Synthesis, Biological Evaluation and in Silico Computational Studies of 7-Chloro-4-(1 H-1,2,3-triazol-1-yl)quinoline Derivatives: Search for New Controlling Agents against Spodoptera frugiperda (Lepidoptera: Noctuidae) Larvae

Article abstract of DOI:10.1021/acs.jafc.9b01067

The insecticidal and antifeedant activities of five 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinoline derivatives were evaluated against the maize armyworm, Spodoptera frugiperda (J.E. Smith). These hybrids were prepared through a copper-catalyzed azide alkyne

Full text of DOI:10.1021/acs.jafc.9b01067

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Agricultural and Environmental Chemistry
Synthesis, Biological Evaluation and in silico Computational
Studies of 7-Chloro-4-(1H-1,2,3-triazol-1-yl)quinoline
Derivatives. Search for new controlling agents against
Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae
Doris Natalia Rosado-Solano, Pedro Luis Sanabria-Florez, Mario Alberto Barón-
Rodríguez, Luz Karime Luna-Parada, Carlos E. Puerto Galvis, Andrés Felipe
Zorro-González, Vladimir V. Kouznetsov, and Leonor Yamile Vargas-Méndez
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01067 • Publication Date (Web): 07 Aug 2019
Downloaded from pubs.acs.org on August 9, 2019
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GA
Synthesis, Biological Evaluation and in silico Computational Studies of 7-Chloro-4-(1H-1,2,3-triazol-1-
yl)quinoline Derivatives. Search for new controlling agents against Spodoptera frugiperda (Lepidoptera:
Noctuidae) larvae
Doris Natalia Rosado-Solano, Mario Alberto Barón-Rodríguez, Pedro Luis Sanabria Flórez, Luz Karime Luna-
Parada, Carlos Eduardo Puerto-Galvis, Andrés Felipe Zorro-González, Vladimir V. Kouznetsov, and Leonor
Yamile Vargas-Méndez
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Synthesis, Biological Evaluation and in silico Computational Studies of 7-Chloro-
4-(1H-1,2,3-triazol-1-yl)quinoline Derivatives. Search for new controlling agents
against Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae
Doris Natalia Rosado-Solanoa, Mario Alberto Barón-Rodrígueza, Pedro Luis Sanabria
Floreza, Luz Karime Luna-Paradab, Carlos Eduardo Puerto-Galvisb,c, Andrés Felipe Zorro-
Gonzáleza,Vladimir V. Kouznetsovb,* and Leonor Yamile Vargas-Méndeza,*
a Grupo de Investigaciones Ambientales para el Desarrollo Sostenible, Facultad de Química
Ambiental, Universidad Santo Tomás, A. A. 1076, Bucaramanga, Colombia.
b Laboratorio de Química Orgánica y Biomolecular, CMN, Parque Tecnológico Guatiguará, Km 2
vía refugio, Universidad Industrial de Santander, Piedecuesta, A.A. 681011, Colombia.
c Laboratorio de Química Orgánica Aplicada, Universidad Manuela Beltrán, Cl. 33 # 26-34,
Bucaramanga, A.A. 680002, Colombia.
E-mail: kouznet@uis.edu.co; leyavar@gmail.com
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Abstract
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The insecticidal and antifeedant activities of five 7-chloro-4-(1H-1,2,3-triazol-1-
yl)quinoline derivatives were evaluated against the maize armyworm, Spodoptera
frugiperda (J.E. Smith). These hybrids were prepared through a copper–catalyzed azide
alkyne cycloaddition (CuAAC, known as a click reaction) and displayed larvicidal
properties with LD50 values below 3 mg/g insect, and triazolyl-quinoline hybrid 6 showed
an LD50 of 0.65 mg/g insect, making it two-fold less potent than methomyl, which was used
as a reference insecticide (LD50 = 0.34 mg/g insect). Compound 4 was the most active
antifeedant derivative (CE50 =162.1 μg/mL) with a good antifeedant index (56-79%) at
concentrations of 250-1000 µg/mL. Additionally, triazolyl-quinoline hybrids 4-8 exhibited
weak inhibitory activity against commercial acetylcholinesterase from Electrophorus
electricus (electric-eel AChE) (IC50 = 27.7 µg/mL) as well as low anti-ChE activity on S.
frugiperda larvae homogenate (IC50 = 68.4 µg/mL). Finally, molecular docking simulations
suggested that hybrid 7 binds to the catalytic active site (CAS) of this enzyme and around
the rim of the enzyme cavity, acting as a mixed (competitive and noncompetitive) inhibitor
like methomyl. Triazolyl-quinolines 4-6 and 8 inhibit AChE by binding over the perimeter
of the enzyme cavity, functioning as noncompetitive inhibitors. The results described in this
work can help to identify lead triazole structures from click chemistry for the development
of insecticide and deterrent products against S. frugiperda and related insect pests.
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21 Keywords 1,2,3-triazolyl-quinoline hybrids; Spodoptera frugiperda; insecticidal activity;
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antifeedant activity; acetylcholinesterase inhibitory activity; molecular docking simulations,
Lipinski’ parameters; Tice’s rule; “ag-likeness”
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25 1. Introduction
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Currently, most methods for pest control in agriculture are based on chemical
insecticides derived from specific classes of organic compounds such as organophosphates,
carbamates, pyrethroids and benzoylureas. These latter compounds still play an important
role in modern agricultural pest management. The main disadvantages of their use are the
progressive reduction of efficiency due to the increased resistance by pest insects and their
high negative impact on the beneficial insect population and agricultural ecosystems.1-5 To
address these serious problems, scientists have been dedicated to developing novel potent
and eco-friendly insecticides with new mechanisms of action. These innovative insecticides
could be designed and developed using a molecular hybridization strategy based on the
combination of the pharmacophores of different bioactive substances to produce a new
hybrid molecule with improved biological profiles, similar to what is done in drug
development.6-8 Quinolines and triazoles are two important classes of small heterocyclic
molecules that have a wide array of agricultural uses; both moieties can be found in
herbicides, fungicides and insecticides.9-15 Thus, the combination (fusion or conjugation) of
these privileged rings in a single hybrid molecule could represent a substantial advance in
agricultural chemistry, especially in insecticide research (Figure 1).
