Rational Design, Synthesis and Evaluation of Indole Nitrogen Hybrids as Photosystem II Inhibitors

Article abstract of DOI:10.1111/php.13295

We report the synthesis of twelve indole derivatives bearing nitro or amide groups via Fischer indole methodology followed by reduction/acetylation and amidation reactions. After thorough characterization, these indoles were subjected to a number of studies in order to evaluate their bioactive potential as photosynthesis and plant growth inhibitors. Firstly, these molecular hybrids were evaluated as photosystem II (PSII) inhibitors through chlorophyll a (Chl a) fluorescence measurement. In this study, 6-chloro-8-nitro-2,3,4,9-tetrahydro-1H-carbazole (15a) and 5-chloro-2,3-dimethyl-7-nitro-1H-indole (15b) showed the best results by reducing the phenomenological parameters of reaction centers ABS/RC, TR₀/RC and ET₀/RC of PSII. Electron chain blockage by these compounds may lead to diminished ATP synthesis and CO₂ fixation which interrupt the plant development. The compounds 15a and 15b both act as postemergent herbicides, reducing the dry biomass of Ipomoea grandifolia and Senna alata weeds by an average of 40% and 37%, respectively, corroborating the fluorescence results. Additionally, the molecular docking study revealed that the presence of strong electron-withdrawing groups at the indole phenyl ring is important for the ligand’s interaction with the binding pocket of protein D1 on PSII. The optimization of these molecular features is the goal of our research group in further understanding and development of new potent herbicides.

Full text of DOI:10.1111/php.13295

DR. LUCAS CAMPOS CURCINO VIEIRA (Orcid ID : 0000-0002-1138-9117)
Article type
: Research Article
Rational Design, Synthesis and Evaluation of Indole Nitrogen Hybrids
as Photosystem II Inhibitors
Jéssica Maria de Souza1, Bruno Rodrigues Fazolo1, Jhully Wellen Ferreira Lacerda1, Mariana
de Souza Moura1, Arielly Celestino Rodrigues dos Santos1, Leonardo Gomes de
Vasconcelos1, Paulo Teixeira de Sousa Junior1, Evandro Luiz Dall’Oglio1, Akbar Ali2, Olívia
Moreira Sampaio1, Lucas Campos Curcino Vieira*3
1 Department of Chemistry, Federal University of Mato Grosso, Cuiabá, 78060-900, Brazil
2 Department of Chemistry, University of Malakand, 18800, Pakistan
3 Engineering Faculty, Federal University of Mato Grosso, Várzea Grande, 78060-900, Brazil
*Corresponding author e-mail: lucascurcino@ufmt.br (Lucas Campos Curcino Vieira)
This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences
between this version and the Version of Record. Please cite this article as doi: 10.1111/PHP.13295
This article is protected by copyright. All rights reserved

ABSTRACT
We report the synthesis of twelve indole derivatives bearing nitro or amide groups via Fischer indole
methodology followed by reduction/acetylation and amidation reactions. After thorough
characterization these indoles were subjected to a number of studies in order to evaluate their bioactive
potential as photosynthesis and plant growth inhibitors. Firstly, these molecular hybrids were evaluated
as photosystem II (PSII) inhibitors through chlorophyll a (Chl a) fluorescence measurement. In this
study, 6-Chloro-8-nitro-2,3,4,9-tetrahydro-1H-carbazole (15a) and 5-chloro-2,3-dimethyl-7-nitro-1H-
indole (15b) showed the best results by reducing the phenomenological parameters of reaction centres
ABS/RC, TR0/RC and ET0/RC of PSII. Electron chain blockage by these compounds may lead to
diminished ATP synthesis and CO2 fixation which interrupt the plant development. The compounds
15a and 15b both act as post-emergent herbicides, reducing the dry biomass of Ipomoea grandifolia
and Senna alata weeds average of 40% and 37%, respectively, corroborating the fluorescence results.
Additionally, the molecular docking study revealed that the presence of strong electron withdrawing
groups at the indole phenyl ring are important for the ligand’s interaction with the binding pocked of
protein D1 on PSII. The optimization of these molecular features is the goal of our research group in
further understanding and development of new potent herbicides.
This article is protected by copyright. All rights reserved

INTRODUCTION
Herbicides are a pesticide class that affect several plant physiological processes, including metabolic
energy transfer, which inhibit plant development and growth. Among the most common effects of
herbicides on plant physiology are the photosynthetic apparatus stress on photosystem II (PSII) and
plant growth inhibition (1–3). In last few years, weed control in agriculture has been carried out
principally via the use of herbicides. Continual and repetitive use of herbicides leads to increased
occurrence of herbicide-resistant weeds as well as continuous damage to the environment and to
human health, making the development of novel, environmentally benign herbicidal agents for the
replacement of currently commercialized herbicides an indispensable area of research (4–8).
Intensive efforts aimed at developing new herbicidal compounds or modifying existing ones are
ongoing (9–16). A wide variety of chemical compounds have demonstrated plant management activity
including the indole derivatives 1 and 2 (12) which demonstrate photosynthesis inhibitory activity as
well as compounds 3 (17) and 4 (18) which are plant growth inhibitors (Fig. 1).
<Figure 1>
In addition to privileged scaffolds such as indole, the presence of electron donating and/or withdrawing
groups may improve the herbicidal activity by modifying physicochemical properties such as molecular
size, hydrogen bonding, ionization state, lipophilicity and aqueous solubility (19, 20). In this context,
compounds bearing nitro or amide groups have demonstrated improved herbicidal activity compared to
unfunctionalized adducts. Compounds containing nitro groups have been studied as photosynthesis
inhibitors, for example compound 5, which is an inhibitor of carbon dioxide fixation (21) as well as
compound 6, which blocks electron transport at the quinone B level by interacting at the D1 protein
(22). In addition, the commercial herbicides pendimethalin (7) and trifluralin (8) inhibit root and shoot
growth (23, 24). Furthermore, herbicidal activity has been reported for a number of amide derivatives
as photosynthetic electron transport inhibitors (9-11) (25–27) as well as for naptalam (12) which
inhibits both basipetal auxin transport and root elongation (Fig. 1) (28).
This article is protected by copyright. All rights reserved

The development of new herbicides based on molecular hybridization is an important strategy,
particularly when the active hybrid molecules possess independent modes of action (29–32). Molecular
hybridization is a rational strategy to develop new prototypes based on the fusion of two bioactive
compounds and also the chemical modification of bioactive molecules. This method provides new
molecules which might present improved activity relative to the original molecule the activities of both
parent molecules expressed in a single hybrid structure (33–37).
Our aims in this work are the synthesis of an array of indole derivatives containing amide or nitro
groups (Fig. 1), evaluation and determination of the effects of these compounds on photosynthetic
apparatus and plant development through root and stems growth and dry biomass assays.
MATERIALS AND METHODS
General Information. Unless otherwise noted, all commercially available reagents were purchased
from Aldrich Chemical Co. and used without purification. Column chromatography was performed
using Merck Silica Gel (230-400 mesh). Thin layer chromatography (TLC) was performed using
Merck Silica Gel GF254, 0.25 mm thickness. For visualization, TLC plates were either placed stained
with iodine vapor or acidic vanillin. Nuclear magnetic resonance (1H NMR) spectra and (13C NMR)
spectra were recorded on a Bruker 500 MHz Nuclear Magnetic Resonance. The coupling constants (J)
are given in hertz (Hz) and chemical shifts are denoted in parts per million (ppm) downfield from
internal standard, tetramethylsilane (TMS). The abbreviations s, bs, d, t, q, m, and dd refer to singlet,
broad singlet, doublet, triplet, quartet, multiplet, and doublet of doublets, respectively. All NMR
spectra were obtained with CDCl3 or DMSO-d6. Fourier transform infrared spectroscopy (FTIR)
analysis was performed on Shimadzu Iraffinity-1 Spectrophotometer. KBr pellets were prepared
mixing the compounds (1 mg) with pure and anhydrous KBr (100 mg). The high resolution mass
spectra (HRMS) data were recorded on UFLC/DAD/MicroTOF.Melting points were recorded on a
PFM-II / MS Tecnopon melting point apparatus.
This article is protected by copyright. All rights reserved

