunarꢀTimofei, S.; Borota, A.; Crisan, L. Combined molecular docking and QSAR
study of fused heterocyclic herbicide inhibitors of D1 protein in photosystem II of
plants. Mol. Divers. 2017, 21, 437–454.
(40) Broser, M.; Glöckner, C.; Gabdulkhakov, A.; Guskov, A.; Buchta, J.; Kern, J.; Müh,
F.; Dau, H.; Saenger, W.; Zouni, A. Structural basis of cyanobacterial photosystem
II inhibition by the herbicide terbutryn. J. Biol. Chem. 2011, 286, 15964–15972.
(41) Singh, D. V.; Agarwal, S.; Kesharwani, R. K.; Misra, K. Molecular modeling and
computational simulation of the photosystemꢀII reaction center to address
isoproturon resistance in Phalaris minor. J. Mol. Model. 2012, 18, 3903–3913.
(42) Czarnota, M. A.; Paul, R. N.; Daynan, F. E.; Nimbal, C. I.; Weston, L. A. Mode of
action, localization of production, chemical nature, and activity of sorgoleone: a
potent PSII inhibitor in Sorghum spp. root exudates. Weed Technology, 2001, 15,
813ꢀ825.
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 30
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
CAPTIONS TO FIGURES
Figure 1. The structure of natural abenquines AꢀD, 1ꢀ4, respectively, and some previously
synthesized analogues (5ꢀ8). The micromolar concentrations of these compounds that
inhibited by 50% the growth of the cyanobacterial strain Synechococcus elongatus PCC
6301 are also reported in parenthesis.26
Figure 2. Preparation of abenquine analogues 9ꢀ22.
Figure 3. Effects increasing concentrations of compounds 10, 14, 15, 21 on ferricyanide
reduction under basal, uncoupling or phosphorylating conditions.
Figure 4. Comparison of the effects of compounds 10, 14, 15, 21 on the whole
chloroplastic electron transport chain and on a partial electron flow involving photosystem
II only.
Figure 5. Binding models of abenquine analogues 14 and 15 with the pocket site of PSII
from Spinacia oleracea (PDB ID: 3JCU).
22
ACS Paragon Plus Environment

Page 23 of 30
Journal of Agricultural and Food Chemistry
Table 1. Natural Abenquines (1ꢀ4) and Analogues (5ꢀ8) as Inhibitor of the Lightꢀ
Dependent Ferricyanide Reduction in Spinach Chloroplasts.
IC50 (µM)
Compound
IC50 (µM)
279 ± 81
1487 ± 987
n.a.
Compound
1 Abenquine A
2 Abenquine B
3 Abenquine C
4 Abenquine D
1.1 ± 0.1
540 ± 258
56.9 ± 3.1
26.0 ± 1.9
5
6
7
8
n.a.
n.a.ꢀ not active in the range tested (0.1 – 100 µM).
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 30
Table 2. Abenquine Analogues (9ꢀ22) Able to Inhibit the LightꢀDependent Ferricyanide
Reduction in Spinach Chloroplasts.
Compound
IC50 (µM)
13 ± 2
Compound
IC50 (µM)
0.38 ± 0.02
2.6 ± 0.4
43 ± 14
9
17
18
19
20
21
22
23b
0.33 ± 0.02
0.37 ± 0.02
45 ± 6
10
11
12
13
14
15
16
n.a.a
4.7 ± 0.5
0.18 ± 0.01
0.19 ± 0.02
3.6 ± 0.5
0.27 ± 0.01
0.95 ± 0.48
n.a.c
a n.a. ꢀ not active in the range tested (0.01 – 100 µM).
b Analogue to 5 having a benzoyl instead of acetyl group.26
c 24% inhibition at 100 ꢀM.
24
ACS Paragon Plus Environment

Page 25 of 30
Journal of Agricultural and Food Chemistry
Figure 1
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 30
Figure 2
26
ACS Paragon Plus Environment

Page 27 of 30
Journal of Agricultural and Food Chemistry
Figure 3
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 30
Figure 4
Figure 5
28
ACS Paragon Plus Environment

Page 29 of 30
Journal of Agricultural and Food Chemistry
29
ACS Paragon Plus Environment