One of most destructive pests of many economically important small-grain crops
(maize, cotton, rice and sorghum) is the fall armyworm moth Spodoptera frugiperda (J. E.
Smith).16 To date, the most common methods for controlling this pest depend on the use of
conventional insecticides such as methomyl, carbaryl, and cypermethrin. Unfortunately,
their efficacy is consistently decreasing due to the development of resistance.17-20 In
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addition, they exhibit a high general toxicity. Therefore, new tools, including novel active
and nontoxic phosphate(carbamate)-free organic molecules for pest management are
urgently required.21,22
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Recently, 1,2,3-triazoles linked to quinoline derivatives have emerged as one of the
most interesting and attractive biological models in medicinal and agricultural
chemistry.23,24 The design and development of triazolyl-quinolines could also be improved
by pesticide-likeness analysis.25-29 This approach is mainly based on Lipinski’s “Rule–of–
Five”30 for pharmaceutical discovery and was adopted later by Tice for agrochemical
applications.31
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According to the statements described above and taking into account our current
interest in developing new insecticide agents able to control S. frugiperda,32 this research
was focused on preparing five triazolyl-quinoline hybrids, 7-chloro-4-(1H-1,2,3-triazol-1-
yl)quinolines, and determining their in vivo larvicidal, antifeedant and anti-
acetylcholinesterase activities. Furthermore, the physicochemical profiles (Lipinski
parameters and Tice criteria) and molecular docking simulations for the prepared molecules
were determined and are discussed.
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65 2. Materials and Methods
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2.1 General Experimental Procedures. Melting points were determined with a
Fisher–Johns melting point apparatus and are not corrected. Infrared spectra were recorded
using KBr pellets on a Shimadzu, model IRA Affinity-1 FT-IR spectrophotometer. Nuclear
magnetic resonance spectra were measured on a Bruker Avance-400 (400 MHz)
spectrometer. Chemical shifts are reported in ppm with the solvent resonance as the internal
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standard (CDCl3: δ 7.26 ppm; DMSO-d6: δ 2.50 ppm). Chemical shifts () and coupling
constants (J) are reported in ppm and Hz, respectively. Mass spectra were recorded on an
ESI-IT Amazon X (Bruker Daltonics) with direct injection. Data were acquired in full scan
mode at 300°C and a capillary voltage of 4500 V using nitrogen as the nebulizer gas at a
flow rate of L/min at 30 psi. A Hewlett Packard 5890a Series II Gas Chromatograph
interfaced with an HP MS ChemStation Data System was also used for MS identification at
70 eV using
a
60
m
capillary column coated with HP-5 [5%
phenylpoly(dimethylsiloxane)]. Elemental analyses were performed on a Thermo Scientific
CHNS-O analyzer (Model Flash 2000), and the experimental results were within ± 0.4 of
the theoretical values. Thin-layer chromatography (TLC) separations were performed using
Merck silica gel 60 F254 precoated plates (0.25 mm). Column chromatography separations
were performed using silica gel 60 (0.063 – 0.200 mm) 70-230 mesh with mixtures of
hexane-ethyl acetate as the eluents.
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Methomyl (analytical standard), cypermethrin (analytical standard), acetylthiocholine
iodide (ATCh) (≥ 98% purity), acetylcholinesterase from Electrophorus electricus (EC
3.1.1.7, Type VI-S), 5,5’-dithiobis-(2-nitro)benzoic acid (DTNB) (99%), sodium hydrogen
phosphate (98%), Tween® 20 for molecular biology, 4,7-dichloroquinoline (97%), sodium
azide (99%), phenylacetylene (98%), 1-ethynyl-4-methylbenzene (97%), 1-ethynyl-4-
methoxybenzene (97%), propargyl alcohol (99%), 2-methyl-3-butyn-2-ol (98%) and
tetrahydrofuran (99%) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Di-
potassium hydrogen phosphate (98% purity) was acquired from Panreac (Barcelona,
Spain). Sodium chloride (99.5% purity) was obtained from Mallinckrodt (St. Louis,
Missouri, USA). Dimethyl sulfoxide, absolute ethanol, acetone, hexane, ethyl acetate, ethyl
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ether, silica gel 60 (230-400 mesh), CDCl3 and DMSO-d6 were acquired from Merck
(Darmstadt, Germany).
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2.2 Recollection and mass-rearing of S. frugiperda. Field populations of S.
frugiperda larvae were collected from a crop of corn during harvest time in the
municipality of Girón, Santander, Colombia. Larvae were transported to the laboratory in a
plastic container with enough plant material to prevent cannibalism. Subsequently, they
were placed in plastic containers (one larva per vessel) with enough food corn. They were
reared in an environmental chamber at 25 ± 1 °C with a 16:8 h light:dark photoperiod
before they were used for experiments.
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2.3 Synthetic Chemistry.
Preparation of 4-azido-7-chloroquinoline (2)33
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4,7-Dichloroquinoline (10 g, 0.05 mol) was dissolved in 50 mL of DMSO, and sodium
azide (4.88 g, 0.075 mol) was added in small portions at room temperature. The resulting
mixture was stirred at room temperature for 12 h. The reaction mixture was treated with
water and extracted with Et2O (3 x 25 mL). The organic extracts were washed with water,
dried over Na2SO4, concentrated, and then purified by column chromatography on silica gel
using a hexane-ethyl acetate mixture (1:1) as the eluent to give colorless product 2. Yield
95%, m.p.: 115-116 °C. IR (KBr): 2125, 1568, 1490, 1421, 1305, 1278, 1203, 815 cm-1. 1H
NMR (CDCl3): δ 7.11 (1H, d, J = 4.8 Hz; 3-H), 7.46 (1H, dd, J = 8.8, 2.0 Hz; 6-H), 7.97
(1H, d, J = 8.8 Hz; 5-H), 8.04 (1H, d, J = 2.0 Hz; 8-H), 8.81 (1H, d, J = 4.8 Hz; 2-H) ppm.
