l -Proline as a Valuable Scaffold for the Synthesis of Novel Enantiopure Neonicotinoids Analogs

Article abstract of DOI:10.1021/acs.jafc.0c05997

In this research, six neonicotinoid analogs derived from l-proline were synthesized, characterized, and evaluated as insecticides against Xyleborus affinis. Most of the target compounds showed good to excellent insecticidal activity. To the best of our knowledge, this is the first report dealing with the use of enantiopure l-proline to get neonicotinoids. These results highlighted the compound 9 as an excellent candidate used as the lead chiral insecticide for future development. Additionally, molecular docking with the receptor and compound 9 was carried out to gain insight into its high activity when compared to dinotefuran. Finally, the neurotoxic evaluation of compound 9 showed lower toxicity than the classic neonicotinoid dinotefuran.

Full text of DOI:10.1021/acs.jafc.0c05997

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Article
L‑Proline as a Valuable Scaffold for the Synthesis of Novel
Enantiopure Neonicotinoids Analogs
́
̃
Israel Bonilla-Landa, Ulises Cuapio-Munoz, Axel Luna-Hernandez, Alfonso Reyes-Luna,
Alfredo Rodríguez-Hernandez, Arturo Ibarra-Juarez, Gabriel Suarez-Mendez, Felipe Barrera-Mendez,
́
́
́
́
Nadia Caram-Salas, J. Francisco Enríquez-Medrano, Ramon E. Díaz de Leon,
́
and Jose Luis Olivares-Romero*
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ABSTRACT: In this research, six neonicotinoid analogs derived from L-proline were synthesized, characterized, and evaluated as
insecticides against Xyleborus affinis. Most of the target compounds showed good to excellent insecticidal activity. To the best of our
knowledge, this is the first report dealing with the use of enantiopure L-proline to get neonicotinoids. These results highlighted the
compound 9 as an excellent candidate used as the lead chiral insecticide for future development. Additionally, molecular docking
with the receptor and compound 9 was carried out to gain insight into its high activity when compared to dinotefuran. Finally, the
neurotoxic evaluation of compound 9 showed lower toxicity than the classic neonicotinoid dinotefuran.
KEYWORDS: neonicotinoids, nitroguanidines, chiral, enantiopure compound, insecticidal activity, docking
public health usages.8,9 These are a type of insecticides that
acts selectively on the central nervous system of the insect
INTRODUCTION
■
Chirality plays an important role in all living organisms and it
and can be efficient ligands for the nicotinic acetylcholine
is quite clear that diastereomer discrimination abounds in the
receptors (nAChRs) of insects.10 However, with the increase
domain of medicinal chemistry and pharmacology.1 None-
of neonicotinoids used on the crop protection for a long
theless, most of the neonicotinoids used so far are nonchiral
time, the problems of cross-resistance11 and bee toxicity12
or administered as a racemic mixture.2,3 Since the naturally
have received more attention, which calls for a new strategy
occurring amino acids are the L-enantiomer, here, it is
of molecular design to find new leading compounds. The
interesting to synthesize and evaluate a series of neonicoti-
neuro-insecticides selectively act on the insect central nervous
noids that merge an L-amino acid such as L-proline, which is
system (CNS) as an agonist of the postsynaptic nicotinic
cheap and commercially available. On the other hand, the
acetylcholine receptors (nAChRs) and present a higher
redbay ambrosia beetle, Xyleborus glabratus, is the vector of
selectivity when compared to the vertebrates.13−15 The
the laurel wilt disease fungal pathogen, Raffaellea lauricola.
great attributes of the neonicotinoids are their novel mode
Since the vector’s initial detection in the U.S.A. in the early
of action, low mammalian toxicity, broad insecticidal
of 2000s, laurel wilt has killed millions of redbay, Persea
spectrum, and good systemic properties.16 Another important
borbonia trees, and other members of the plant family
property of neonicotinoids is their environmental footprint
Lauraceae.4 In this context, avocado (Persea Americana Mill.)
which allows the replacement of the more toxic and
is the most important agricultural crop susceptible to laurel
nonselective organophosphorus, pyrethroid, and carbamate
wilt.5 As the disease continues to move to the south and west
insecticides. The reported neonicotinoids so far can be
from its original focus, it has caused significant concern as
classified according to the pharmacophore as N-nitro-
Florida, California and other avocado-producing areas such as
guanidines, (imidacloprid, thiamethoxam, clothianidin, and
Mexico. In the absence of effective control measures,
dinotefuran), nitromethylenes (nitenpyram) and N-cyano-
monetary losses caused by laurel wilt could eventually
amidines (acetamiprid and thiacloprid) (Figure 1).
range up to 54 million in the U.S.A., and even greater
All of these compounds are characterized by their high
loses might occur if the disease moves elsewhere.6
insecticidal activities against insects and relative safety toward
Management is currently focused on monitoring, sanitation,
and direct control using contact or systemic insecticides.7
Indeed, efficacious and cost-effective measures are urgently
needed to protect avocado from laurel wilt. In this context,
neonicotinoids are an important type of compounds with
Received: September 18, 2020
Revised: December 10, 2020
Accepted: January 15, 2021
Published: January 26, 2021
potent insecticidal activity. Since their introduction in the
1980s, these compounds have been established as the
insecticides of choice for agricultural, animal health, and
https://dx.doi.org/10.1021/acs.jafc.0c05997
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Figure 1. Classic neonicotinoids.
mammals and aquatic life.17 Encouraged by this, we hereby
introduce a new structure developing strategy, using
dinotefuran as the lead compound, and a series of novel
neonicotinoids derivatives were designed by introducing the
enantiopure amino acid L-proline. Therefore, in search of
improvement, the length of the carbon chain that connected
the nitroguanidine and the L-proline was evaluated against the
beetle Xyleborus affinis. Furthermore, the interactions
between these new compounds and nAChR were also
investigated by molecular docking. To the best of our
knowledge, this is the first report that describes the
incorporation of the enantiopure amino acid L-proline into
the nitroguanidine core to get highly active insecticides
against Xyleborus affinis.
Experimental Procedures. Preparation of Compound 1. First,
using an ice-salt bath to keep the temperature between 0 and −5
°C, 30 mL of concentrated H2SO4 was slowly added to 14 mL of
concentrated HNO3 (warning: the temperature increases suddenly).
Then 10 g (71.84 mmol) of S-methylisothiourea hemisulfate salt
was added in one portion and the mixture was stirred for 6 h
keeping the temperature at 0 °C. The mixture was poured in
crushed ice and a white solid appeared, which was recovered by
filtration, washed with distilled water and dried in high vacuum.
Data for Compound 1. Yield: 74%, white solid. Spectral data
matched with previous reports,18 the H NMR data is provided here
1
for convenience:. 1H NMR (500 MHz, DMSO-d6) δ: 9.15 (brs,
2H), 2.40 (s, 3H).
Preparation of Compound 3. A 18.7 g sample of compound 2
(92.5 mmol) was added over 30 min at 0 °C to a stirred solution of
5 g (36.9 mmol) S-methylnitroisothiourea in 50 mL of pyridine and
used as a solvent. After the mixture was stirred for 10 min, it was
poured in crushed ice, diluted with 100 mL of concentrated HCl,
and the white solid was collected by filtration; then the solid was
crystallized in EtOH to obtain 6.8 g (65%) of white crystals which
were suspended in 70 mL of acetonitrile and cooled at 0 °C.
Afterward, 13.4 mL (0.83g, 26.9 mmol) of methylamine solution in
methanol (2.0 M) was added over 1 h. The reaction was stirred for
30 min at room temperature, and then the solid phthalimide was
discarded by filtration and the filtrate was evaporated. The residue
had enough purity to use in the next reaction without further
purification.