General Method for the Synthesis of Compounds 14a-b. A solution of substituted phenyl amine
(13a-b) (0.16 mol) in HCl 37% (6.5 mL) was cooled to 0˚C. To the resulting cooled solution was
added sodium nitrite (7.24 mmol in 4.0 mL water) dropwise under stirring at the same temperature and
stirred for 10 min. Subsequently, a solution of SnCl2.2H2O (0.026 mol in 3.5 mL HCl 37%) was added
dropwise. Then, the resulting suspension was stirred at 0 ˚C for 30 min. The resulting residue was
filtered, washed with diethyl ether (2 × 10 mL) and dried under vacuum. The crude product was used
for further reactions without purification (38).
General Method for the Synthesis of Compounds 15a-d. Using a modified literature procedure
(39), to a suspension of substituted phenylhydrazine hydrochloride (14a-b) (1.50 mmol) and
cyclohexanone or 2-butanone (1.50 mmol) in water (8 mL) was added H2SO4 (0.7 mmol). The mixture
was heated at 100˚C for 30 min. After completion of the reaction, the mixture was cooled to room
temperature and cold water (10 mL) was added. The precipitate was filtered, washed with water (3 x
10 mL) and dried under reduced pressure to provide the hydrazone intermediate.
To a suspension of hydrazone intermediate in acetic acid (8 mL) was added ZnCl2 (0.01 mol, 1.36 g).
The mixture was heated at 118ºC for 12 h. After completion of the reaction, the mixture was cooled to
0°C, the precipitate was filtered off, washed with cold water (10 mL) and dried under reduced pressure
to provide the crude product. Further purification was achieved by flash column chromatography on a
silica gel employing hexane:ethyl acetate (9:1) to give the desired products.
6-chloro-8-nitro-2,3,4,9-tetrahydro-1H-carbazole (15a). Yield: 32% (120 mg). Orange solid. Melting
point: 212 ºC (lit.: 215 ºC) (40). IR (KBr pellet, cm-1): 3417, 2931, 2846, 1512. NMR 1H (DMSO-d6,
500 MHz) δ: 1.76-1.85 (m, 4H), 2.62-2.64 (m, 2H), 2.76-2.78 (m, 2H), 7.88 (d, J = 1.8 Hz, 1H), 7.91
(d, J = 1.91 Hz, 1H). NMR 13C (DMSO-d6, 125 MHz) δ: 20.0, 22.3, 22.8, 110.0, 116.0, 121.8, 124.4,
126.8, 131.5, 132.7, 140.6.
5-chloro-2,3-dimethyl-7-nitro-1H-indole (15b). Yield: 30% (101 mg). Orange solid. Melting point:
201-205 ºC (lit.: 202 ºC) (40). IR (KBr pellet, cm-1): 3387, 3093, 2924, 2854, 1512, 1365, 1307,
1
1296, 894. NMR H (DMSO-d6, 500 MHz) δ: 2.17 (s, 3H), 2.39 (s, 3H), 7.91 (d, J = 1.9 Hz, 1H),
This article is protected by copyright. All rights reserved

7.93 (d, J = 1.8 Hz, 1H), 11.67 (br s, 1H). NMR 13C (DMSO-d6, 125 MHz) δ: 8.40, 11.70, 107.82,
116.54, 122.34, 125.21, 126.92, 131.90, 134.86, 138.24.
8-chloro-6-nitro-2,3,4,9-tetrahydro-1H-carbazole (15c). Yield: 44% (165 mg). Yellow solid. Melting
point: 220-225 ºC (lit.: 218 ºC) (40). IR (KBr pellet, cm-1): 3348, 2931, 2854, 1512, 1481, 1327,
1
1296, 887. NMR H (DMSO-d6, 500 MHz) δ: 1.78-1.87 (m, 4H), 2.67-2.70 (m, 2H), 2.74-2.76 (m,
2H), 7.94 (d, J = 1.9 Hz, 1H), 8.30 (d, J = 1.9 Hz, 1H), 11.87 (br s, 1H). NMR 13C (DMSO-d6, 125
MHz) δ: 20.70, 22.78, 22.84, 23.17, 113.14, 113.76, 115.19, 115.61, 128.16, 136.02, 140.66.
7-chloro-2,3-dimethyl-5-nitro-1H-indole (15d): Yield: 36% (121 mg) Yellow solid. Melting point:
245-248 ºC (lit.: 230 ºC) (40). IR (KBr pellet, cm-1): 3348, 3093, 2924, 2854, 1519, 1481, 1327, 887.
NMR 1H (DMSO-d6, 500 MHz) δ: 2.22 (s, 3H), 2.36 (s, 3H), 7.93 (d, J = 1.98 Hz, 1H), 8.33 (d, J =
13
1,98 Hz, 1H), 11.84 (br s, 1H). NMR C (DMSO-d6, 125 MHz) δ: 8.14, 11.19, 109.88, 113.53,
114.59, 114.90, 129.24, 135.01, 137.26, 140.21.
General Method for the Synthesis of Compounds 16a-d: Using modified literature procedures (41,
42), to a solution of nitro derivative (15a-d) (0.20 mmol) in ethyl acetate (3 mL) was added
SnCl2.2H2O (1.4 mmol, 0.316 g). The mixture was stirred at room temperature for 48 h. After
completion of the reaction, an NH4OH solution (2 mL) was added, and the precipitate was filtered and
washed with ethyl acetate (3 x 10 mL). The organic layers were combined and the solvent was
removed under reduced pressure to provide amine intermediate.
To a solution of the amine intermediate in acetic acid (3.0 mL) was added acetic anhydride (10.6
mmol, 1.00 mL) and 10% aqueous solution of sodium acetate (6.0 mL). The mixture was stirred at
room temperature for 4 h. After completion of the reaction, water (10 mL) was added to the mixture
and extracted with dichloromethane (2 x 5.0 mL). The organic layers were combined, washed with
saturated aqueous solution of NaHCO3, brine and solvent was removed under reduced pressure.
Further purification was achieved by flash column chromatography on a silica gel employing
hexane:ethyl acetate (1:2) to give the desired products.
This article is protected by copyright. All rights reserved

N-(6-chloro-2,3,4,9-tetrahydro-1H-carbazol-8-yl)acetamide (16a): Yield: 24% (12 mg). Purple solid.
Melting point: 230 ºC. IR (KBr pellet cm-1): 3317, 2931, 2854, 1651, 1535, 1481, 1411, 725. NMR
1H (DMSO-d6, 500 MHz) δ: 1.75-1.85 (m, 4H), 2.12 (s, 3H), 2.56-2.58 (m, 2H), 2.71-2.73 (m, 2H),
7.13 (d, J = 1.7 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 9.64 (s, 1H), 10.49 (s, 1H). NMR 13C (DMSO-d6,
125 MHz) δ: 20.39, 22.64, 22.73, 23.73, 108.75, 112.10, 112.58, 122.39, 123.74, 125.81, 129.09,
135.80, 168.41. HR-ESI-MS: C14H16ClN2O [M+H]+ m/z found 263.0958, calculated 263.0951.
N-(5-chloro-2,3-dimethyl-1H-indol-7-yl)acetamide (16b): Yield: 35% (18 mg). Green solid. Melting
1
point: 254-260 ºC. IR (KBr pellet, cm-1): 3294, 2924, 2854, 1658, 1535, 1465, 1373, 894. NMR H
(DMSO-d6, 500 MHz) δ: 2.10 (s, 3H), 2.11 (s, 3H), 2.31 (s, 3H), 7.14 (d, J = 1.84 Hz, 1H), 7.46 (d, J
= 1.83 Hz, 1H), 9.62 (br s, 1H), 10.47 (br s, 1H). NMR 13C (DMSO-d6, 125 MHz) δ: 8.21, 11.24,
23.76, 105.83, 111.93, 112.75, 122.39, 123.51, 125.30, 130.73, 132.74, 168.44. HR-ESI-MS:
C12H14ClN2O [M+H]+ m/z found 237.0799, calculated: 237.0795.
N-(8-chloro-2,3,4,9-tetrahydro-1H-carbazol-6-yl)acetamide (16c): Yield: 38% (20 mg). Grey solid.
Melting point: 290-295 ºC. IR (KBr pellet, cm-1): 3294, 3116, 2931, 2854, 1658, 1558, 1450, 1381,
1
864. NMR H (DMSO-d6, 500 MHz) δ: 1.76-1.81 (m, 4H), 2.00 (s, 3H), 2.54-2.56 (m, 2H), 2.67-
2.69 (m, 2H), 7.33 (d, J = 1.68 Hz, 1H), 7.48 (d, J = 1.52 Hz, 1H), 9.78 (br s, 1H), 10.84 (br s, 1H).
NMR 13C (DMSO-d6, 125 MHz) δ: 20.59, 22.63, 22.77, 22.81, 23.88, 106.64, 109.30, 112.64,
114.25, 128.66, 129.13, 131.54, 136.98, 167.71. HR-ESI-MS: C14H16ClN2O [M+H]+ m/z found
263.0968, calculated 263.0951.
N-(7-chloro-2,3-dimethyl-1H-indol-5-yl) acetamide (16d): Yield: 26% (17 mg). Blue solid. Melting
1
point: 210 ºC. IR (KBr pellet, cm-1): 3209, 3078, 2924, 2854, 1627, 1581, 1489, 894, 717. NMR H
(DMSO-d6, 500 MHz) δ: 2.01 (s, 3H), 2.09 (s, 3H), 2.30 (s, 3H), 7.32 (d, J = 1.54 Hz, 1H), 7.50 (d, J
= 1.32 Hz, 1H), 9.79 (br s, 1H), 10.84 (br s, 1H). NMR 13C (DMSO-d6, 125 MHz) δ: 8.41, 11.15,
23.88, 106.30, 106.83, 112.58, 113.97, 128.64, 130.20, 131.50, 133.93, 167.69. HR-ESI-MS:
C12H13ClN2NaO [M+Na]+ m/z found 259.0662, calculated 259.0614.
This article is protected by copyright. All rights reserved