13C NMR (CDCl3): δ 108.7, 119.9, 123.7, 127.5, 128.1, 136.5, 146.3, 149.5, 151.2 ppm.
GC-MS: tR = 15.63 min, m/z (EI): 162 (M+. - 42). Elemental analysis: calcd for C9H5ClN4:
C, 52.83; H, 2.46; N, 17.33; found: C, 52.55; H, 2.77; N, 17.18.
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General procedure for the synthesis of triazolyl-quinolines 4-8:
The
alkyne
(phenylacetylene,
1-ethynyl-4-methylbenzene,
1-ethynyl-4-
methoxybenzene, propargyl alcohol, or 2-methyl-3-butyn-2-ol) (0.015 mol) was dissolved
in methanol (12 mL), and 4-azido-7-cloroquinolina (0.01 mol), copper sulfate (0.25 mmol),
sodium ascorbate (0.50 mmol), water (2 mL) and tetrahydrofuran (THF) (8 mL) were
added. The resulting mixture was stirred at room temperature for 12 h and then treated with
NaHCO3 solution (5%, 10 mL). The products were extracted with ethyl acetate (3 x 20
mL). The organic extracts were washed with water, dried over Na2SO4, concentrated, and
then purified by column chromatography on silica gel using hexane and ethyl acetate as the
eluents to afford hybrids 4-8 as stable colorless powders.
7-Chloro-4-(4-phenyl-1H-1,2,3-triazol-1-yl)quinoline (4).34 Yield 67%, colorless
powder, m.p.: 157-158 °C. IR (KBr): 1606, 1597, 1562, 1483, 1234, 1022, 879, 767, 694
cm-1. 1H NMR (CDCl3): δ 7.26 (1H, t, J = 7.4 Hz; 4-HAr), 7.34 (2H, t, J = 7.6 Hz; 3,5-HAr),
7.40 (1H, d, J = 4.6 Hz; 3-H), 7.45 (1H, dd, J = 9.1, 2.1 Hz; 6-H), 7.79 (2H, d, J = 7.8 Hz;
2,6-HAr), 7.91 (1H, d, J = 9.1 Hz; 5-H), 8.096 (1H, d, J = 2.4 Hz; 8-H), 8.099 (1H, s, 5-
HTA), 8.92 (1H, d, J = 4.6 Hz; 2-H) ppm. 13C NMR (CDCl3): δ 115.9, 120.6, 121.1, 124.6,
125.9 (2C), 128.8, 128.9, 129.1 (2C), 129.4, 129.4, 136.9, 140.9, 148.5, 150.2, 151.4 ppm.
GC-MS: tR = 11.33 min, m/z (EI): 306 (M+.), 280, 278, 243, 214, 162. Elemental analysis:
calcd for C17H11ClN4: C, 66.56; H, 3.61; N, 18.26; found: C, 66.43; H, 3.86; N, 18.09.
7-Chloro-4-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)quinoline (5). Yield 47%,
colorless powder, m.p.: 160-161 °C. IR (KBr): 1612, 1562, 1494, 1452, 1440, 1252, 1020,
875, 786, 678 cm-1. 1H NMR (CDCl3): δ 3.86 (3H, s, -OCH3), 7.00 (2H, d, J = 8.8 Hz; 3,5-
HAr), 7.53 (1H, d, J = 4.6 Hz; 3-H), 7.59 (1H, dd, J = 9.1, 2.1 Hz; 6-H), 7.86 (2H, d, J = 8.8
Hz; 2,6-H), 8.07 (1H, d, J = 9.1 Hz; 5-H), 8.15 (1H, s, 5-HTA), 8.23 (1H, d, J = 2.1 Hz; 8-
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H), 9.05 (1H, d, J = 4.6 Hz; 2-H) ppm. 13C NMR (CDCl3): δ 55.3, 114.4 (2C), 115.8, 120.3,
120.6, 122.0, 124.7, 127.3 (2C), 128.9, 129.4, 136.9, 141.0, 148.3, 150.1, 151.4, 160.1
ppm. GC-MS: tR = 16.15 min, m/z (EI): 336 (M+.), 310, 308, 293, 237, 203, 175, 154.
Elemental analysis: calcd for C18H13ClN4O: C, 64.20; H, 3.89; N, 16.64; found: C, 64.32;
H, 4.03; N, 16.38.
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7-Chloro-4-(4-methylphenyl)-1H-1,2,3-triazol-1-yl)quinoline (6).34 Yield 97%, white
powder, m.p.: 169-170 °C. IR (KBr): 1597, 1562, 1436, 1230, 1029, 1016, 877, 817 cm-1.
1H NMR (DMSO-d6): δ 2.37 (3H, s, -CH3), 7.34 (2H, d, J = 7.9 Hz; 3,5-HAr), 7.80 (1H, dd,
J = 9.1, 2.2 Hz; 6-H), 7.89 (2H, d, J = 8.1 Hz; 2,6-HAr), 7.92 (1H, d, J = 4.7 Hz; 3-H), 8.16
(1H, d, J = 9.1 Hz; 5-H), 8.30 (1H, d, J = 2.1 Hz; 8-H), 9.18 (1H, d, J = 4.7 Hz; 2-H), 9.26
(1H, s, 5-HTA) ppm. 13C NMR (DMSO-d6): δ 20.8, 116.7, 120.1, 123.1, 125.4 (2C), 125.6,
126.9, 128.0, 128.9, 129.5 (2C), 135.5, 137.9, 140.4, 147.1, 149.4, 152.3 ppm. GC-MS: tR
= 16.15 min, m/z (EI): 336 (M+.), 310, 308, 293, 237, 203, 175, 154. Elemental analysis:
calcd for C18H13ClN4O: C, 64.20; H, 3.89; N, 16.64; found: C, 64.32; H, 4.03; N, 16.38.