MATERIALS AND METHODS
■
General Information. Nuclear magnetic resonance (1H, 13C,
DEPTQ135) was recorded in a Bruker Avance III HD equipped
with a BBO probe. The solvents used were CDCl3 or DMSO-d6, the
chemical shifts (δ) were expressed in parts per million and coupling
constants (J) in Hertz. The following abbreviations were used to
explain NMR multiplicities: s = singlet, t = triplet, q = quartet, d =
doublet, dd = double of doublets, ddd = double of doublet of
doublets, dt = doublet of triplets, m = multiplet, and brs = broad
signal. Spectra were processed using TopSpin 4.0.7 from Bruker
BioSpin. Melting points were measured on a Stuart SMP10
apparatus using open glass capillaries and the values were
uncorrected. Reactions were monitored by thin layer chromatog-
raphy (TLC) performed on silica gel 60 F254 plates (Merck) and
visualization was carried out with ammonium molybdate, 2,4-
dinitrophenylhidrazine, phosphomolybdic acid, potassium permanga-
nate or UV. High-resolution mass spectra (HRMS) were obtained in
a Q-TOF mass spectrometer equipped with an electrospray
ionization (ESI) interface Synapt G2-Si, Waters Inc. All of the
mixture sensitive reactions were performed under nitrogen
atmosphere. Anhydrous THF and Et2O were dried with
benzophenone and sodium. All other commercial reagents were
purchased with Sigma-Aldrich and used without further additional
purification.
Data for Compound 3. Yield: 80%, white solid. Spectral data
matched with previous reports;18 however, the 1H NMR data is
1
provided here for convenience. H NMR (500 MHz, DMSO-d6) δ:
7.83 (brs, 1H), 2.91 (brs, 3H), 2.44 (s, 3H).
Preparation of Compound 5. A 5.0 g (43.4 mmol) sample of of
L-proline and 9.38 g of KOH (167.2 mmol) were dissolved in 2-
propanol (30 mL); then 7.5 mL of BnCl (8.25 g, 65.17 mmol) was
added dropwise for over 2 h at 40 °C, and the reaction was stirred 6
h at the same temperature. The mixture was acidified until pH of
5−6 with concentrated HCl and 15 mL of chloroform was added;
this solution was stirred overnight at room temperature. The solid
that formed was filtered and washed with plenty of DCM. The
filtrate was evaporated under reduced pressure, the residue was
solubilized with acetone, and the mixture was left to cool in an ice
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bath; a white solid appeared, and it was filtered and washed with
cool acetone.
crude was purified using FCC (DCM/2-propanol = 9.5:0.5) and
after that was recrystallized in DCM/hexane.
Data for Compound 9. Yield: 65%, crystalline white solid, mp
Data for Compound 5. Yield: 70%, white solid. Spectral data
matched with previous reports;19 the H NMR data is provided here
1
130−133 °C, [α]23.5 = −65.2 (c 1, MeOH). H NMR (500 MHz,
1
for convenience. 1H NMR (500 MHz, CDCl3) δ: 7.51−7.36 (m,
5H), 4.31 (d, J = 12.8 Hz, 1H), 4.08 (d, J = 12.8 Hz, 1H), 3.87 (t, J
= 7.8 Hz, 1H), 3.32−3.24 (m, 1H), 2.97 (q, J = 8.8 Hz, 1H), 2.35−
2.22 (m, 1H), 2.00−1.89 (m, 2H), 1.85−1.71 (m, 1H).
DMSO-d6, 60 °C) δ: 8.70 (b, 1H), 7.80 (s, 2H), 7.37−7.21 (m,
5H), 4.00 (d, J = 13.3 Hz, 1H), 3.41−3.30 (m, 2H), 3.27−3.22 (m,
1H), 2.87−2.77 (m, 2H), 2.23 (td, J = 9.2, 7.2 Hz, 1H), 1.91 (dt, J
= 12.2, 8.2 Hz, 1H), 1.72−1.52 (m, 3H). 13C NMR (125 MHz,
DMSO-d6, 60 °C) δ: 160.5, 139.8, 129.0, 128.5, 127.2, 62.1, 58.4,
Preparation of Compound 6. The solution of 6.4 g (31.18
mmol) N-benzyl-S-proline in 64 mL of THF was treated with 1.77 g
(37.95 mmol) of LiAlH4 at 0 °C. The mixture was stirred at room
temperature for 12 h, and KOH (10% in water) was added until a
white precipitate appeared. The mixture was filtered and the residue
was washed with THF, then the filtrate was dried over Na2SO4 and
evaporated. The crude was used without further purification.
Data for compound 6. Yield: 83%, slightly yellow oil. Spectral
+
54.0, 43.8, 28.4, 23.0. HRMS (ESI): calculated for C13H20N5O2 [M
+ H]+, 278.1612; found, 278.1617 (−2.9).
Preparation of Compound 10. A 4.1 mL sample of Et3N (2.97
g, 29.35 mmol) was added in a single portion to a stirred solution of
7 (4.33 g (22.63 mmol) in 57 mL DCM); afterward, the mixture
was cooled at 0 °C and 2.27 mL (3.37 g, 29.41 mmol) of
methanesulfonyl chloride was slowly added, and the mixture was
stirred for 2 h at 0 °C. Afterward, 15 mL of water was added, and
the organic phase was separated; the aqueous phase was extracted
with 30 mL of DCM, the organic extracts were washed with brine,
dried over Na2SO4, filtered, and evaporated in a rotatory evaporator
to obtain the crude mesylate. This was dissolved in 33 mL of
DMSO, 4 Å molecular sieve (1.85 g), KI 0.75 g (11.43 mmol), and
KCN 3.38g (51.9 mmol) were successively added, and the reaction
was stirred during 4 h at 60 °C. The reaction was diluted with water
and extracted with AcOEt; the organic phase was washed with
aqueous saturated solution of NaHCO3 and brine, dried over
Na2SO4, filtered, and evaporated with a rotatory evaporator. The
residue was purified by FCC (hexane/AcOEt = 9:1).
1
1
data matched with previous reports; the H NMR data is provided
1
here for convenience. H NMR (500 MHz, CDCl3) δ: 7.35−7.23
(m, 5H), 3.97 (d, J = 13.0 Hz, 1H), 3.66 (dd, J = 10.7, 3.4 Hz, 1H),
3.43 (dd, J = 10.8, 2.1 Hz, 1H), 3.35 (d, J = 13.0 Hz, 1H), 2.97
(ddd, J = 9.4, 6.2, 3.1 Hz, 1H), 2.79 (s, 1H), 2.77−2.70 (m, 1H),
2.29 (td, J = 9.4, 7.6 Hz, 1H), 1.94 (dq, J = 12.7, 8.8 Hz, 1H),
1.88−1.80 (m, 1H), 1.75−1.63 (m, 2H).