General Method for the Synthesis of Compounds 20a-b. Using a modified literature procedure
(43), to a solution of compounds 19a or 19b (0.7 mmol) in ethanol (5 mL) was added dropwise H2SO4
(5.72 mmol, 0.305 mL). The mixture was refluxed for 16 h. After completion of the reaction, water
(10 mL) was added to the mixture and extracted with diethyl ether (3 x 10 mL). The organic layers
were combined, washed with saturated aqueous solution of NaHCO3 (3 x 10 mL), brine and the
solvent removed under reduced pressure. Further purification was achieved by flash column
chromatography on a silica gel employing hexane:ethyl acetate (9:1) to give the desired products.
Ethyl 2,3,4,9-tetrahydro-1H-carbazole-8-carboxylate (20a): Yield: 70%. White solid. Melting point:
1
84-85 ºC (lit.: 76 ºC).(43) IR (KBr pellet, cm-1): 3402, 2985, 1674, 1265. NMR H (DMSO-d6, 500
MHz) δ: 1.42 (t, J = 7.17 Hz, 3H), 1.84-1.94 (m, 4H), 2.69-2.71 (m, 2H), 2.75-2.77 (m, 2H), 4.42(q,
J = 7.17 Hz, 2H), 7.07 (t, J = 7.63 Hz, 1H), 7.63 (d, J = 7.63 Hz, 1H), 7.78 (d, J = 7.63 Hz, 1H), 9.39
13
(br s, 1H). NMR C (DMSO-d6, 125 MHz) δ: 14.46, 20.80, 23.11, 23.19, 23.20, 60.59, 109.97,
111.81, 118.18, 122.94, 123.10, 129.02, 135.37, 135.80, 167.80.
Ethyl 2,3-dimethyl-1H-indole-7-carboxylate (20b): Yield: 62%. White solid. Melting point: 144-146
1
ºC (lit.: 104-106 ºC).(44) IR (KBr pellet, cm-1): 3402, 2978, 1674, 1265. NMR H (DMSO-d6, 500
MHz) δ: 1.43(t, J = 7.15 Hz, 3H), 2.23 (s, 3H), 2.39 (s, 3H), 4.43 (q, J = 7.15 Hz, 2H), 7.08 (t, J =
7.61 Hz, 1H), 7.85 (d, 7.77 Hz, 1H), 7.78 (dd, J = 7.61 and 0.9 Hz, 1H), 9.39 (s, 1H). NMR 13C
(DMSO-d6, 125 MHz) δ: 8.36, 11.54, 14.45, 60.57, 106.92, 111.55, 118.10, 122.88, 123.30, 130.62,
131.94, 135.32, 167.77.
General Method for the Synthesis of Compounds 21a-b. Modified literature procedure (45), a
suspension of 20a or 20b (0.206 mmol), 40% aqueous CH3NH2 (0.600 mL) in ethanol (1 mL) was
stirred at r.t. for 5 days. After completion of the reaction, the mixture was extracted with ethyl acetate
(3 x 10 mL). The organic layers were combined, washed with brine and the solvent removed under
reduced pressure. Further purification was achieved by flash column chromatography on a silica gel
employing dichloromethane:methanol (95:5) to give the desired products 21a or 21b.
This article is protected by copyright. All rights reserved

N-methyl-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (21a): Yield: 19%. White solid. Melting
point: 224-225 ºC. IR (KBr pellet, cm-1): 3394, 3309, 3055, 1643. NMR 1H (DMSO-d6, 500 MHz) δ:
1.84-1.93 (m, 4H), 2.69-2.71 (m, 2H), 2.74-2.77 (m, 2H), 3.04 (s, 3H), 6.36 (br s, 1H), 7.03 (t, J =
7.63 Hz, 1H), 7.23 (dd, J = 0.6 and 7.48 Hz, 1H), 7.58 (dd, J = 0.6 and 7.79 Hz, 1H), 9.83 (s, 1H).
NMR 13C (DMSO-d6, 125 MHz) δ: 20.96, 23.24, 23.32, 23.39, 26.60, 109.56, 115.07, 117.69,
118.03, 121.66, 129.57, 135.39, 136.10. 168.97. HR-ESI-MS: C14H17N2O [M+H]+ m/z found
229.1347, calculated 229.1341.
N,2,3-trimethyl-1H-indole-7-carboxamide (21b): Yield: 21%. White solid. Melting point: 190-193
1
ºC. IR (KBr pellet, cm-1): 3441, 3325, 2954, 1620. NMR H (DMSO-d6, 500 MHz) δ: 2.22 (s, 3H),
2.37 (s, 3H), 3.04 (s, 3H), 6.33 (br s, 1H), 7.04 (t, J = 7.63 Hz, 1H), 7.22 (dd, J = 0.46 and 7.48 Hz,
1H), 7.59 (d, J = 7.48 Hz, 1H), 9.82 (br s, 1H). NMR 13C (DMSO-d6, 125 MHz) δ: 8.50, 11.67,
26.57, 106.53, 114.81, 117.64, 117.96, 121.87, 131.20, 132.73, 134.93, 168.96. HR-ESI-MS:
C12H15N2O [M+H]+ m/z found 203.1186, calculated 203.1184.
Chlorophyll a fluorescence measurements (semi in vivo assay). According to a modified
literature procedure (12), ten spinach leaves were cut into 1.0 cm diameter discs and placed with 20
mL of modified Krebs solution into a Petri dish (9 cm diameter), the solution consisted of NaCl (115
mM), KCl (5.9 mM), MgCl2 (1.2 mM), KH2PO4 (1.2 mM), Na2SO4 (1.2 mM), CaCl2 (25 mM), and
NaHCO3 (25 mM, pH = 7.4). After 12 h of incubation at room temperature, compounds 15a-d, 16a-d,
20a-b and 21a-b were added in dimethyl sulfoxide (DMSO) solutions at 100 µM for each Petri dish
and incubated for 6 additional hours. Then, the discs were adapted in the dark for 30 min. Finally,
chlorophyll a (Chl a) fluorescence transients were measured using a Hansatech Handy- Plant Efficient
Analyzer; a three LEDs array allowed for illumination with 650 nm continuous light. DMSO was used
as negative control in order to consider the solvent effect. OJIP transients were analysed according to
the JIP test (see Supporting Information). The experiment was performed in triplicate and the results
were expressed as mean and standard error.
Plant material for in vivo assays. Commercially available seeds of Ipomoea grandifolia, Senna
alata and Sida rhombifolia weeds were placed in plastic pots (top diameter, 10.5 cm; bottom diameter,
This article is protected by copyright. All rights reserved