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1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methanol (7). Yield 55%, white powder,
m.p.: 164-165 °C. IR (KBr): 3257, 1612, 1591, 1440, 1230, 1058, 1033, 879, 844 cm-1. 1H
NMR (DMSO-d6): δ 4.71 (2H, d, J = 5.4 Hz, -CH2-), 5.44 (1H, t, J = 5.6 Hz; -OH), 7.77
(1H, dd, J = 9.1, 2.2 Hz; 6-H), 7.82 (1H, d, J = 4.7 Hz; 3-H), 8.02 (1H, d, J = 9.1 Hz; 5-H),
8.26 (1H, d, J = 2.2 Hz; 8-H), 8.69 (1H, s, 5-HTA), 9.13 (1H, d, J = 4.7 Hz; 2-H) ppm. 13C
NMR (DMSO-d6): δ 54.8, 116.8, 120.3, 124.9, 125.4, 128.0, 128.8, 135.2, 140.5, 148.8,
149.3, 152.3 ppm. GC-MS: tR = 5.69 min, m/z (EI): 262 (M+.), 260, 217, 215, 203, 168,
162. Elemental analysis: calcd for C12H9ClN4O: C, 55.29; H, 3.48; N, 21.49; found: C,
55.46; H, 3.64; N, 21.67.
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2-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propan-2-ol (8).34 Yield 91%, white
powder, m.p.: 148-149 °C. IR (KBr): 3356, 2978, 1612, 1595, 1564, 1438, 1238, 1168,
879, 821 cm-1. 1H NMR (CDCl3): δ 1.78 (6H, s, (-CH3)2), 5.29 (1H, s, -OH), 7.47 (1H, d, J
= 4.6 Hz; 3-H), 7.56 (1H, d, J = 9.1, 2.0 Hz; 6-H), 7.96 (1H, s, 5-HTA), 7.99 (1H, d, J = 9.1
13
Hz; 5-H), 8.21 (1H, d, J = 2.1 Hz; 8-H), 9.02 (1H, d, J = 4.6 Hz; 2-H) ppm. C NMR
(CDCl3): δ 30.5 (2C), 68.7, 115.9, 120.5, 121.2, 124.6, 128.9, 129.3, 136.9, 141.0, 150.1,
151.3, 156.5 ppm. GC-MS: tR = 5.41 min, m/z (EI): 290 (M+.), 288, 247, 245, 205, 203,
167, 162. Elemental analysis: calcd for C14H13ClN4O: C, 58.24; H, 4.54; N, 19.40; found:
C, 58.39; H, 4.76; N, 19.58.
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2.4 Insecticidal assay. Compounds 4-8 were evaluated by topical application following
a previously described bioassay protocol.35 First, groups of 10 larvae were selected and
weighed to determine the average weight of each group (500 ± 50 mg). The experimental
compounds and reference insecticide (methomyl) were each tested at doses of 1000, 500,
250, 100, 50 and 1 µg/larva using acetone as the solvent.
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After selecting the group for each concentration of the test compounds 4-8, the larvae
were placed in Petri dishes (one larva per box to avoid cannibalism). Then, 1 L of solution
was applied to the 2nd and 3rd mesothoracic segments of the larvae, leaving a considerable
time between applications. Assays were performed in triplicate. The mortality rates (%)
were determined 2, 4, 6, 8, 24, 48 and 72 h after treatment, considering death as when the
larvae do not respond to mild twinges. Finally, the lethal concentration, the concentration at
which 50% of the population dies (LC50), was calculated using the following equation:
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2.5 Antifeedant assay. The antifeedant effect was estimated through a no-choice
assay.35 S. frugiperda larvae with an average weight of 500 ± 50 mg and five acetone
solutions with concentrations of 1000, 500, 250 100 and 50 µg/mL were used.
Cypermethrin was employed as a reference compound. Solutions of experimental
compounds 4-8 were prepared in 1 mL of acetone and 100 µL of these solutions were
added to disks with an average weight of maize of 400 ± 50 mg, and 100 µL of an acetone
solution of cypermethrin was used as the control.
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Larvae selected for the bioassay were stored in individual containers without food for
six hours before the bioassay. Then, a corn disk with the experimental compounds was
given to the larvae. Ten larvae were used for each concentration. The food was weighed
every 24 h for 72 h, and the antifeedant effect (% AI) was calculated with the following
equation:
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%AI = [(C-T) / (C+T)] x 100%
(2)
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where C represents the weight of the diet consumed by the control larvae, and T is the
weight of the diet consumed by the treatment larvae.
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2.6 In vitro acetylcholinesterase activity. Acetylcholinesterase (AChE, EC 3.1.1.7,
Type VI-S) inhibition was assessed by the Ellman method modifying by scaling
microplates based on the reaction of released thiocholine to give a colored product with a
chromogenic reagent.36 The assay was performed in liquid medium in a 96-well microplate
with a final volume of 200 µL. Solutions of the reference compound (methomyl) or
experimental compounds 4-8 (at serial concentrations from 1x10-3 M to 4.88 x 10-7 M)
prepared in phosphate–buffered saline (pH 7.5, 100 L) and 50 µL of the AChE solution
(0.25 U/mL) were placed in each well.
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The plate was incubated at 25 °C for 30 min and then 100 µL of the substrate solution
(DTNB and ATCh at pH 7.5) was added. After five minutes, the absorbance was
determined at 405 nm using a Biochrom EZ 400 UV-Visible spectrophotometer. The IC50
is defined as the concentration of the studied compound corresponding to exactly 50% of
the maximum inhibitory effect against AChE. This value (AChEI, %) was calculated from
the data acquired using SoftMax Pro 5.2 software from Molecular Devices based on the
following equation:
AChEI (%) = 100-[(AS-AB)/(AC-AB)] x 100
(3)
where AS is the measured absorbance, AB is the absorbance of the blank, and AC is the
absorbance of the control, and this was used to determine the enzyme activity without an
inhibitor.