Preparation of Compound 7. A 1.37 g sample of triphenyl-
phosphine (5.23 mmol) and 0.92 g (6.27 mmol) of phthalimide
were dissolved in 15 mL of anhydrous THF; then a mixture of 1.0 g
(5.23 mmol) of 6 in 4 mL of THF was added, and the reaction was
stirred for 10 min. Then a solution of diethyl azodicarboxylate (40%
wt in toluene) 2.27 mL (0.91 g, 5.23 mmol) was added dropwise,
and the mixture was refluxed for 8 h. The reaction was allowed to
cool at room temperature, and the solvents were evaporated under
reduced pressure; the residue was dissolved in diethyl ether (50 mL)
and stirred for 1 h after which a precipitate appeared. The
precipitate was filtered and discarded, and the filtrate was evaporated
under reduced pressure. The residue was passed through a short pad
of silica (hexane/AcOEt = 7:3) in order to remove the excess of
triphenylphosphine oxide; in this way, the reaction yielded 1.36 g
(85%) of the corresponding intermediate as a colorless oil. This was
dissolved in EtOH (40 mL) and 1.1 mL (0.56 g, 11.18 mmol) of
hydrazine monohydrate (64 wt %) was added, and the reaction was
refluxed for 2 h; after this time, HCl (6 mL of 1.0 M in water) was
added and the heating was continued for 30 min. The reaction was
filtered, and the filtrate was evaporated. The residue was dissolved in
NaOH (10 mL of solution 1.0 M in water) and extracted with
DCM (×3), then organic phases were combined, dried over
Na2SO4, and evaporated under reduced pressure.
Data for Compound 10. Yield: 57%, colorless oil, [α]23.5
=
1
−81.5 (c 1, MeOH). H NMR (500 MHz, CDCl3) δ: 7.36−7.29
(m, 4H), 7.28−7.23 (m, 2H), 3.90 (d, J = 13.1 Hz, 1H), 3.47 (d, J
= 13.0 Hz, 1H), 3.05−2.97 (m, 1H), 2.87−2.80 (m, 1H), 2.48−2.34
(m, 2H), 2.30 (td, J = 9.4, 7.0 Hz, 1H), 2.17−2.04 (m, 1H), 1.91−
1.80 (m, 1H), 1.78−1.70 (m, 2H). 13C NMR (125 MHz, CDCl3) δ:
139.0, 128.6, 128.3, 127.1, 118.4, 59.7, 58.7, 54.4, 30.9, 23.4, 22.5.
+
HRMS (ESI): calculated for C13H17N2 [M + H]+, 201.1391; found,
201.1394 (1.0). IR (ATR) υmax: 2953, 2797, 2246, 1453 cm−1.
Preparation of Compound 11. To a stirred solution of 10 (1.2 g
(6 mmol)) in 20 mL of anhydrous diethyl ether was slowly added a
suspension of LiAlH4 (0.68g (17.97 mmol)) in 20 mL of diethyl
ether at 0 °C. The mixture was stirred for 12 h at room temperature
before the reaction was quenched with NaOH (10% in water); the
suspension was filtered over Celite, the solid residue was washed
with DCM, and then the filtrate was dried over Na2SO4 and
evaporated under reduced pressure. The residue was obtained with a
sufficient purity thus purification was not needed.
Data for Compound 11. Yield: 82%, colorless oil, [α]23.3
=
1
Data for Compound 7. Yield: 68%, colorless oil, spectral data
−81.5 (c 1, MeOH). H NMR (500 MHz, CDCl3) δ: 7.34−7.28
(m, 4H), 7.26−7.21 (m, 1H), 4.06 (d, J = 12.7 Hz, 1H), 3.16 (d, J
= 12.7 Hz, 1H), 2.90 (ddd, J = 9.8, 7.4, 2.8 Hz, 1H), 2.84 (ddd, J =
12.2, 8.9, 5.7 Hz, 1H), 2.72 (ddd, J = 12.2, 8.7, 6.2 Hz, 1H), 2.42
(qd, J = 8.0, 3.4 Hz, 1H), 2.09 (td, J = 9.3, 8.1 Hz, 1H), 1.98−1.91
(m, 1H), 1.91−1.81 (m, 1H), 1.75−1.62 (m, 2H), 1.58−1.46 (m,
4H). 13C NMR (125 MHz, CDCl3) δ: 139.5, 129.0, 128.2, 126.8,
62.4, 58.7, 54.1, 39.5, 37.7, 30.2, 22.1. HRMS (ESI): calculated for
C13H20N2Na+ [M + H]+, 227.1524; found, 227.1525 (0.4)
Preparation of Compound 12. This compound was prepared
following the same procedure described for 8. In this case, 0.6 g
(2.93 mmol) of 11 was reacted with 0.52 g (3.52 mmol) of S,-N-
dimethylnitroisothiourea in DCM (15 mL). The crude was purified
FCC (gradient from DCM/2-propanol = 9.5:0.5 to DCM/2-
propanol = 8:2).
matched with previous reports;20 the H NMR data is provided here
1
for convenience. 1H NMR (500 MHz, CDCl3) δ: 7.34−7.28 (m,
4H), 7.25−7.21 (m, 1H), 3.97 (d, J = 13.1 Hz, 1H), 3.30 (d, J =
13.1 Hz, 1H), 2.98−2.91 (m, 1H), 2.77 (dd, J = 12.9, 5.4 Hz, 1H),
2.71 (dd, J = 12.9, 3.4 Hz, 1H), 2.59−2.52 (m, 1H), 2.24−2.16 (m,
1H), 1.94−1.81 (m, 2H), 1.74−1.63 (m, 2H), 1.60−1.51 (m, 2H).
Preparation of Compound 8. A 1.0 g (5.25 mmol) sample of 7
and 0.94 g (6.30 mmol) of S-methyl-N-methylnitroisothiourea were
dissolved in 20 mL of DCM and stirred at room temperature for 18
h. The crude was purified FCC (DCM/2-propanol = 9.5:0.5).
Data for Compound 8. Yield: 43%, colorless oil, [α]23.5= −78.4
1
(c 1, MeOH). H NMR (500 MHz, DMSO-d6, 60 °C) δ: 8.76 (b,
1H), 7.87 (b, 1H), 7.33−7.20 (m, 5H), 3.98 (d, J = 13.2 Hz, 1H),
3.36 (d, J = 13.3 Hz, 1H), 3.34−3.29 (m, 1H), 3.23 (dt, J = 13.5,
4.3 Hz, 1H), 2.88−2.80 (m, 2H), 2.75 (d, J = 4.3 Hz, 3H), 2.23 (q,
J = 8.4 Hz, 1H), 1.96−1.84 (m, 1H), 1.71−1.53 (m, 3H). 13C NMR
(125 MHz, DMSO-d6, 60 °C) δ: 158.9, 139.8, 128.9, 128.5, 127.2,
62.0, 58.3, 54.0, 44.0, 28.5, 28.4, 23.1. HRMS (ESI): calculated for
Data for Compound 12. Yield: 69%, colorless oil, [α]23.4= −60.6
(c 1, MeOH). 1H NMR (500 MHz, DMSO-d6) δ: 7.77 (s, 1H),
7.34−7.16 (m, 6H), 3.95 (d, J = 13.2 Hz, 1H), 3.36−3.17 (m, 3H),
2.87−2.71 (m, 4H), 2.55−2.49 (m, 1H), 2.20−2.06 (m, 1H), 1.98−
1.80 (m, 2H), 1.69−1.55 (m, 3H), 1.54−1.45 (m, 1H). 13C NMR
(125 MHz, DMSO-d6) δ: 158.2, 139.8, 129.2, 128.5, 127.1, 61.6,
58.2, 53.7, 38.8, 29.9, 28.6, 22.4. HRMS (ESI): calculated for
+
C14H22N5O2 [M + H]+, 292.1768; found, 292.1773 (1.4).
Preparation of Compound 9. A 1.25 g (6.56 mmol) sample of 7
and 1.06 g (7.88 mmol) of S-methylnitroisothiourea were dissolved
in 20 mL of DCM and stirred at room temperature for 18 h. The
+
C14H22N5O2 [M + H]+, 306.1925; found, 306.1930 (−1.3).