7.5 cm; height, 7.0 cm) filled with approximately 100 g of a mixture of 80:20 w/w soil/vermiculite
(plant growth medium). All pots were watered every day, maintained in a greenhouse at 25-30°C and
under normal day/night illumination. Plants were selected by uniformity size after 20 days of growth S.
rhombifolia and 4 days for I. grandifolia and S. alata. Plants of similar size were separated in three
groups: negative control (DMSO), positive control (DCMU) and experimental treated with
compounds 15a-b and 21b at 50 and 100 µM
Determination of Chl a fluorescence in intact leaves. Chl a fluorescence was measured in leaves
of the control plant and those treated with compounds 15a-b and 21b at 50 and 100 µM. After 1 h and
72 h of treatment, the plants adapted in the dark for 30 min were excited by light from an array of three
light-emitting diodes delivering 3000 µmol m–2 s–1 of red light (650 nm). The Chl a fluorescence
induction curves were measured at room temperature with a portable Hansatech Fluorescence Handy
PEA (plant efficiency analyser) apparatus.
Plant growth assay. To evaluate the effect of 15a-b and 21b in I. grandifolia, S. alata and S.
rhombifolia plant growth, the plant length and dry biomass was measured after 10 days of treatment.
Dry biomass was determined by drying all components in an oven at 60˚C for 15 days. Negative and
positive controls were performed using DMSO and DCMU, respectively. PDW was measured using an
analytical balance (2).
Molecular docking. Molecular docking simulations were performed to analyse interactions of the
compounds 15a-d, 16a-d, 20a-b and 21a-b with the active site of spinach photosystem II (PDB
deposited code: 3JCU) using the AutoDock 4.2 software (46). Chemical structures of the ligands were
drawn in the ChemDraw Professional 15.0 software followed by MM2 energy minimization.
According to a modified procedure (47), the target enzyme was prepared for molecular docking using
the chain A of the protein, followed by removal of all water molecules, addition of all hydrogens,
Gasteiger charges calculations, and lastly, all nonpolar hydrogens were merged to carbon atoms. A grid
box size of 40ꢀ×ꢀ40ꢀ×ꢀ40 points in x, y and z directions and -41.788, 3.115, -18.728 grid centre was set
for the enzyme. The box was centred based on the cognate ligand with a spacing of 0.486 Å. Cluster
This article is protected by copyright. All rights reserved

analysis was performed on the docked results using a root mean square deviation (RMSD) tolerance of
2 Å (48). 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
iterated 100 times, yielding 100 docked conformations. Best docking pose was selected on the basis of
key interacting residues: cluster RMS, number of hydrogen bonds and minimum binding energy.
PyMOL software (PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC) was employed
to show binding pose of indole derivatives in protein D1 (see Supporting Information).
Statistical analysis. Chl a fluorescence measurement (PIabs parameter) and plant growth assays were
performed in a completely randomized design. Data were analysed by means of analysis of variance
(ANOVA), and treatment means were compared by Tukey test (Honestly significant differences, HSD)
(5 and 1 %) was used to estimate least significant range between means (12).
RESULTS AND DISCUSSION
Indole derivative synthesis
The synthesis of nitro-indole derivatives 15a-d was based on Fischer indole methodology.
Substituted anilines (13a-b) were diazotized employing sodium nitrite in hydrochloric acid followed by
the reduction of the resultant diazonium chloride salts using stannous chloride in hydrochloric acid to
furnish 14a-b in a good yields (38). Aqueous solutions of phenyl hydrazine hydrochlorides 14a-b were
refluxed with cyclohexanone or 2-butanone in the presence of sulfuric acid to provide the hydrazone
intermediates, which were reacted with ZnCl2 in acetic acid to afford indoles 15a-d (39). Then, the
nitro groups were reduced employing SnCl2.2H2O and thereafter acetylation reaction with acetic
anhydride provides the amide derivatives 16a-d (Scheme 1) (41, 42).
<Scheme 1>
This article is protected by copyright. All rights reserved

To obtain compounds 21a-b, the carboxylic acid derivatives 19a-b (12) were refluxed in
ethanol using sulfuric acid as catalyst providing the ester derivatives 20a-b in moderate to good
chemical yields (43). Thus, compounds 20a-b were reacted with methylamine aqueous solution to
furnish the amide derivatives 21a-b (Scheme 2) (45).
<Scheme 2>
Effect of indole derivatives on Chl a fluorescence transient
Semi in vivo assays. The performance index per absorption (PIabs) has been considered the most
sensitive parameter for plant stress detection on PSII (49), relating the efficiency of excitation energy
absorption, electron capture and transfer by PSII, providing valuable information about environmental
stress on the photosynthetic apparatus (50–52). Almost all of the investigated hybrid molecules showed
appreciable activity decreasing PIabs parameter on Chl a fluorescence (Fig. 2).
<Figure 2>
The lowest values for PIabs were obtained when the leaf discs were exposed to 15a-b and 21b
(p  0.01). Nitro-indole derivatives 15a-d reduced PIabs parameter from 40% to 56%, however the
conversion of the nitro to an amide group (16a-d) decreased the inhibitory activity and there was no
statistically significant difference among them compared to the control (p > 0.05). The amides 21a-b
showed better inhibitory activity than the corresponding esters 20a-b, which increased PIabs by 13%
and 16%, respectively.
Additionally, the reduction of the parameters related to the reaction centre (RC) such as
energy fluxes for absorption (ABS/RC), electron trapping (TR0/RC), electron transport (ET0/RC) and
energy dissipation (DI0/RC) as well as the quantum yield parameters (PHI(P0), PSI0 and PHI(E0))
corroborate the inhibitory activity on PSII (Fig. 3) (49).
This article is protected by copyright. All rights reserved

<Figure 3>
The plant physiological vitality can be evaluated through the variation of PIabs and Vj
fluorescence parameters, which can corroborate the compound activity on active site of PSII.(52) The
J-level increase (2 ms) is characteristic of the transient OJIP fluorescence curve that describes the
effects of compounds by blocking the electron transport chain on PSII beyond the QA site. The indole
derivatives 15a-b and 21b at 50 µM and 100 µM increased the Vj parameter by 100% to 150%,
confirming the blockage of electron passage in the quinone pool, which corresponds to the QA re-
oxidation kinetics by electron transfer to QB (53).
Compound 15a reduced ET0/RC, PSI0 and PHI(E0) by 30%, 20% and 25%, respectively, at 50
µM and 100 µM (Fig. 3(A)). This variation shows the electron flow blockage through the transport
chain, indicating that PSII active reaction centres become heat sinks or silent reaction centres because
the energy is not employed to reduce QA (54). Compound 15b demonstrated better results at 50 µM
reducing TR/RC, ET0/RC and PHI(E0) parameters by 20%, 34% and 22%, respectively (Fig. 3 (B)).
This decrease shows the absorbed energy was trapped, reducing the electron flow in active reaction
centres. Additionally, the PsbS protein of the photosystem II increases the thermal dissipation of the
energy absorbed by the photosystem (52).
Compound 21b demonstrated the best result by blocking the electron flow. The ABS, TR0,
ET0, and DI0 parameters by RC were reduced by 38%, 38%, 52% and 37% respectively, at 100 µM
(Fig.3 (C)). The energy absorption reduction by the antenna complex directly influences the electron
transport and system energy dissipation, resulting in the photosynthesis Z-scheme not occurring
properly (55). The decrease of PSI0 and PHI(E0) parameters by 30% and 35%, respectively
corroborating the reduction of PSII quantum efficiency by these compounds, suggesting a decrease in
-
electron excitation and transfer by the electron transport chain after QA .
In vivo assays. Compounds 15a-b and 21b which showed the best activity in semi in vitro assays were
evaluated in in vivo experiments employing Ipomoea grandifolia, Senna alata and Sida rhombifolia
This article is protected by copyright. All rights reserved

weeds at 50 µM and 100 µM. The effects of 15a-b and 21b on PIabs parameter were evaluated after 1
and 72 hours after treatment with the active compounds (Fig. 4 (A-C)).
<Figure 4>
All compounds impacted the photosynthetic apparatus over the course of the experiment. The
effects of indole derivatives on I. grandifolia and S. alata plants were observed only after 72 hours of
treatment, potentially due to aspects of the plant’s physiology which increase the time taken by the
bioactive compounds to reach the PSII on the thylakoid membrane. The best results against I.
grandifolia were observed with compounds 15b and 21b at 50 µM, which decreased the PIabs
parameter by 20% and 11%, respectively. In the S. alata assay, compounds 15a-b at 50 µM reduced
PIabs parameter by 18% and 19%, respectively. The improved activity observed of compounds 15b
(ClogP 3.954) and 21b and (ClogP 1.991) compared to 15a (ClogP 4.577) at low concentration can be
rationalized by the reduced lipophilicity coefficient (ClogP), as the ClogP increases, water solubility of
the compound is decreased, which affects the biological activity.
The PIabs parameter was decreased by an average of 77% to 87% by compounds 15a-b and 21b
at both concentrations after 1 hour of treatment for S. rhombifolia plant. However, after 72 hours of
treatment, PIabs parameter increased by an average of 44% to 136% compared to control. These results
are related to the low photosynthetic capacity and initial stress adaptation caused by the indole
derivatives, indicating the efficiency of the photosynthetic process in the plant is impacted by reversible
stress effects on PSII (16).
The effects of 15a-b and 21b on photosynthetic quantum parameters in I. grandifolia, S. alata
and S. rhombifolia plants are displayed in Fig. 5. In I. grandifolia plants (Fig. 5 (A)), compounds 15b
and 21b reduced ET0/RC, PHI(E0), PSI0 and (PHI(P0) parameters by an average of 10%. The electron
transport and quantum yield reductions suggest an interruption of the photosynthesis process.
Additionally, the lower maximum quantum yield of the primary photochemistry and the electron
-
transport chain after QA indicates that the effect was at the quinone pool on the acceptor side of PSII.
This article is protected by copyright. All rights reserved