2.7 Ex vivo AChE activity on larvae homogenate. For the acetylcholinesterase
assay, pools of twenty heads of S. frugiperda larvae (500 ± 5 mg) were quickly manually
macerated in 10 mL of 0.1 M sodium phosphate buffer (pH 8.0, cold ice). The homogenate
was immediately centrifuged (4500 rpm, 5 °C) for 45 min following the known protocol.
The resulting supernatant was decanted and centrifuged for 15 min to better separate the
mitochondrial enzyme solution. The final supernatant was used as the enzymatic source. To
quantify the amount of acetylcholinesterase enzyme present in the obtained extract, the
standard addition method was used. Briefly, 20 μL of the enzymatic extract was added to 0,
100, 200, 300 and 400 μL of an enzyme standard solution with a known concentration (0.50
U/mL) of electric-eel AChE and brought to 1 mL with 0.1 M sodium phosphate buffer (pH
8.0). To evaluate the ex vivo inhibitory capacity of the triazolyl-quinoline derivatives 4-8
for AChE, the procedure described in Section 2.6 was followed using the extract obtained
from S. frugiperda heads as the enzymatic source.
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2.8 eeAChE tertiary structure prediction. The structure of Electrophorus electricus
acetylcholinesterase (eeAChE) was predicted by bioinformatic tools as indicated below.
The sequence of eeAChE hosted on UniProt (code 042275) was submitted to the I-
TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/).37-39 The predicted
model of the tertiary structure with the highest score and serine hydrolase properties was
selected as the preliminary tertiary structure for performing the simulations. The structure
was optimized in aqueous media by energy minimization in a cubic simulation cell with
side lengths of 14.559 nm. Explicit water molecules, such as TIP4P,40 were added to
simulate the solvation. Several optimizations were run with the Steepest descent method
and the amber99sb force field41 implemented in GROMACS.42-44 Then, 20 ns molecular
dynamics (MD) simulations were performed to determine the most conformation of the
enzyme monomer under laboratory conditions. The MD simulations were run at 298 K and
1 atm with periodic boundary conditions with a leap-frog method implemented in
GROMACS.42-44 The enzyme structures with a RMSD of the backbone less than 0.09 nm
were clustered together. All docking calculations were performed using the protein
structure chosen from the largest cluster.
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2.9 Docking calculations. Molecular docking calculations of the interactions between
hybrids 4-8 and eeAChE were performed to elucidate the possible in vitro molecular
inhibition mechanism of eeAChE by triazolquinolines. Additionally, a molecular docking
simulation of eeAChE – E-methomyl binding was performed as a reference. The docking
simulation was run with an amber94 force field45 implemented in Autodock Vina.46
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2.10 Statistical analysis. All experiments were carried out in triplicate. The
concentration giving 50% inhibition (IC) was calculated by nonlinear regression using
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Prism GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA,
USA). The dose–response curve was obtained by plotting the percentage inhibition versus
the concentration. The lethal concentration (LC50) is expressed as the standard error of the
mean (SEM) of three different experiments in triplicate, and the analyses were performed
using regression probit analysis with SPSS for Windows version 19.0.
263 3. Results and discussion
3.1 Chemistry. The desired 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinoline
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derivatives (4-8) were obtained in moderate to good yields through a CuAAC click reaction
between 4-azido-7-chloroquinoline (2)33 and corresponding alkynes 3 (Scheme 1).23-24
The crude reaction products were generally purified by column chromatography. The
yields for these click reactions were in the range of 50-97%. The final products were stable,
colorless powders with well-defined melting points (see the SI, Table SI1). The structures
and chemical purities of hybrids 4-8 were confirmed on the basis of spectrometric data (IR
1
13
spectroscopy, GC-MS, and H and C NMR spectroscopy) and comparison with literature
data.34 The proton at the C-5 position of the triazole ring of these hybrids appeared as a
singlet at  8.03-8.30 ppm indicating the 1,4-regioisomers were obtained (Scheme 1).
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3.2 Biological evaluation of insecticidal and antifeedant properties. The
insecticidal activities of prepared triazolyl-quinolines 4-8 against S. frugiperda larvae
weighing 500  50 mg indicated that the tested molecules were lethal to 50% of the
population at concentrations below 3 mg/g insect (Table 1). The most active compound was
4-(4-methylphenyl)-1H-1,2,3-triazolyl-quinoline (hybrid 6), which showed an LD50 of 0.65
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mg/g insect making it two-fold less potent than methomyl, a reference insecticide (LD50 =
0.34 mg/g insect). 4-Methoxyphenyl-triazolyl quinoline (5) was less active as an insecticide
with an LD50 value of 2.83 mg/g insect, while hybrid 7 was the most potent compound with
an LD50 value of 1.68 mg/g insect. Within this range, 4-phenyl-1H-1,2,3-triazolyl quinoline
(4, LD50 = 1.99 mg/g insect) and hybrid 8 (LD50 = 1.80 mg/g insect), with hydroxyalkyl
chains at C-5 of the triazole moiety, showed good insecticidal activities (Table 1).
Compounds 4-8 were tested as potential antifeedant agents using a no-choice assay in
which corn disks with compounds 4-8 preabsorbed were administered to S. frugiperda
larvae. The results of this experiment (Table 1) showed that three of the hybrids, 4, 5 and 8
had moderate deterrent effects on S. frugiperda larvae (CE50 < 300 µg/mL).