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Preparation of Compound 13. This compound was prepared
following the same procedure described for 9. In this case, 0.25 g
(1.22 mmol) of 11 was reacted with 0.19 g (1.46 mmol) of S-
methylnitroisothiourea. The crude was purified using FCC (DCM/
2-propanol/MeOH = 8.5:1.5:0.1).
rotamers that were observed under the conditions in which the
spectrum was acquired, and the asterisk indicates the minor one. H
1
NMR (500 MHz, CDCl3) δ 6.87−6.78 (m, 1H), 5.85−5.80 (m,
1H), 4.51* (brs, 1H), 4.22−4.14 (m, 2H), 3.46−3.36 (m, 2H),
2.13−2.08 (m, 1H), 1.9−1.83 (m, 2H), 1.8−1.75 (m, 1H), 1.46* (s,
9H), 1.42 (s, 9H), 1.31, 1.29 (m, 3H). 13C NMR (125 MHz,
CDCl3) δ 166.5, 154.3*, 154.3, 148.5, 148.2*, 120.4, 120.0*, 79.7,
79.6*, 60.3, 60.0*, 57.8, 57.5*, 46.6*, 46.2, 31.7, 30.8*, 28.5*, 28.4,
23.5*, 22.9, 14.25. HRMS (ESI): calculated for C14H23NNaO4 [M +
Na]+, 292.1524; found, 292.1525 (3.4 ppm).
Data for Compound 13. Yield: 56%, colorless oil, [α]23.5
=
1
−63.4 (c 1, MeOH). H NMR (500 MHz,CDCl3) δ: 8.91 (s, 1H),
8.43 (s, 2H), 7.38−7.27 (m, 5H), 3.87 (d, J = 12.7 Hz, 1H), 3.46−
3.20 (m, 3H), 2.97 (ddd, J = 10.8, 7.4, 3.7 Hz, 1H), 2.84−2.73 (m,
1H), 2.38 (q, J = 9.8, 9.2 Hz, 1H), 2.02−1.93 (m, 1H), 1.92−1.82
(m, 2H), 1.81−1.66 (m, 2H), 1.65−1.55 (m, 1H). 13C NMR (125
MHz, CDCl3) δ: 160.2, 138.0, 129.2, 128.6, 127.6, 62.0, 59.2, 53.5,
Preparation of Compound Int-17. In a round-bottom flask
equipped with a magnetic stirrer was prepared a solution of 16
(1.73g (6.42 mmol)) in 20 mL of ethanol, and subsequently 173 mg
of Pd(OH) (10% w/w) was added. Then the reaction was purged
using vacuum and then refilled with a balloon of hydrogen; this
heterogeneous solution was stirred for 2 h. Finally, the mixture was
filtered over a small pad of Celite and silica, the solid was washed
with DCM, and the filtrate was evaporated under reduced pressure.
Data for Compound Int-17: Yield. 95%, slightly yellow oil,
[α]23.5 = −48.5 (c 0.1, CHCl3). Some signals are duplicated due to
two rotamers that were observed under the conditions in which the
spectrum was acquired, the asterisk indicates the minor one. 1H
NMR (500 MHz, CDCl3) δ: 4.11 (q, J = 7.0 Hz, 2H), 3.88−3.73
(m, 1H), 3.48−3.24 (m, 2H), 2.28 (q, J = 8.0, 7.4 Hz, 2H), 2.08−
1.75 (m, 4H), 1.73−1.56 (m, 2H), 1.45 (s, 9H), 1.24 (t, J = 7.0 Hz,
3H). 13C NMR (125 MHz, CDCl3) δ 173.7, 173.4*, 155.9*, 154.7,
79.3*, 78.9, 60.3, 56.6, 46.4*, 46.1, 31.5*, 31.2, 30.7, 29.9, 29.6*,
28.5, 28.4*, 24.8, 23.7*, 23.0. HRMS (ESI): calculated for
+
38.1, 31.5, 29.2, 22.5. HRMS (ESI) calculated for C14H22N5O2 [M
+ H]+, 292.1768; found, 292.1773 (0).
Preparation of Compound 14. A sample of 2.48 g of LiAlH4
(65.14 mmol) was suspended in 65 mL of THF; afterward 5.0 g
(43.43 mmol) of S-proline was added at 0 °C. The mixture was
refluxed for 2 h under nitrogen atmosphere. After cooling the
reaction, a solution of KOH (10% in water) was added; next the
mixture was filtered and the residue was refluxed with 50 mL of
THF for 1 h and then filtered again. The organic extracts were
combined, dried over Na2SO4, and evaporated under reduced
pressure at ∼40 °C. The residue was used in the next reaction
without further purification.
Data for Compound 14. Yield: 83%, brown oil. Spectral data
matched with previous reports;21 the H NMR data is provided here
1
for convenience. 1H NMR (500 MHz, CDCl3) δ: 3.60−3.54 (m,
1H), 3.39−3.32 (m, 2H), 3.18−3.04 (m, 3H), 3.01−2.87 (m, 2H),
1.90−1.67 (m, 2H), 1.50−1.39 (m, 1H).
+
C14H25NNaO4 [M + Na]+, 294.1681; found, 294.1684 (1.0 ppm).
Preparation of Compound 15. A 6.2 mL sample of triethylamine
(4.5 g, 44.48 mmol) was slowly added to a stirred solution of 14
(3.0 g (29.6 mmol)) in a 45 mL mixture of 1,4-dioxane/water 1:1,
and the reaction was stirred for 15 min. Di-tert-butyldicarbonate (8.4
g (38.55 mmol)) in 10 mL of 1,4-dioxane was added dropwise at 0
°C. The reaction was stirred during 12 h. The reaction was diluted
with AcOEt (100 mL) and the organic phase was washed with
brine, dried over Na2SO4, and evaporated. The residue was purified
using FCC (hexane/AcOEt = 1:1).
Preparation of Compound 17. A 4.2 mL (0.18 g, 8.39 mmol)
sample of a solution of 2.0 M LiBH4 in THF was added dropwise to
a stirred solution of Int-17 (1.74 g (6.46 mmol)) in anhydrous THF
(30 mL) at room temperature; the stirring was continuous for 12 h.
Next, the reaction was quenched by the careful addition of 3 mL of
MeOH, and volatiles were evaporated under reduced pressure; the
residue was partitioned in AcOEt/water (3:1) (50 mL), the organic
phase was separated, and the remaining aqueous phase was extracted
with additional AcOEt. The combined organic extracts were dried
over Na2SO4, filtered, and evaporated. The residue was purified by
FCC (Hexane/AcOEt = 1:1).
Data for Compound 15. Yield: 90%, colorless oil. Spectral data
matched with previous reports;21 the H NMR data is provided here
1
for convenience. 1H NMR (500 MHz, CDCl3) δ: 4.85−4.77 (m,
1H), 4.02−3.94 (m, 1H), 3.69−3.55 (m, 2H), 3.52−3.42 (m, 1H),
3.36−3.27 (m, 1H), 2.07−1.97 (m, 1H), 1.80 (dh, J = 26.5, 6.5 Hz,
2H), 1.57−1.50 (m, 1H), 1.47 (s, 9H).