The increase of DI0/RC and Vj parameters by 15% and 20% respectively, corroborate the electron
flow blockage described previously, due to an excess of energy being dissipated in a non-
photochemical way and that the detrimental effect was at the polyphasic level J (2 ms) in the quinone
redox process.
<Figure 5>
Compounds 15a-b showed the best results against S. alata plants (Fig. 5 (B)), at 50 µM after 72
hours of treatment. Compounds 15a and 15b decreased the phenomenological parameters by reaction
centre ABS/RC, TR0/RC and ET0/RC by an average of 25% and 10%, respectively. The electron
transport chain blockage on PSII was confirmed by the action site location through an increase of Vj
parameter by 28% (15a) and 18% (15b).
Compounds 15a-b and 21b showed appreciable results on S. rhombifolia plants after 1 hour of
contact and completely lost their activity after 72 h, potentially due to a self-protection mechanism of
the S. rhombifolia weed by the stressed conditions induced by the compound (Fig. 5 (C)). The indole
derivatives showed similar activity on PSII, increasing ABS/RC, TR0/RC and DI0/RC parameters by an
average of 17% to 31%, 10% to 18% and 32% to 56%, respectively, demonstrating that the
compounds lead to inhibition of the QA redox process via energy accumulation in reaction centres. The
reduction of quantum parameters PHI(E0), PSI0 and PHI(P0) by 22%-40%, 17%-34% and 10%,
respectively, confirm the electron transport chain was affected in the primary photochemical process
-
leading to an accumulation of QA at PSII.
The effects caused by 15a-b and 21b in the in vivo studies are not significant compared to the
semi in vivo assay. Compound penetration into the living plant is impeded by several factors, including
membrane permeability, compound solubility, subcellular compartmentalization, and a number of
complicating physiological process. These can completely overturn in vivo inhibitory activity for a
given compound (56).
This article is protected by copyright. All rights reserved

Effect of indole derivatives on plant growth
The phytotoxic activity of 15a-b and 21b was evaluated through plant growth assays
employing I. grandifolia, S. alata and S. rhombifolia weeds. Plant length and dry biomass were
evaluated after 6 days of treatment with 15a-b and 21b at 50 μM and 100 μM. DMSO was used as
negative control and DCMU was employed as positive control at 50 μM (Fig. 6).
<Figure 6>
All active compounds showed same behaviour; decreasing plant length and dry biomass, except
for the S. rhombifolia weed which showed no variation in plant growth (Fig. 6 (C)).
On the I. grandifolia weed (Fig. 6 (A)), indole derivative 15a showed the best results at 100
µM reducing the plant length and dry biomass by 24% and 40%, respectively. Compound 21b
decreased plant length by 30% and dry biomass by 31% at 50 μM and 100 µM, respectively.
Compound 15b affected the plant growth by reducing the plant length by 30% at 100 µM; however,
the dry biomass was not reduced.
These results suggest that the photosynthesis inhibitory activity diminishes the synthesis of ATP
and CO2 fixation which disturbs plant development. The best result was achieved for 15b in S. alata at
50 µM which demonstrated a similar activity to commercial herbicide DCMU, reducing the dry
biomass by 37% at 50 µM compared to the control (Fig.6 (B)). These results corroborate the PSII
inhibitory activities demonstrated by the Chl a fluorescence assay, indicating that 15a and 15b have a
selective action as a post-emergent herbicide, blocking the electron transfer process at PSII in the
plants. These compounds appear to be potential herbicides with similar symptoms such as chlorosis and
necrosis of the plant tissue exhibited by the photosynthesis inhibitor as with DCMU.
Molecular docking
This article is protected by copyright. All rights reserved

The docking was carried out for all synthesized compounds employing the protein D1 of the
PSII reaction centre from Spinacia oleracea (PDB ID: 3JCU). In addition, herbicides that inhibit PSII
prefer the position near the loop made by Ser264 and Phe265 at protein D1. The same orientation for
indole derivatives and DCMU in the binding site was observed (57–59). Figure 7 exhibits the best
docking pose of compounds 15a and 15b including hydrogen bonding interactions, with binding
energies of -8.47 kcalmol-1 and -7.39 kcalmol-1, respectively.
<Figure 7>
The nitro group of 15a-d forms strong hydrogen bond interactions with the OH group of
Ser264 and the NH group of His252 with an individual bond-length range of 1.799–1.971Å. These
hydrogen-bonding interactions play a key role in determining the 3D space position of the nitro
derivatives in the binding pocket of protein D1. Additionally, hydrophobic interactions were observed
with Phe211, Met214, His215, Leu218, Val219 and Leu271. For compounds 15c and 15d, the
exchange of a nitro for a chlorine group on the indole scaffold disfavours hydrogen bonds and
hydrophobic interactions at the protein D1 active site, reducing their binding energies to -7.62 kcalmol-
1 and -6.82 kcalmol-1, respectively.
In this pocket, the amide group of compounds 16a-d acts as hydrogen bond acceptor with the
backbone OH of Ser264 and the imidazole NH of His252. Furthermore, the amide group acts as an
electron donating group, increasing the indole ring’s electron density, disfavouring Pi-sigma
interactions with the residues Leu218 and Leu271, decreasing the affinity with the binding site. The
difference observed between the binding mode of the ester derivatives 20a-b and amide derivatives
21a-b is associated to the strong hydrogen-bonding interaction that the amide group of 21a-b forms in
the site pocket with His252, with a distance bond of 1.888 Å for 21b.
These results suggest that hydrogen bond acceptors and electron withdrawing groups on the
indole phenyl ring are important for ligand interactions via hydrogen bond formation with Ser264 and
This article is protected by copyright. All rights reserved

His252 as well as the hydrophobic interactions with Leu218 and Leu271, potentially leading to an
inhibition of protein D1 on PSII.
CONCLUSION
This work demonstrates the synthesis of indole derivatives based on the principle of molecular
hybridization and their evaluation as photosynthesis and plant growth inhibitors. The best results were
obtained for the compounds 6-chloro-8-nitro-2,3,4,9-tetrahydro-1H-carbazole (15a) and 5-chloro-2,3-
dimethyl-7-nitro-1H-indole (15b), which decreased the phenomenological parameters ABS/RC,
TR0/RC and ET0/RC of PSII. The compound 15a and 15b act as the post-emergent herbicide
prototype since the dry biomass was reduced through plant growth assays employing I. grandifolia, S.
alata and S. rhombifolia weeds. The best result was achieved for 15b in S. alata at 50 µM which
demonstrated similar activity to commercial herbicide DCMU reducing the dry biomass by 37% at 50
µM compared to the control, corroborating the fluorescence results.
The nitro indole derivatives showed better results than amide indole derivatives indicating the
presence of strong electron withdrawing groups at indole phenyl ring is important for the ligand’s
interaction with the binding pocked of protein D1 on PSII as showed by molecular docking studies.
Moreover, optimization of these molecular features is a goal of our group in further understanding and
development of new herbicides.
ACKNOWLEDGEMENTS- This work has been supported in part by FAPEMAT (grant
0217830/2017 and 0250685/2017), CNPq (grant 430089/2016-3) and UFMT for financial support and
fellowships. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior - Brasil (CAPES) - Finance Code 001.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the
end of the article:
This article is protected by copyright. All rights reserved

Table S1. Formulae and explanation of the technical data of the OJIP curves and the selected JIP-test
parameters used in this work.
Table S2. Binding interaction of indole derivatives (15a-d, 16a-d, 20a-b and 21a-b) and DCMU with
the pocket site of PSII of Spinacia oleracea L., and ClogP.
Figure S1. Models of the binding interaction of 15a with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S2. Models of the binding interaction of 15b with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S3. Models of the binding interaction of 15c with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S4. Models of the binding interaction of 15d with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S5. Models of the binding interaction of 16a with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S6. Models of the binding interaction of 16b with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S7. Models of the binding interaction of 16c with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S8. Models of the binding interaction of 16d with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S9. Models of the binding interaction of 20a with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S10. Models of the binding interaction of 20b with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S11. Models of the binding interaction of 21a with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
Figure S12. Models of the binding interaction of 21b with the pocket site of PSII of Spinacia oleracea
(PDB ID: 3JCU).
This article is protected by copyright. All rights reserved