The antifeedant activity of cypermethrin, which is widely used to control armyworm,
afforded better results (CE50 = 23.9 µg/mL). The least active compound in this test was
hybrid 7 (CE50 = 557.4 µg/mL). Based on their antifeedant index values (% AI values)
(Table 2), almost of the prepared hybrids (comp. 4-6 and 8) (AI = 71-79%) showed
excellent antifeedant activities at a concentration of 1000 µg/mL relative to the antifeedant
index of cypermethrin (AI = 78%). Out of all derivatives, triazolyl-quinoline 4 was the best
deterrent compound with an index of 79%, while hybrid 7 was the least active antifeedant
molecule (AI = 61%). Notably, triazolyl-quinoline 4 was sufficiently active at a
concentration of 250 µg/mL (AI = 56%). The antifeedant effects of this compound are
shown in Figure 2.
Because AChE is the target enzyme for inhibition by different insecticides,34-36 the
inhibitory effects of hybrids 4-8 on AChE were determined based on eeAChE and the
AChE of the insect crude extract. The results (Table 3) revealed that all the tested hybrids
showed weak AChE inhibitory effects (27-34 µg/mL < IC50 < 68-75 µg/mL) and similar
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structure-activity relationship (SAR) trends, e.g., compound 4 inhibited eeAChE and S.
frugiperda larvae ChE with IC50 values 52.1 and 73.9 µg/mL, respectively, and so on.
However, in general, the test compounds more efficiently inhibited eeAChE than they did
crude S. frugiperda larvae ChE homogenate, and the anti-eeAChE activity of quinoline
hybrids 4-8 tended to increase as follows: 7>6>8>4>5, while their inhibitory effects on
crude S. frugiperda larvae ChE fell in the following order: 6>8>7>4>5. Notably, the two
most active enzyme inhibitors, 6 and 7, showed interesting behavior against both enzymes,
whereas hybrid 7 bearing a hydroxymethyl substituent (hydrophilic moiety) on the triazole
ring, presented the strongest inhibitory effect on eeAChE (IC50 = 27.7 µg/mL), hybrid 6,
with a 4-methylphenyl fragment (lipophilic moiety) on the triazole ring, revealed the best
inhibition of crude S. frugiperda larvae AChE homogenate (IC50 = 34.6 µg/mL).
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Considering that compound 6 possesses a good insecticidal activity (LD50 = 0.65 μg/g
insect) and that compound 7 is inactive (LD50 = 1.68 μg/g insect), the observed effects
seem to be interesting and may be, significant. Therefore, to better understand these results,
we performed in silico computational studies.
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3.3 In silico computational studies using the DataWarrior program. Currently,
the initial assessment of a potential bioactive compound starts with the prediction of its
possible biological activities and with an understanding of its possible mechanism of
action.47,48 For this purpose, we first determined whether compounds 4-8 have chemical and
physical properties that would make them potential pharmacological agents. Thus, using
the stand-alone DataWarrior program (Osiris),49 we established that molecular hybrids 4-8
fulfil Lipinski “rule–of–five”; they snow i) octanol/water partition coefficients (cLogP
values) ≤ 5; ii) molecular weights (MWs) ≤ 500 Da; iii) have ≤ 10 hydrogen bond acceptors
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(HBA); iv) have ≤ 5 hydrogen bond donors (HBD); v) have ≤ 10 rotatable bonds (ROTB);
and vi) have topological polar surface areas (TPSA values) ≤ 140 (Table 4).50
Nevertheless, due to the biodiversity of pests and the environmental conditions, another
set of rules (physicochemical properties) beyond Lipinski’s “rule–of–five” should be
considered in agrochemical development and discovery including in the design of
insecticides.27,29 This rule is Tice’s criteria, which established specific and primary
parameters for agrochemical agents, and these include: i) a cLogP between 0 and 5; ii) an
MW between 150 and 500 Da; iii) an HBA between 1 and 8; iv) an HBD ≤ 2; and v) a
ROTB < 12.31 Therefore, hybrids 4-8 and the reference compounds, methomyl (Met) and
cypermethrin (Cyp), have good agrochemical-likeness (“ag-likeness”) because none of
these derivatives breaks or exceeds Tice’s rules (Table 4). However, analyzing the main
molecular descriptors of these hybrids and methomyl, it is clear that methomyl, a small
molecule, potent insectcide (LD50 = 0.34 mg/g insect), is hydrophilic (clogP = 0.61) and
highly water soluble (clogS = -1.35), while the most active hybrid 6, LD50 = 0.65 mg/g
insect, is the most lipophilic molecule of this series (clogP = 3.49) and is poorly water
soluble (clogS = -4.90). Moreover, hybrid 7, which showed weak insecticidal properties
(LD50 = 1.68 mg/g insect), possesses a similar hydrophilicity (clogP = 0.85, clogS = -2.69)
to that of methomyl.
Applied topically to the dorsal of the larvae, the molecules must penetrate the cuticle,
an integument that protects the inner and outer surfaces of insects.51 As the insect
integument can be considered a two-phased structure, with lipophilic (epi- and exocuticles
containing lipids, lipoproteins and proteins) and hydrophilic (endocuticle, a chitin-protein
complex) layers,52-54 both lipophilic molecules (hybrids 4-8) and hydrophilic compounds
(methomyl) can generally pass through this active biochemical barrier into the hemolymph
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to reach specific targets. Indeed, earlier Gerolt’s tests with methomyl showed this
insecticide can pass through the integument of adult housefly at a high rate, i.e., at 8-10%
(depending on dose) over a period of 19 h.55,56 Thus, the insecticidal activity of a molecule
is the result of a series of multifarious interactions between the compound and the insect's
tissues, and penetration is an important initial step, but is still poorly studied. Based on
these factors, this situation results in highly complex structural activity relationships. In
addition to Tice’s parameters, the insecticidal activities of new compounds are also related
to their size, shape, stability, and pKa values.28,29
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Regarding the final step, i.e., the interaction between the toxicant and its possible
targets, e.g., AChE enzyme, enzymatic inhibitory activity is more accurately predicted by
molecular docking studies.