Data for Compound 17. Yield: 95%, colorless oil, [α]23.5
=
−47.14 (c 0.1, CHCl3). Some signals are duplicated due to two
rotamers that were observed under the conditions in which the
spectrum was acquired; the asterisk indicates the minor one. 1H
NMR (500 MHz, CDCl3) δ: 3.67 (brs, 2H), 3.44−3.23 (m, 2H),
2.01 (brs, 1H), 1.97−1.76 (m, 4H), 1.70−1.51 (m, 3H), 1.46 (s,
9H), 1.39 (m, 1H). 13C NMR (125 MHz, CDCl3) δ: 154.8, 79.1,
62.7*, 62.4, 57.0*, 56.6, 46.5, 46.1*, 30.8, 30.2, 29.5*, 29.1, 28.6,
Preparation of Compound 16. A solution of 15 in 11 mL of
DCM was added to a stirred solution of oxalyl chloride 0.71 mL
(1.06g, 8.35 mmol) in 11 mL of DCM cooled at −78 °C, then a
solution of DMSO 0.54 mL (0.59g, 7.55 mmol) in 11 mL of DCM
was slowly added. After 5 min. The reaction was stirred 20 min and
4.84 mL of Et3N (3.51g, 34.68 mmol) was added in a single
portion; the mixture was stirred for another 10 min and then
allowed to warm to room temperature. The mixture was diluted
with additional DCM and afterward was washed with saturated
aqueous solution of NH4Cl and brine; the organic phase was dried
over Na2SO4 and evaporated under reduced pressure. The crude was
used in the next reaction without any purification.
+
23.7, 23.0*. HRMS (ESI): calculated for C12H23NNaO3 [M +
Na]+, 252.1575; found, 252.1584 (3.2 ppm).
Preparation of Compound 18. The procedure used to synthesize
18 was identical to that described to obtain N-Boc-S-prolinal. In this
case 1.39 g (6.06 mmol) of 17, 0.56 mL (0.85 g, 6.66 mmol) of
oxalyl chloride, 0.51 mL (0.57 g, 7.28 mmol mmol) of DMSO, and
4.22 mL (3.06 g, 30.24 mmol) were used to perform the reaction.
The crude was purified in FCC (hexane/AcOEt = 8:2).
Afterward, a stirred solution of triethyl phosphonoaceate (1.66
mL (1.87g, 8.36 mmol)) in anhydrous THF (10 mL) was added
dropwise in a suspension of NaH (0.2 g (8.36 mmol)) in 5 mL of
anhydrous THF at 0 °C; after the addition, the reaction was stirred
at room temperature for 30 min, then the mixture was cooled at 0
°C and the crude N-Boc-S-prolinal was added dropwise in
anhydrous THF (10 mL) to the mixture reaction. The reaction
was stirred for 8 h at room temperature. MeOH (1 mL) was added
to quench the reaction, solvents were evaporated under reduced
pressure, and the residue was solubilized in AcOEt, washed with
brine, dried over Na2SO4, filtered, and evaporated. The crude was
purified by FCC (hexane/AcOEt = 7:3).
Data for Compound 18. Yield: 86%, colorless oil, [α]25.2
=
−7.82 (c 0.1, CHCl3). Some signals are duplicated due to two
rotamers that were observed under the conditions in which the
spectrum was acquired; the asterisk indicates the minor one. 1H
NMR (500 MHz, CDCl3) δ: 9.77 (s, 1H), 3.93−3.75 (m, 1H),
3.50−3.25 (m, 2H), 2.58−2.37 (m, 2H), 2.05−1.80 (m, 3H), 1.78−
1.69 (m, 1H), 1.67−1.57 (m, 2H), 1.46 (s, 9H). 13C NMR (125
MHz, CDCl3) δ: 202.3, 201.8*, 155.1*, 154.9, 79.5, 79.2*, 56.4,
46.6, 46.2*, 40.9, 40.7*, 30.7, 30.4*, 30.2, 28.5, 28.4*, 27.0, 23.7,
+
23.01*, 14.4. HRMS (ESI): calculated for C12H21NNaO3 [M +
Na]+ 250.1419, found 250.1422 (1.2 ppm).
Preparation of Compound 19. A 0.56 mL (0.57 g, 2.89 mmol)
sample of N,N-dibenzylamine was added to a stirred solution of 0.6
g (2.63 mmol) of 18 and 0.48 mL of Et3N (0.34 g, 3.36 mmol) in
Data for Compound 16. Yield: 81%, slightly yellow oil, [α]23.5
−28.9 (c 0.1, CHCl3). Some signals are duplicated due to two
=
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1,2-DCE (20 mL) at 0 °C, and then the mixture was stirred for 5
min. Afterward, 1.12 g (5.28 mmol) of sodium triacetoxyborohy-
dride was added in one portion. After the mixture was stirred for 16
h at room temperature, saturated aqueous sodium bicarbonate
solution was added, the organic phase was separated, and the
aqueous phase was extracted with AcOEt. The organic extracts were
combined, dried over Na2SO4, filtered, and evaporated. The residue
was purified in FCC (hexane/AcOEt = 8.5:1.5).
2H), 3.03 (ddd, J = 10.4, 7.3, 3.2 Hz, 1H), 2.76 (d, J = 4.8 Hz,
3H), 2.72−2.64 (m, 1H), 2.32 (dd, J = 9.1 Hz, 1H), 2.04−1.95 (m,
1H), 1.83−1.53 (m, 6H). 13C NMR (125 MHz, CDCl3) δ: 158.3,
137.3, 129.5, 128.5, 127.7, 64.3, 59.0, 54.4, 41.9, 29.7, 29.5, 28.1,
+
25.0, 22.1. HRMS (ESI): calculated for C16H26N5O2 [M + H]+,
320.2086; found, 320.2087 (0.9).
Preparation of Compound 24. A 0.44 g (3.29 mmol) sample of
S-methylnitroisothiourea was added in one portion to a stirred
solution of N-Boc-(S)-prolinamine 0.55 g (2.80 mmol) in DCM (20
mL) at room temperature. The reaction was stirred for 12 h, and
the volatiles were evaporated under reduced pressure. Finally, the
residue was purified using FCC (hexane/AcOEt = 4:6).
Data for Compound 24. Yield: 33%, white solid, [α]26 = −17.43
Data for Compound 19. Yield: 93%, colorless oil, [α]25.4
=
−32.36 (c 0.1, CHCl3). Some signals are duplicated due to two
rotamers that were observed under the conditions in which the
spectrum was acquired; the asterisk indicates the minor one. 1H
NMR (500 MHz, CDCl3) δ: 7.39−7.35 (m, 4H), 7.34−7.29 (m,
4H), 7.27−7.21 (m, 2H), 3.81−3.62 (m, 1H), 3.61−3.50 (m, 4H),
3.44−3.23 (m, 2H), 2.50−2.37 (m, 2H), 1.93−1.74 (m, 3H), 1.61−
1.55 (brs, 2H), 1.54−1.39 (m, 11H), 1.34−1.21 (m, 2H). 13C NMR
(125 MHz, CDCl3) δ: 154.6, 139.8, 128.7, 128.1, 126.7, 78.9, 58.2,
57.2, 53.6*, 53.4, 46.4*, 46.0, 32.5, 31.8*, 30.7, 29.9*, 28.5, 26.9*,
1
(c 1, MeOH). H NMR (500 MHz, CDCl3) δ: 9.18 (brs, 1H), 8.78
(brs, 2H), 3.84−3.73 (m, 1H), 3.44−3.26 (m, 3H), 3.23−3.13 (m,
1H), 2.11−2.02 (m, 1H), 1.98−1.82 (m, 3H), 1.47 (s, 9H). 13C
NMR (125 MHz, CDCl3) δ: 159.3, 154.8, 79.6, 55.4, 45.5, 43.2,
+
28.4, 27.4, 22.5. HRMS (ESI): calculated for C11H21N5NaO4 [M +
+
23.93, 23.09. HRMS (ESI): calculated for C26H37N2O2 [M + H]+,
Na]+, 310.1941; found, 310.1490 (−0.3).
409.2855; found, 409.2866 (2.7 ppm).