Figure S13. Models of the binding interaction of DCMU with the pocket site of PSII of Spinacia
oleracea (PDB ID: 3JCU).
Figure S14. 1H NMR spectrum of compound 15a (DMSO-d6, 500 MHz).
Figure S15. 13C NMR spectrum of compound 15a (DMSO-d6, 125 MHz).
Figure S16. 1H NMR spectrum of compound 15b (DMSO-d6, 500 MHz).
Figure S17. 13C NMR spectrum of compound 15b (DMSO-d6, 125 MHz).
Figure S18. 1H NMR spectrum of compound 15c (DMSO-d6, 500 MHz).
Figure S19. 13C NMR spectrum of compound 15c (DMSO-d6, 125 MHz).
Figure S20. 1H NMR spectrum of compound 15d (DMSO-d6, 500 MHz).
Figure S21. 13C NMR spectrum of compound 15d (DMSO-d6, 125 MHz).
Figure S22. 1H NMR spectrum of compound 16a (DMSO-d6, 500 MHz).
Figure S23. 13C NMR spectrum of compound 16a (DMSO-d6, 125 MHz).
Figure S24. 1H NMR spectrum of compound 16b (DMSO-d6, 500 MHz).
Figure S25. 13C NMR spectrum of compound 16b (DMSO-d6, 125 MHz).
Figure S26. 1H NMR spectrum of compound 16c (DMSO-d6, 500 MHz).
Figure S27. 13C NMR spectrum of compound 16c (DMSO-d6, 125 MHz).
Figure S28. 1H NMR spectrum of compound 16d (DMSO-d6, 500 MHz).
Figure S29. 13C NMR spectrum of compound 16d (DMSO-d6, 125 MHz).
Figure S30. 1H NMR spectrum of compound 20a (CDCl3, 500 MHz).
Figure S31. 13C NMR spectrum of compound 20a (CDCl3, 125 MHz).
Figure S32. 1H NMR spectrum of compound 20b (CDCl3, 500 MHz).
Figure S33. 13C NMR spectrum of compound 20b (CDCl3, 125 MHz).
Figure S34. 1H NMR spectrum of compound 21a (CDCl3, 500 MHz).
Figure S35. 13C NMR spectrum of compound 21a (CDCl3, 125 MHz).
Figure S36. 1H NMR spectrum of compound 21b (CDCl3, 500 MHz).
Figure S37. 13C NMR spectrum of compound 21b (CDCl3, 125 MHz).
This article is protected by copyright. All rights reserved

REFERENCES
1. Green, J. M. (2014) Current state of herbicides in herbicide-resistant crops. Pest Manag. Sci. 70,
1351–1357. https://doi.org/10.1002/ps.3727.
2. Sampaio, O. M., Lima, M. M. de C., Veiga, T. A. M., King-Díaz, B., da Silva, M. F. das G. F. and
Lotina-Hennsen, B. (2016) Evaluation of antidesmone alkaloid as a photosynthesis inhibitor. Pestic.
Biochem. Physiol. 134, 55–62. https://doi.org/10.1016/j.pestbp.2016.04.006.
3. Sampaio, O., Vieira, L., Bellete, B., King-Diaz, B., Lotina-Hennsen, B., da Silva, M., Veiga, T.,
Sampaio, O. M., Vieira, L. C. C., Bellete, B. S., King-Diaz, B., Lotina-Hennsen, B., Da Silva, M. F.
das G. F. and Veiga, T. A. M. (2018) Evaluation of Alkaloids Isolated from Ruta graveolens as
Photosynthesis Inhibitors. Molecules 23, 2693. https://doi.org/10.3390/molecules23102693.
4. Bałczewski, P., Biczak, R., Turek, M., Pawłowska, B., Różycka-Sokołowska, E., Marciniak, B.,
Deska, M. and Skalik, J. (2018) Ammonium 2,2′-thiodiacetates – Selective and environmentally safe
herbicides. Ecotoxicol. Environ. Saf. 163, 408–416.
https://doi.org/https://doi.org/10.1016/j.ecoenv.2018.07.093.
5. Dinehart, S. K., Smith, L. M., McMurry, S. T., Anderson, T. A., Smith, P. N. and Haukos, D. A.
(2009) Toxicity of a glufosinate- and several glyphosate-based herbicides to juvenile amphibians from
the Southern High Plains, USA. Sci. Total Environ. 407, 1065–1071.
https://doi.org/https://doi.org/10.1016/j.scitotenv.2008.10.010.
6. Hoagland, R. E., Boyette, C. D., Weaver, M. A. and Abbas, H. K. (2007) BIOHERBICIDES:
RESEARCH AND RISKS. Toxin Rev. 26, 313–342. https://doi.org/10.1080/15569540701603991.
This article is protected by copyright. All rights reserved

7. Saha, S., Dutta, D., Karmakar, R. and Ray, D. P. (2012) Structure–toxicity relationship of
chloroacetanilide herbicides: Relative impact on soil microorganisms. Environ. Toxicol. Pharmacol.
34, 307–314. https://doi.org/https://doi.org/10.1016/j.etap.2012.04.014.
8. Federico, C., Motta, S., Palmieri, C., Pappalardo, M., Librando, V. and Saccone, S. (2011)
Phenylurea herbicides induce cytogenetic effects in Chinese hamster cell lines. Mutat. Res. Toxicol.
Environ. Mutagen. 721, 89–94. https://doi.org/https://doi.org/10.1016/j.mrgentox.2010.12.013.
9. Ye, F., Zhai, Y., Kang, T., Wu, S.-L., Li, J.-J., Gao, S., Zhao, L.-X. and Fu, Y. (2019) Rational
design, synthesis and structure-activity relationship of novel substituted oxazole isoxazole
carboxamides as herbicide safener. Pestic. Biochem. Physiol. 157, 60–68.
https://doi.org/https://doi.org/10.1016/j.pestbp.2019.03.003.
10. Hernández-Romero, Y., Acevedo, L., de Los Ángeles Sánchez, M., Shier, W. T., Abbas, H. K. and
Mata, R. (2005) Phytotoxic Activity of Bibenzyl Derivatives from the Orchid Epidendrum rigidum. J.
Agric. Food Chem. 53, 6276–6280. https://doi.org/10.1021/jf0508044.
11. Rios, M. Y., Córdova-Albores, L. C., Ramírez-Cisneros, M. Á., King-Díaz, B., Lotina-Hennsen,
B., León Rivera, I. and Miranda-Sánchez, D. (2018) Phytotoxic Potential of Zanthoxylum affine and
Its Major Compound Linarin as a Possible Natural Herbicide. ACS Omega 3, 14779–14787.
https://doi.org/10.1021/acsomega.8b02020.
12. Mendes, M. C. da S., Fazolo, B. R., De Souza, J. M., De Vasconcelos, L. G., De Sousa Junior, P.
T., Dall’Oglio, E. L., Soares, M. A., Sampaio, O. M. and Vieira, L. C. C. (2019) Synthesis and
evaluation of indole derivatives as photosynthesis and plant growth inhibitors. Photochem. Photobiol.
Sci. 18, 1350–1358. https://doi.org/10.1039/c8pp00506k.
13. Zhang, Y.-Y., Gao, S., Liu, Y.-X., Wang, C., Jiang, W., Zhao, L.-X., Fu, Y. and Ye, F. (2020)
Design, Synthesis, and Biological Activity of Novel Diazabicyclo Derivatives as Safeners. J. Agric.
Food Chem. 68, 3403–3414. https://doi.org/10.1021/acs.jafc.9b07449.
14. Zhao, L.-X., Jiang, M.-J., Hu, J.-J., Zou, Y.-L., Cheng, Y., Ren, T., Gao, S., Fu, Y. and Ye, F.
This article is protected by copyright. All rights reserved