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3.4 Molecular docking studies. To perform the docking simulations with the
eeAChE, it was necessary to predict its tertiary structure, which was not reported in the
Protein Data Bank (PDB, http://www.rcsb.org/pdb). Thus, using known protocols, the
secondary and tertiary structures of this enzyme were predicted from its amino acid
sequence (code 042275), which is available in the UniPro database.37-39 The constructed
tertiary structure of eeAChE displayed a globular shape with a catalytic active site (CAS)
containing an acyl pocket (Phe312, Phe315 and Phe355), a choline binding side (Trp107)
and a catalytic triad (Glu223, Ser224 and His493) (Figure 2A-C), and a peripheral anionic
site (PAS) at the rim of the cavity, involving Tyr93, Asp95, Thr96, Ser97, Tyr98, Glu302,
Ser364, Asp366 and Tyr358 residues; these are important properties of serine hydrolases.57
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The docking experiments showed interesting results. First, the molecular docking of
eeAChE – E-methomyl revealed spontaneous interactions of between this carbamate and
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the CAS and the two binding zones (I and II) on the enzyme surface, and binding energies
between -5.2 and -4.5 kcal/mol were observed (Figure 3A, Table 5).
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Our binding model indicates that methomyl may act as a competitive inhibitor of
eeAChE as it reaches the CAS (Figure 3B, C) and forms multiple dipole-dipole interactions
with the hydroxyl groups of Tyr145 and Tyr354, and the guanidine group of Arg258. At the
same time, however, this carbamate binds Val153 and Asp155 residues in the backbone of
binding zone I via dipole-dipole interactions, while it binds zone II via hydrogen bonds and
multiple dipole-dipole interactions (Data not shown). Thus, these results confirmed that E-
methomyl is a mixed inhibitor, as reported in the literature.58 Regarding the analysis of
eeAChE – triazolyl-quinoline 4-8 interactions, we found that among the five hybrids, only
hybrid 7 could occupy the CAS of the enzyme with a binding energy of -7.1 kcal/mol
(Table 5). The - interactions between the choline binding site of the enzyme (indole
group of Trp107 and phenolic hydroxyl group of Tyr354) and the quinoline ring of this
hybrid, and the hydrogen bond of the guanidine group of Arg258 in the acyl pocket and the
hydroxyl group of triazolyl-quinoline 7 are important for the spontaneity of the binding.
Additionally, the conformation suggests a dipole-dipole interaction between the triazole
ring of the compound and the hydrogens of the benzyl groups of Phe312 and Phe355
(Figure 4). This result indicates that hybrid 7 may be a competitive inhibitor of
acetylcholine hydrolysis by AChE, as predicted by the E-methomyl – eeAChE molecular
docking results. No other hybrids reached the CAS in the molecular modeling studies,
which is consistent with the inhibitory potential of triazolyl-quinoline 7, being the best
AChE inhibitor (LD50 = 27.7 µg/mL) (Table 3). The molecular volume data (Table 5) also
indicated that compounds 4-6 and 8 may be too large to fit in the CAS of the enzyme.
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Overall, the molecular docking results with tested triazolyl-quinoline derivatives 4-8
suggested that all hybrids, including compound 7, could bind to two zones outside the
enzyme cavity (Figure 5), the same spots as E-methomyl (Figure 3), with multiple binding
modes. The more spontaneous binding modes of hybrids 4-8 to zone I (Figure 5A, C)
suggested a dipole-dipole interaction with the enzyme; moreover, these modes could be
stabilized via - stacking interactions with several aromatic residues around the cavity. On
the other hand, in binding zone II, two main interactions were observed, and they are
important in triazoles recognition (Figure 5B, D). The π-π stacking interactions between the
aromatic rings of hybrids 4 and 5 and the benzyl group of Phe312 formed a T-shape.
Additionally, dipole-dipole interactions are formed between the triazole rings of
compounds 7 and 8 and the guanidine groups of Arg268 and Arg313.
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The proximity of binding zones I and II to the rim of the enzyme cavity and the
spontaneous binding of the tested hybrids could stabilize an enzyme conformation and
destroy the flexibility necessary to carry out its catalytic action, indicating that these
hybrids could work as noncompetitive inhibitors of eeAChE.
412 4. Conclusions
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The larvicidal, antifeedant, and anti-AChE activities of five triazolyl-quinoline
hybrids 4-8 were evaluated against the maize armyworm, Spodoptera frugiperda (J.E.
Smith). We found that 4-(4-methylphenyl)-1H-1,2,3-triazolyl-quinoline, hybrid 6, was the
most promising compound, and it exhibited insecticidal (LD50 = 0.65 mg/g insect),
antifeedant (antifeedant index 56-79% at 250-1000 µg/mL) and AChE activities on both
commercial eeAChE (27.7 µg/mL) and supernatant ChE enzyme from S. frugiperda larvae
head homogenate (53.1 µg/mL).
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Additionally, to understand these in vivo results, in silico agrochemical-likeness
evaluations and molecular docking studies were performed. We established that an inactive
insecticidal and hydrophilic hybrid, such as 7 (clogP = 0.85), binds to the CAS of AChE (-
7.1 kcal/mol) in zones outside the enzyme cavity, working as a mixed (competitive and
noncompetitive) inhibitor, while the most potent insecticidal and lipophilic hybrid (6, clogP
= 3.49) did not reach the CAS of AChE. Therefore, the in vivo insecticidal activity of
hybrid 6 could be attributed to its inhibition of AChE, while the insecticidal inactivity of
hybrid 7, the most active AChE inhibitor, can be explained by its strong hydrophilicity and
relative large molecular volume, which prevents this compound from reaching the AChE
target.
All this information can help to identify lead triazole structures derived from click
chemistry for the development of insecticide and deterrent products against S. frugiperda
and related insect pests.
ASSOCIATED CONTENT
Supporting Information
436 Physicochemical properties of hybrids 4-8; copies of the NMR and IR spectra of
437 triazolylquinolines 4-8 and azido-quinoline precursor (2); GC-MS data of
438 triazolylquinolines 4-8; biological information related to compounds 4-8.