Preparation of Compound 25. A 0.4 mL (0.44 g, 5.57 mmol)
sample of acetyl chloride in DCM (1 mL) was added dropwise to a
stirred solution of 24 (0.4 g (1.4 mmol)) in a blend of DCM/
MeOH 2:1 (3 mL) at 0 °C. After the mixture was stirred for 2 h,
the solvents were evaporated, and the residue was purified using
FCC (AcOEt/MeOH = 8:2).
Preparation of Compound 20. A 48 mg sample of Pd/C (10%
w/w) was added to a solution of 19 (0.48 g (1.17 mmol)) in
methanol (15 mL), and the heterogeneous mixture was stirred
during 48 h under H2 atmosphere. The mixture was filtered through
a pad of Celite, the solid was washed with DCM, the filtrate was
evaporated under reduced pressure to obtain the amine which was
used in the next reaction without any further purification.
Data for Compound 20. Yield: 93%, colorless oil, [α]22 = −39.9
(c 0.1, CHCl3). Some signals are duplicated due to two rotamers
that were observed under the conditions in which the spectrum was
Data for Compound 25. Yield: 77%, yellow oil, [α]21 = 12.8 (c
1
1, MeOH). H NMR (500 MHz, DMSO-d6, 50 °C) δ: 8.19 (brs,
4H), 3.71−3.62 (m, 1H), 3.57−3.45 (m, 2H), 3.24−3.11 (m, 2H),
2.12−2.01 (m, 1H), 2.00−1.81 (m, 2H), 1.64 (dq, J = 12.8, 8.1 Hz,
1H). 13C NMR (125 MHz, DMSO-d6, 50 °C) δ: 159.8, 58.5, 45.5,
1
+
acquired; the asterisk indicates the minor one. H NMR (500 MHz,
42.2, 27.9, 23.5. HRMS (ESI): calculated for C6H14N5O2 [M +
H]+, 188.1147; found, 188.1151 (2.1).
DMSO-d6, 50 °C) δ: 3.69−3.59 (m, 1H), 3.30−3.23 (m, 1H),
3.23−3.13 (m, 1H), 2.66−2.55 (m, 2H), 1.94−1.69 (m, 3H), 1.60
(brs, 2H), 1.40 (s, 9H), 1.36 (m, 3H). 13C NMR (125 MHz,
DMSO-d6, 50 °C) δ: 154.0, 78.5, 57.0, 46.5, 41.4, 32.1*, 31.5,
30.7*, 29.9, 28.9, 28.7*, 28.6, 23.7, 23.0*. HRMS (ESI): calculated
Biological Assay. The insecticidal activities of title compounds
against Xyleborus affinis were tested according to the previously
reported procedure of the contact toxicity on filter paper.20
A
Whatman grade 42, ashless filter paper (5 cm diameter) was placed
in a glass Petri dish (50 × 17 mm diameter). An aliquot of 0.25 mL
of the solution of a 0.05 M compound in dimethyl sulfoxide/water
(1:1) was applied uniformly to the filter paper disc; the mixture of
solvents was used as the negative control, and the solution of
dinotefuran at the same concentration was used as positive control.
The solvent was allowed to distribute evenly for 5 min prior to the
introduction of five adult insects into each dish. Since the boiling
point of dimethyl sulfoxide and water are high enough to be
volatilized at room temperature, it is not necessary to replenish the
solvent mixture. According to statistical requirements, each treat-
ment was replicated three times at 25 °C 1 °C with the organism
grown in the laboratory. Xyleborus affinis were reared in an artificial
media according to Biedermann et al.22 with some modifications.
Rearing media were maintained in a climatic chamber at 26 °C and
60% of RH in complete dark. Adults were obtained at 30 days after
female inoculation by dissecting the media culture. Insect mortalities
were recorded after 12 h. Insects were presumed dead if they
remained immobile and did not respond to three probings with a
blunt dissecting probe after a 5 min recovery period.
Docking Study. The high nAChR inhibitory activity of
compound 9 was chosen to perform the ligand−protein interaction,
and AutoDock 4.2 was used to carry out the molecular docking
simulations. Because the amino acids forming the active pockets are
both structurally and functionally consistent in the diverse nAChRs
and AchBPs, the crystal structure of the Aplysia californica binding
protein (aChBP) complex with the neonicotinoid imidacloprid
(PDB: 3C79) was used as the template to construct the models.23,24
The receptor was prepared for docking, and the lower energy
conformations of each compound were optimized by the semi-
empirical method PM3. The compound 9 and dinotefuran were
flexibly docked automatically in the active site of nAChR. The
structure was prepared using the UCSF-Chimera software by
removing waters and cocrystallized ligands, and then adding
Gasteiger charges and polar hydrogens. The search space in the
+
for C12H25N2O2 [M + H]+, 229.1916; found, 229.1918 (0.9).
Preparation of Compound 21. This compound was prepared
following the same procedure described for 9. In this case 0.6 g
(2.62 mmol) of 20 was reacted with 0.47 g (3.15 mmol) of S-
methyl-N-methylnitroisothiourea in DCM (15 mL). The crude was
purified in FCC (hexane/AcOEt = 6:4).
Data for Compound 21. Yield: 62%, colorless oil, [α]22.7
=
+12.12 (c 0.1,CHCl3). Some signals are duplicated due to the two
rotamers that were observed under the conditions in which the
spectrum was acquired; the asterisk indicates the minor one. 1H
NMR (500 MHz, DMSO-d6) δ: 9.07 (brs, 1H), 7.25 (brs, 1H), 3.63
(brs, 1H), 3.30−3.11 (m, 4H), 2.77 (s, 2H), 1.95−1.69 (m, 3H),
1.60 (s, 2H), 1.53−1.42 (m, 2H), 1.39 (s, 9H), 1.34−1.20 (m, 2H).
13C NMR (125 MHz, DMSO-d6) δ: 159.5, 158.3, 79.7*, 79.5, 55.1,
46.5, 46.4*, 41.6, 41.3*,40.9, 31.2, 30.7*, 28.5, 28.4, 25.4, 23.5,
+
23.3*. HRMS (ESI): calculated for C14H27N5NaO4 [M + Na]+,
352.1960; found, 352.1956 (−1.4).
Preparation of Compound 23. A 0.43 mL (0.47 g, 6.0 mmol)
sample of acetyl chloride in DCM (1 mL) was added dropwise at 0
°C to a stirred solution of 21 (0.5 g (1.51 mmol)) in a blend of
DCM/MeOH 2:1 (3 mL). The reaction was stirred for 2 h. Then,
the solvents were evaporated under reduced pressure to obtain the
intermediate hydrochloride (0.40 g (1.5 mmol)), which was
dissolved in acetonitrile (20 mL). Next, tetrabutylammonium iodide
(0.61 g (1.65 mmol)), potassium carbonate (0.52 g (3.76 mmol)),
and benzyl bromide (0.18 mL (0.27 g, 1.57 mmol)) were added
sequentially. After the reaction was refluxed during 8 h, the mixture
was filtered and the remaining solid was washed with DCM and
evaporated. The residue was purified using FCC (DCM/2-
propanol/NH4OH = 8:3:0.2).