(2020) Design, Synthesis, and Herbicidal Activity of Novel Diphenyl Ether Derivatives Containing Fast
Degrading Tetrahydrophthalimide. J. Agric. Food Chem. 68, 3729–3741.
https://doi.org/10.1021/acs.jafc.0c00947.
15. Ye, F., Zhai, Y., Guo, K.-L., Liu, Y.-X., Li, N., Gao, S., Zhao, L.-X. and Fu, Y. (2019) Safeners
Improve Maize Tolerance under Herbicide Toxicity Stress by Increasing the Activity of Enzymes in
Vivo. J. Agric. Food Chem. 67, 11568–11576. https://doi.org/10.1021/acs.jafc.9b03587.
16. Digrado, A., De La Motte, L. G., Bachy, A., Mozaffar, A., Schoon, N., Bussotti, F., Amelynck,
C., Dalcq, A. C., Fauconnier, M. L., Aubinet, M., Heinesch, B., Du Jardin, P. and Delaplace, P. (2018)
Decrease in the photosynthetic performance of temperate grassland species does not lead to a decline
in the gross primary production of the ecosystem. Front. Plant Sci. 9, 1–16.
https://doi.org/10.3389/fpls.2018.00067.
17. King, R. R., Lawrence, C. H. and Gray, J. A. (2001) Herbicidal Properties of the Thaxtomin Group
of Phytotoxins. J. Agric. Food Chem. 49, 2298–2301. https://doi.org/10.1021/jf0012998.
18. Xiao, L., Du, F., Zhou, Y. and Wang, Z. (2018) Isolation of chlamydosporin indole derivative from
Fusarium chlamydosporum QJP1 fermentation useful as herbicide. CN 2018-10125578.
19. Tice, C. M. (2001) Selecting the right compounds for screening: Does Lipinski’s rule of 5 for
pharmaceuticals apply to agrochemicals? Pest Manag. Sci. 57, 3–16. https://doi.org/10.1002/1526-
4998(200101)57:1<3::AID-PS269>3.0.CO;2-6.
20. Clarke, E. D. and Delaney, J. S. (2006) Physical and Molecular Properties of Agrochemicals: An
Analysis of Screen Inputs, Hits, Leads, and Products. Chim. Int. J. Chem. 57, 731–734.
https://doi.org/10.2533/000942903777678641.
21. Szigeti, Z., Tóth, E. and Paless, G. (1982) Mode of action of photosynthesis inhibiting 4-hydroxy-
benzonitriles containing nitro group. Photosynth. Res. 3, 347–356.
https://doi.org/10.1007/BF00034115.
This article is protected by copyright. All rights reserved

22. Varejão, J. O. S., Barbosa, L. C. A., Varejão, E. V. V., Maltha, C. R. A., King-Díaz, B. and
Lotina-Hennsen, B. (2014) Cyclopent-4-ene-1,3-diones: A new class of herbicides acting as potent
photosynthesis inhibitors. J. Agric. Food Chem. 62, 5772–5780. https://doi.org/10.1021/jf5014605.
23. Bajwa, A. A., Ullah, A., Farooq, M., Chauhan, B. S. and Adkins, S. (2019) Chemical control of
parthenium weed (Parthenium hysterophorus L.) in two contrasting cultivars of rice under direct-
seeded conditions. Crop Prot. 117, 26–36.
https://doi.org/https://doi.org/10.1016/j.cropro.2018.11.009.
24. Deligios, P. A., Carboni, G., Farci, R., Solinas, S. and Ledda, L. (2019) The influence of herbicide
underdosage on the composition and diversity of weeds in oilseed rape (Brassica napus L. var. oleifera
D.C.) mediterranean fields. Sustain. 11, 1653. https://doi.org/10.3390/su11061653.
25. Musiol, R., Tabak, D., Niedbala, H., Podeszwa, B., Jampilek, J., Kralova, K., Dohnal, J., Finster,
J., Mencel, A. and Polanski, J. (2008) Investigating biological activity spectrum for novel quinoline
analogues 2: Hydroxyquinolinecarboxamides with photosynthesis-inhibiting activity. Bioorg. Med.
Chem. 16, 4490–4499. https://doi.org/https://doi.org/10.1016/j.bmc.2008.02.065.
26. Castelo-Branco, P. A., Dos Santos, F. J. L., Rubinger, M. M. M., Ferreira-Alves, D. L., Piló-
Veloso, D., King-Díaz, B. and Lotina-Hennsen, B. (2008) Inhibition and uncoupling of photosynthetic
electron transport by diterpene lactone amide derivatives. Zeitschrift fur Naturforsch. - Sect. C J.
Biosci. 63, 251–259. https://doi.org/10.1515/znc-2008-3-415.
27. Dolezal, M., Zitko, J., Osicka, Z., Kunes, J., Vejsova, M., Buchta, V., Dohnal, J., Jampilek, J. and
Kralova, K. (2010) Synthesis, antimycobacterial, antifungal and photosynthesis-inhibiting activity of
chlorinated N-phenylpyrazine-2-carboxamides. Molecules 15, 8567–8581.
https://doi.org/10.3390/molecules15128567.
28. Rashotte, A. M., Brady, S. R., Reed, R. C., Ante, S. J. and Muday, G. K. (2000) Basipetal Auxin
Transport Is Required for Gravitropism in Roots of Arabidopsis. Plant Physiol. 122, 481 LP – 490.
https://doi.org/10.1104/pp.122.2.481.
This article is protected by copyright. All rights reserved

29. Cojocaru, O. A., Shamshina, J. L., Gurau, G., Syguda, A., Praczyk, T., Pernak, J. and Rogers, R.
D. (2013) Ionic liquid forms of the herbicide dicamba with increased efficacy and reduced volatility.
Green Chem. 15, 2110–2120. https://doi.org/10.1039/C3GC37143C.
30. Wang, T., Bing, G., Zhang, X., Qin, Z., Yu, H., Qin, X., Dai, H., Miao, W., Wu, S. and Fang, J.
(2010) Synthesis and herbicidal activities of 2-cyano-3-benzylaminoacrylates containing thiazole
moiety. Bioorg. Med. Chem. Lett. 20, 3348–3351.
https://doi.org/https://doi.org/10.1016/j.bmcl.2010.04.027.
31. Ma, H.-J., Li, Y.-H., Zhao, Q.-F., Zhang, T., Xie, R.-L., Mei, X.-D. and Ning, J. (2010) Synthesis
and Herbicidal Activity of Novel N-(2,2,2)-Trifluoroethylpyrazole Derivatives. J. Agric. Food Chem.
58, 4356–4360. https://doi.org/10.1021/jf9042166.
32. Wang, D.-W., Lin, H.-Y., Cao, R.-J., Chen, T., Wu, F.-X., Hao, G.-F., Chen, Q., Yang, W.-C. and
Yang, G.-F. (2015) Synthesis and Herbicidal Activity of Triketone–Quinoline Hybrids as Novel 4-
Hydroxyphenylpyruvate Dioxygenase Inhibitors. J. Agric. Food Chem. 63, 5587–5596.
https://doi.org/10.1021/acs.jafc.5b01530.
33. Kakwani, M. D., Suryavanshi, P., Ray, M., Rajan, M. G. R., Majee, S., Samad, A., Devarajan, P.
and Degani, M. S. (2011) Design, synthesis and antimycobacterial activity of cinnamide derivatives: A
molecular hybridization approach. Bioorg. Med. Chem. Lett. 21, 1997–1999.
https://doi.org/https://doi.org/10.1016/j.bmcl.2011.02.022.
34. Barbosa, T. P., Sousa, S. C. O., Amorim, F. M., Rodrigues, Y. K. S., de Assis, P. A. C., Caldas, J.
P. A., Oliveira, M. R. and Vasconcellos, M. L. A. A. (2011) Design, synthesis and antileishmanial in
vitro activity of new series of chalcones-like compounds: A molecular hybridization approach. Bioorg.
Med. Chem. 19, 4250–4256. https://doi.org/https://doi.org/10.1016/j.bmc.2011.05.055.
35. Karthikeyan, C., Solomon, V. R., Lee, H. and Trivedi, P. (2013) Design, synthesis and biological
evaluation of some isatin-linked chalcones as novel anti-breast cancer agents: A molecular
hybridization approach. Biomed. Prev. Nutr. 3, 325–330.
This article is protected by copyright. All rights reserved