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442
ACKNOWLEDGEMENTS
This research was supported by the University of Santo Tomas and Governance of
Santander-Colombia (contract A-833, GIFQCAAMBPJOV12016), Patrimonio Autónomo
Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación,
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Francisco José de Caldas (contract RC-007-2017, 11027455897), Young Researchers and
Innovators (D.N.R.S.), the doctoral program Colciencias 567 (L.K.L.P.) and the doctoral
program Colciencias 617 (C.E.P.G.).
CONFLICTS OF INTEREST
The authors declare no conflicts of interest, financial or otherwise.
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39. Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y. Nat. methods 2015, 12, 7–8.
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40. Horn, H. W.; Swope, W. C.; Pitera, J. W.; Madura, J. D.; Dick, T. J.; Hura, G. L.;
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546
547
548
549
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556 SCHEMES
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R
N
N
N
N
R
N
Cl
N
N
3
NaN3
.
CuSO4 5H2O
sodium ascorbate (1:2)
MeOH/H2O/THF(6:1:4)
rt, 12 h
DMSO
65 oC, 6 h
Cl
Cl
N
Cl
N
2
4-8
1
95 %
50-97 %
Me
OMe
Me
Me
OH
OH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Cl
N
Cl
N
Cl
N
Cl
N
Cl
N
67 %
4
50 %
5
97 %
6
55 %
7
80 %
8
557
558
Scheme 1. Straightforward preparation of 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinolines 4-8 from
559
560
4,7-dichloroquinoline.
561
562
563
564
565
566
567
568
569
570 TABLES
571
572
Table 1. Insecticidal and antifeedant activities of triazolyl-quinoline molecules 4-8 and reference
agrochemicals.a,b
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573
574
575
576
577
Tested comp.
Insecticidal activity
Antifeedant activity
2
2
LD50, mg/g insect
(CI)
CE50, µg/mL
(CI)
162.1
(155.3 - 168.9)
296.3
(287.6 - 305.0)
356.0
(345.9 - 366.2)
557.4
(529.4 - 585.4)
286.5
4
1.99
(1.84 - 1.20)
2.83
(2.74 - 2.92)
0.65
(0.66 - 0.79)
1.68
(1.67 - 1.69)
1.80
(1.78 - 1.82)
0.34
0.98
0.97
0.92
0.99
0.99
0.85
--
0.77
0.89
0.90
0.92
0.79
--
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
5
6
7
8
(282.1 - 290.9)
Methomyl
Cypermethrin
ntc
(0.31 - 0.37)
ntc
23.9
(22.5 - 25.4)
0.77
a Expressed in LD50 values as well as CE50 values and shown as the mean (n > 3) values;
95% confidence interval (CI) values for the respective compounds; b 2 Chi-squared test;
c Not tested.
596
597
598
599
Table 2. Antifeedant index (% AI) of triazolyl-quinolines 4-8 and
cypermethrin, as a reference deterrent agrochemical.
Concentration, µg/mL
Tested comp
1000
79
75
71
61
500
70
58
53
49
63
72
250
56
44
41
33
49
66
100
44
32
31
20
30
61
50
33
15
15
13
12
56
4
5
6
7
8
73
78
Cypermethrin
600
601
602
603
604
605
606
Table 3. Inhibition potencies of tested molecules 4-8 against eeAChE and crude S.
frugiperda larvae AChE homogenate.
AChE inhibition propertiesa
Tested
comp.
eeAChE
S. frugiperda larvae ChE homogenate
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2
2
IC50, µg/mL
(CI)
IC50, µg/mL
(CI)
4
5
6
7
8
52.1
(51.3 - 52.9)
68.4
(65.3 - 71.4)
29.5
(28.5 - 30.5)
27.7
(27.5 - 27.9)
0.81
0.81
0.71
0.78
0.77
0.73
73.9
(71.5 - 76.2)
75.8
(75.6 - 76.1)
34.6
(33.8 - 35.4)
53.1
(52.8 - 53.5)
0.67
0.66
0.59
0.49
0.62
0.59
32.3
(31.8 - 32.9)
44.3
(43.3 - 45.2)
Met.b
0.31
0.41
(0.304 - 0.316)
(0.407 - 0.409)
a
607
608
609
610
611
IC50 values are expressed in µg/mL and shown as the mean (n > 3) values; 95% confidence interval (CI)
values for the respective compounds; 2 Chi-squared test; b Methomyl.
612
613
614
615
Table 4. Molecular descriptors calculated for molecules 4-8 according to the DataWarrior
program and analysis of Tice’s criteria.
Comp.
MWa
cLogPb cLogSc HBAd HBDe ROTBf TPSAg
Tice’s rule violations
4
5
6
7
8
306.755 3.1462
336.781 3.0762
320.782 3.4901
260.683 0.8527
288.370 1.7259
-4.560
-4.578
-4.904
-2.690
-3.175
-1.349
-6.688
4
5
4
5
5
4
4
0
0
0
1
1
1
0
2
3
2
2
2
3
7
43.60
52.83
43.60
63.83
63.83
75.99
59.32
0
0
0
0
0
0
0
Met.h 162.212 0.6131
Cyp.i 416.303 5.3412
616
617
a
b
c
Molecular Weight (g/mol); Logarithm of the partition coefficient between n-octanol and water;
d
e
618 Logarithm of the solubility measured in mol/L; Number of hydrogen-bond acceptors; Number of
f
619 hydrogen-bond donors; Number of rotatable bonds (calculated using the Molinspiration Cheminformatics
620 software); g Polar surface area (Å2); h Methomyl; i Cypermethrin.
621
622
623
624
625
Table 5. Binding energy of eeAChE – triazolyl-quinolines 4-8 interactions
∆G, kcal mol-1
MVa
(Å3)
Comp.
Binding zone I
Active site
Binding zone II
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