Data for Compound 23. Yield: 54%, colorless oil, [α]24.5
−39.96 (c 0.1,CHCl3). H NMR (500 MHz, CDCl3) δ: 9.32 (br s,
1H), 7.38−7.27 (m, 5H), 6.43 (br s, 1H), 4.02 (d, J = 12.7 Hz,
1H), 3.48 (br s, 1H), 3.34 (d, J = 12.7 Hz, 1H), 3.31−3.21 (m,
=
1
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a
Scheme 1. Synthesis of Target Molecules 8 and 9
a
(A) (i) HNO3, H2SO4, 6 h, 0 °C. (B) CH3NH2 2 M, CH3CN, 0 °C to r.t., 24 h. (C) (i) KOH, BnCl, i-PrOH, r.t., 40 °C, 6 h; (ii) LiAlH4, THF, 0
°C to room temperature, 12 h; (iii) Phtalamide, PPh3, DEAD, THF, reflux, 8 h; (iv) Hidrazine, EtOH, r.t., reflux, 2 h; (v) and (vi) CH2Cl2, r.t, 18
h.
a
Scheme 2. Synthesis of Compounds 12 and 13
a
Reagents and conditions: (i) MsCl, Et3N, CH2Cl2, 0 °C, 2 h; (ii) NaCN, DMSO, 60 °C, 4 h; (iii) LiAlH4, r.t., 12 h; (iv) and (v) CH2Cl2, r.t., 12 h.
protein (grid-box) was calculated using the AutoDockTools and
AutoGrid softwares.
The molecular docking was performed on AutoDock4.2 software
using 100 runs and 5 million energy evaluations; the remaining
parameters were set as default.
For each cluster, the conformation with the lowest binding energy
in the binding site was chosen for further analysis and comparison.
AccelrysDS visualizer 2.5 [Accelrys Inc., San Diego, CA (2009)] was
used for molecular modeling to determine their binding orientations
and interactions.
and compound 9. The guidelines of the Official Mexican Standard
(NOM-062-ZOO-1999), as well as the protocol and norms of the
international and universal bioethics committee were followed to
care and use the laboratory animals. The animals were in an area
with a regulated temperature of 25 2 °C, a relative humidity of 50
15%, and dark light cycles of 12 h each.
Animals were divided into three groups: The first group was given
a 1000 mg/kg dose of dinotefuran as a positive control, the second
group was given a 1000 mg/kg dose of compound 9, and the third
group was given 2 mL/kg of saline as a control. Studies were
conducted by the EPA using a lethal dose 50 of double (LD50 =
2000 mg/kg).
Neurotoxic Effect. Female rats of the Wistar strain of 220−250
g of weight were used to test the neurotoxic effect of Dinotefuran
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a
Scheme 3. Synthesis of Compounds 21 and 23
a
Reagents and conditions: (i) LiAlH4, THF, reflux, 2 h; (ii) Boc2O, Et3N, 1,4-diox/H2O, 12 h; (iii) (COCl)2, DMSO, Et3N, CH2Cl2, −78 °C, 45
min; (iv) (MeO)2OPCH2CO2Et, NaH, THF, 0 °C, 8 h; (v) H2, Pd/C, EtOH, 2 h then LiBH4, THF, r.t., 12 h; (vi) Dess-Martin periodinane,
CH2Cl2; (vii) NHBn2, NaBH(OAc)3, DCE, r.t; (viii) H2, Pd/C, EtOH, r.t., 12 h; (ix) CH2Cl2, r.t., 8 h; (x) AcCl, MeOH, 0 °C then K2CO3, TBAI,
BnBr, CH3CN.
For animal training, the equipment was adjusted to a constant
speed of 16 rpm. Animals were placed on the rotating rod for 10
Scheme 4. Mechanism of the Horner−Wadsworth−
Emmons Reaction
min and the number of falls and the time in which they had the first
fall were evaluated. This procedure was carried out during three
consecutive days. On the fourth day, the training test was repeated
to make sure the animals were not uncoordinated and healthy.
Thereafter, the test compounds dinotefuran, compound 9, and saline
were orally administered. One hour after the injection, it was
evaluated the number of falls and the time for the first fall. It was
used with the same parameters of speed (16 rpm) and the time (10
min) to identify any alteration in the locomotive activity of the rat,
which indicated a secondary effect in the central nervous system
level. Results were expressed as the average
standard error and
analyzed by a one−way ANOVA followed by a Tukey to compare
differences between groups.
RESULTS AND DISCUSSION
■
Synthesis of Derivatives. The synthesis of the target
molecules 8 and 9 was shown in Scheme 1. First, compound
1 was prepared using a solution of HNO3 and H2SO4 during
6 h at 0 °C, and then compound 3 was prepared using as a
starting material the dioxoisoindoline 2 and a solution of
methylamine in acetonitrile [see Scheme 1A,B]. Then, after
the N-protection of the L-proline, this was reduced with
lithium aluminum hydride to obtain the N-benzyl prolinol 6.
The Mitsunobu type reaction of 6, followed by the reaction
with hydrazine in ethanol delivers the diamine 7. Finally, this
diamine was coupled with the compounds 1 or 3 to obtain
the nitroguanidine 9 and 8, respectively.
diamine 11 in 82% yield (Scheme 2). Finally, this diamine
was coupled with the compounds 1 or 3 to obtain the
nitroguanidines 13 and 12, respectively.
The synthesis of the derivatives with three methylenes is
shown in Figure 4. Here, the L-proline was reduced to the
amino alcohol 14, followed by the protection of the nitrogen
with Boc2O to furnish the N-Boc protected amino alcohol 15.
Then, the alcohol was oxidized by a Swern reaction, which
was followed by a Horner−Wadsworth−Emmons reaction to
allow us to obtain the ethyl acrylate 16. The compound 16
was reduced with lithium borohydride to obtain the alcohol
17. Next, the oxidation of the alcohol 17 with Swern
reaction, followed by reductive amination in the presence of
dibenzyl amine, gave the diamine N-Boc protected 19, which
after catalytic hydrogenation provided the free diamine 20.
The L-proline derivatives with two methylenes between the
nitroguanidine and the pyrrolidine moiety were synthesized
starting from the N-benzyl prolinol 6; this alcohol was
converted to the cyano derivative through an SN2 reaction.
Next, the reduction of the cyano compound 10 gave the
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a
Scheme 5. Synthesis of Compound 25
a
Reagents and conditions: (i) DCM, r.t., 12 h; (ii) DCM/MeOH (2:1), AcCl, 2 h, 0 °C.
Then, after the coupling with compound 3 the deprotection/
protection reaction delivered the compounds 21 and 23,
respectively. Finally, compound 25 was synthesized in three
steps starting from the 2-aminomethyl pyrrolidine (Scheme
3).
Table 1. Bioassay of L-Proline Neonicotinoids Derivatives
a
b
compound
length carbon chain
mortality %
dinotefuran
8
9
12
13
21
23
25
1
1
1
2
2
3
3
1
40
73
93
80
53
33
60
13
Most of the reactions showed in these synthetic procedures
are nucleophilic substitutions, reduction, and oxidation
reactions. Nonetheless, from the mechanistic point of view,
the Horner−Wadsworth−-Emmons reaction is quite interest-
ing. In Scheme 4, it is disclosed the mechanism reaction is
similar to the mechanism of the Wittig reaction. Here, the
stereochemistry is set by steric approach control, where the
antiperiplanar approach of the carbanion B to the carbon of
the carbonyl group of compound A is favored when the
aldehydic hydrogen eclipses the bulky phosphoranyl moiety.
As a result, the ester group is placed syn to the aldehyde
pyrrolidine group, thus the alkene assumes E-orientation of
these groups after rotation to form the oxaphosphetane C.
Lastly, the resulting phosphate byproduct is readily separated
from the desired products by simple washing with water.