https://doi.org/https://doi.org/10.1016/j.bionut.2013.04.001.
36. Gündüz, M. G., Tahir, M. N., Armaković, S. J. S., Özkul Koçak, C. and Armaković, S. J. S.
(2019) Design, synthesis and computational analysis of novel acridine-(sulfadiazine/sulfathiazole)
hybrids as antibacterial agents. J. Mol. Struct. 1186, 39–49.
https://doi.org/https://doi.org/10.1016/j.molstruc.2019.03.010.
37. Reddyrajula, R. and Dalimba, U. (2019) Quinoline–1,2,3-triazole Hybrids: Design and Synthesis
through Click Reaction, Evaluation of Anti-Tubercular Activity, Molecular Docking and In Silico
ADME Studies. ChemistrySelect 4, 2685–2693. https://doi.org/10.1002/slct.201803946.
38. Raker, J., Wang, Y., Pechulis, A. D., Haber, J. C., Lynch, M. A. and Spring, S. L. (2012)
Synthesis of novel benzo[b]pyrazolo[1,5]diazepines. J. Heterocycl. Chem. 49, 221–226.
https://doi.org/10.1002/jhet.878.
39. Czeskis, B. A. and Wheeler, W. J. (2005) Synthesis of β3 adrenergic receptor agonist LY377604
and its metabolite 4-hydroxycarbazole, labeled with carbon-14 and deuterium. J. Label. Compd.
Radiopharm. 48, 407–419. https://doi.org/10.1002/jlcr.935.
40. Deorha, D. and Joshi, S. (1961) Notes- Cyclic Products from Hydrazines. I. Nitroindoles,
Nitrotetrahydrocarbazoles, Nitroindolenines and Nitrotetrahydrocarbazolenines. J. Org. Chem. 26,
3527–3530. https://doi.org/10.1021/jo01067a614.
41. Hadida Ruah, Sara S.; Grootenhuis, Peter D.J.; Van Goor, Frederick; Zhou, Jinglan; Bear, Brian;
Miller, Mark T.; McCartney, Jason; Numa, M. M. J. (2007) Modulators of ATP-binding cassette
transporters. 1–208.
42. Perkin, W. H. and Plant, S. G. P. (1923) LXXXI.—Derivatives of tetrahydrocarbazole. Part II. J.
Chem. Soc. Trans. 123, 676–695. https://doi.org/10.1039/CT9232300676.
43. Collar, W. M. and Plant, S. G. P. (1926) CIX.—Derivatives of tetrahydrocarbazole. Part V.
Carboxylic acids. J. Chem. Soc. 129, 808–810. https://doi.org/10.1039/JR9262900808.
This article is protected by copyright. All rights reserved

44. Brown, U. M., Carter, P. H. and Tomlinson, M. (1958) 374. Formyl compounds. Part II. J. Chem.
Soc. 1843–1849. https://doi.org/10.1039/JR9580001843.
45. Miller, W H; Newlander, K A; Seefeld, M A; Uzinskas, I N; Dewolf, W E Jr; Jakas, D. R. (2002)
Fab I Inhibitors.
46. Allouche, A. (2012) Software News and Updates Gabedit — A Graphical User Interface for
Computational Chemistry Softwares. J. Comput. Chem. 32, 174–182. https://doi.org/10.1002/jcc.
47. Nain-Perez, A., Barbosa, L. C. A., Maltha, C. R. A., Giberti, S. and Forlani, G. (2017) Tailoring
Natural Abenquines to Inhibit the Photosynthetic Electron Transport through Interaction with the D1
Protein in Photosystem II. J. Agric. Food Chem. 65, 11304–11311.
https://doi.org/10.1021/acs.jafc.7b04624.
48. Brozell, S. R., Mukherjee, S., Balius, T. E., Roe, D. R., Case, D. A. and Rizzo, R. C. (2012)
Evaluation of DOCK 6 as a pose generation and database enrichment tool. J. Comput. Aided. Mol.
Des. 26, 749–773. https://doi.org/10.1007/s10822-012-9565-y.
49. Oukarroum, A., El Madidi, S. and Strasser, R. J. (2016) Differential heat sensitivity index in barley
cultivars (Hordeum vulgare L.) monitored by chlorophyll a fluorescence OKJIP. Plant Physiol.
Biochem. 105, 102–108. https://doi.org/https://doi.org/10.1016/j.plaphy.2016.04.015.
50. Dai, Y., Yuan, L., Zhang, S., Wang, J., Xie, S., Zhao, M., Chen, G., Sun, R. and Wang, C. (2019)
Comprehensive evaluation for cold tolerance in wucai (brassica campestris l.) by the performance index
on an absorption basis (PIabs). Agronomy 9, 1–18. https://doi.org/10.3390/agronomy9020061.
51. Paoli, L. and Landi, M. (2013) The photosynthetic performance of sterile and fertile sporophytes in
a natural population of the fern Dryopteris affinis. Photosynthetica 51, 312–316.
https://doi.org/10.1007/s11099-013-0022-5.
52. Chen, S., Kang, Y., Zhang, M., Wang, X., Strasser, R. J., Zhou, B. and Qiang, S. (2015)
Differential sensitivity to the potential bioherbicide tenuazonic acid probed by the JIP-test based on fast
This article is protected by copyright. All rights reserved

chlorophyll fluorescence kinetics. Environ. Exp. Bot. 112, 1–15.
https://doi.org/https://doi.org/10.1016/j.envexpbot.2014.11.009.
53. Kalaji, H. M., Schansker, G., Ladle, R. J., Goltsev, V., Bosa, K., Allakhverdiev, S. I., Brestic, M.,
Bussotti, F., Calatayud, A., Dąbrowski, P., Elsheery, N. I., Ferroni, L., Guidi, L., Hogewoning, S. W.,
Jajoo, A., Misra, A. N., Nebauer, S. G., Pancaldi, S., Penella, C., Poli, D., Pollastrini, M.,
Romanowska-Duda, Z. B., Rutkowska, B., Serôdio, J., Suresh, K., Szulc, W., Tambussi, E.,
Yanniccari, M. and Zivcak, M. (2014) Frequently asked questions about in vivo chlorophyll
fluorescence: practical issues. Photosynth. Res. 122, 121–158. https://doi.org/10.1007/s11120-014-
0024-6.
54. de Carvalho, A. C., Salvador, J. P., Pereira, Thais de M. , Ferreira, P. H. A., Lira, J. C. S. and
Veiga, T. A. M. (2016) Fluorescence of Chlorophyll a for Discovering Inhibitors of Photosynthesis in
Plant Extracts. Am. J. Plant Sci. 07, 1545–1554.
55. McConnell, I., Li, G. H. and Brudvig, G. W. (2010) Energy Conversion in Natural and Artificial
Photosynthesis. Chem. Biol. 17, 434–447. https://doi.org/10.1016/j.chembiol.2010.05.005.
56. Avram, S., Funar-Timofei, S., Borota, A., Chennamaneni, S. R., Manchala, A. K. and Muresan, S.
(2014) Quantitative estimation of pesticide-likeness for agrochemical discovery. J. Cheminform. 6, 42.
https://doi.org/10.1186/s13321-014-0042-6.
57. Sobolev, V. and Edelman, M. (1995) Modeling the quinone-B binding site of the photosystem-II
reaction center using notions of complementarity and contact-surface between atoms. Proteins Struct.
Funct. Bioinforma. 21, 214–225. https://doi.org/10.1002/prot.340210304.
58. Takahashi, R., Hasegawa, K., Takano, A. and Noguchi, T. (2010) Structures and Binding Sites of
Phenolic Herbicides in the QB Pocket of Photosystem II. Biochemistry 49, 5445–5454.
https://doi.org/10.1021/bi100639q.
59. Guo, Y., Cheng, J., Lu, Y., Wang, H., Gao, Y., Shi, J., Yin, C., Wang, X., Chen, S., Strasser, R. J.
and Qiang, S. (2020) Novel Action Targets of Natural Product Gliotoxin in Photosynthetic Apparatus
This article is protected by copyright. All rights reserved

. Front. Plant Sci. 10, 1688.
FIGURE CAPTIONS
Figure 1. Design of amide or nitro indole derivatives from known herbicidal activity compounds.
Figure 2. Performance Index (PIabs) of spinach leaf discs subjected to 15a-d, 16a-d, 20a-b and 21a-b
(100 µM). (*) and (**) indicate significant difference from control (DMSO) with p  0.05 and p 
0.01, respectively.
Figure 3. Radar plots of the effects of (A) 15a, (B) 15b and (C) 21b on Chl a fluorescence
parameters calculated from the OJIP curve in spinach sheet discs at 50 µM and 100 µM.
Figure 4. Performance Index (PIabs) of (A) I. grandifolia at 72 hours, (B) S. alata at 72 hours and (C)
S. rhombifolia at 1 hour treated with 15a-b and 21b (50 µM and 100 µM). (*) and (**) indicate
significant difference from control (DMSO) with p  0.05 and p  0.01, respectively.
Figure 5. Radar plots of the effects of 15a-b and 21b on (A) I. grandifolia at 72 hours, (B) S. alata
at 72 hours and (C) S. rhombifolia at 1 hour on Chl a fluorescence parameters calculated from the
OJIP curve at 50 µM.
Figure 6. Plant length and dry biomass of (A) I. grandifolia, (B) S. alata and (C) S. rhombifolia after
10 days of treatment with 15a-b and 21b at 50 and 100 µM. (*) and (**) indicate significant difference
from control (DMSO) with p  0.05 and p  0.01, respectively.
Figure 7. Binding models of 15a (A) and 15b (B) with the pocket site of PSII from S. oleracea (PDB
code: 3JCU).
This article is protected by copyright. All rights reserved

SCHEME CAPTIONS
Scheme 1. Synthesis of compounds 15a-d and 16a-d.
Scheme 2. Synthesis of compounds 21a-b.
This article is protected by copyright. All rights reserved