Finally, Scheme 5 shows the synthesis of compounds 24
and 25. As a starting material, diamine N-Boc was used to
couple to compound 1, then after the deprotection reaction
compound 25 was obtained.
a
A total of 0.25 mL of compound was used with a concentration 0.05
b
M. The mortality was determined after 12 h.
Insecticidal Activities. In general, all of the compounds
showed from good to excellent insecticidal activity against
Xyleborus affinis. In Table 1, it was shown that compounds 8
and 9 had an outstanding insecticidal activity of 73% and
93%, respectively, while the control dinotefuran had 40%
mortality after 12 h. When the number of methylenes
between the nitroguanidine and the pyrrolidine moiety is
increased by two carbons, the insecticidal activity diminished
to 80% and 53% for compounds 12 and 13, respectively.
The bioassay of compounds 21 and 23 showed that the
increased number of methylenes up to three lessen the
insecticidal activity. Indeed, the percent mortality was
reduced by up to 33%. The protecting group also played
an important role in the insecticidal activity, and it was
observed from Figure 2 that the N-benzyl group was
indispensable to obtain high mortality. The use of Boc and
free amine dramatically diminished the mortality percent up
to 13%.
Figure 2. Effect of the length of carbons atoms on the insecticidal
activity.
Scheme 6. Biological Activity of Opposite Enantiomers of
α-Methylbenzylamine
The analysis of nonmethylated compounds 9, 13, and 25
showed a negative impact on the insecticidal activity if the
number of methylenes was increased. The N-methylated
compounds had a similar behavior except for the compound
8 that had minor insecticidal activity compared to 12 (Figure
2). Finally, compound 9 without the N-benzyl moiety 25 was
also studied; nonetheless, the bioassay showed 13% of
mortality after 12 h.
a
Table 2. Affinity and Inhibition Constant
a
compound
free energy (kcal/mol) “Score” inhibition constant (Ki)
dinotefuran
9
−5.64
−6.57
73.06 μM
15.23 μM
a
To perform the simulations, the parameters of 100 runs with 5
million evaluations were used for each molecular coupling experiment
using a Lamarkian genetic algorithm.
As a part of our research program of biologically active and
enantioenriched compounds, two experiments were run to
demonstrate and gain insight into the importance of chirality
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Figure 3. (a,c) The 2D and 3D representations of the interaction of dinotefuran with the residues of the search cavity in 3C79. (b,d) The 2D
and 3D representations of the interaction of compound 9 with the residues of the search cavity in 3C79.
in the use of new neonicotinoids. Thus, the (S)-(−)- α-
methylbenzylamine S-26 and the (R)-(+)-α-methylbenzyl-
amine R-26 were used to synthesize opposite enantiomers of
neonicotinoids. These two isomers are commercially available
and cheap, and the synthesis to obtain the desired
neonicotinoids is one-pot. The results are disclosed in
Scheme 6. To our delight, the R-enantiomer had higher
mortality than the S-enantiomer, 53 and 27, respectively.
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damaging their acetylcholine receptors.26 In this regard, the
neurotoxic effect of dinotefuran and compound 9 were
analyzed using the RotaRod test with female rats of the
Wistar strain. It was observed in Figure 4 that dinotefuran
was more neurotoxic than compound 9. The elapsed time
required for the first fall and the number of falls was higher
when the rats were administered with dinotefuran. These
results suggest that compound number 9 is safer for
mammalians than the commercially available dinotefuran.
In summary, a series of novel neonicotinoid’s analogs were
designed and synthesized by introducing the amino acid L-
proline. To the best of our knowledge, this is the first report
of neonicotinoids that incorporate an enantiopure L-amino
acid. The bioassay results indicated that compound 9 had an
excellent insecticidal activity. Hence, this sort of novel
compound that emerged can be as a strategic model to
develop new chiral and enantiopure neonicotinoids with high
insecticidal activity and low toxicity to mammalians.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c05997.
Figure 4. Neurotoxic evaluation of compound 9. (A) Number of rat
falls during the period of 10 min. (B) Elapsed time required for the
first rat fall. Data are the mean SEM of six animals. * Significantly
different from the saline group (P ≤ 0.001) and ** significantly
different between dinotefuran and compound 9 (P ≤ 0.001) as
determined by one-way ANOVA followed by the Tukey’s test.
Chemicals and instruments; data on 1H NMR, HRMS,
optical rotation, and melting points of target com-
pounds; complementary information on docking and
neurotoxic effect. (PDF)
These results suggest that both the chirality and the absolute
configuration of bioactive compounds play an essential role in
the response produced in the insect.
AUTHOR INFORMATION
■
Corresponding Author
́
Jose Luis Olivares-Romero − Red de Estudios Moleculares
Docking. The result obtained showed that the score of
the compound 9 was −6.57 kcal/mol, whereas the
dinotefuran was −5.64 kcal/mol; this suggests a better
affinity with the receptor and to our delight this is in
agreement with the experimental result (see Table 2,
compound 9). Another interesting result derived from
docking was the inhibition constant, which was lower for
compound 9 (15.23 uM) when compared to dinotefuran
(73.06); these results suggest that compound 9 is an
improved and more potent inhibitor since the concentration
required to produce half-maximum inhibition is almost five
times lower than dinotefuran (Table 2).
The 3D representation of the molecular recognition with
the residues of the search cavity of the binding protein 3C79
(AChBP) with compound 9 and dinotefuran showed
different sorts of interactions between them (see Figure 3).
For instance, the dinotefuran showed interactions with the
receptor in Phe78 and the oxygen of the nitro moiety, and
the oxygen of the heterocyclic ring interacted with the Thr24
of the receptor. On the other hand, compound number 9
showed a higher degree of interactions between the receptor
and nitroguanidine moiety of the neonicotinoid analog. Also,
an interaction of the cation-π with the aromatic ring of
compound 9 and the Met116 and Val108 was found, which
plays an important role in neonicotinoid’s insecticidal
activity.25
Avanzados, Clúster Científico y Tecnológico BioMimic,
Campus III, Instituto de Ecología, 91073 Xalapa, Veracruz,
México; orcid.org/0000-0001-7389-3514;
Email: jose.olivares@inecol.mx; Fax: +52 (228) 842 18
00
Authors
Israel Bonilla-Landa − Red de Estudios Moleculares
Avanzados, Clúster Científico y Tecnológico BioMimic,
Campus III, Instituto de Ecología, 91073 Xalapa, Veracruz,
México
̃
Ulises Cuapio-Munoz − Red de Estudios Moleculares
Avanzados, Clúster Científico y Tecnológico BioMimic,
Campus III, Instituto de Ecología, 91073 Xalapa, Veracruz,
México
́
Axel Luna-Hernandez − Red de Estudios Moleculares
Avanzados, Clúster Científico y Tecnológico BioMimic,
Campus III, Instituto de Ecología, 91073 Xalapa, Veracruz,
México
Alfonso Reyes-Luna − Red de Estudios Moleculares
Avanzados, Clúster Científico y Tecnológico BioMimic,
Campus III, Instituto de Ecología, 91073 Xalapa, Veracruz,
México
́
Alfredo Rodríguez-Hernandez − Red de Estudios
Moleculares Avanzados, Clúster Científico y Tecnológico
BioMimic, Campus III, Instituto de Ecología, 91073
Xalapa, Veracruz, México
Neurotoxic Effect. Albeit neonicotinoids have proven to
be less toxic to the environment than others insecticidal
compounds, some research had suggested the risk to the
exposure of neonicotinoids to mammals and humans, mainly
Arturo Ibarra-Juarez − Cátedra CONACyT en el, Instituto
de Ecología AC, Xalapa, Veracruz, México; Red de Estudios
Moleculares Avanzados, Clúster Científico y Tecnológico
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AC, Xalapa, Veracruz, México
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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(N’benzylprolyl)amino]benzophenone (BPB) and Ni(II) complexes
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This work was supported by Consejo Nacional de Ciencia y
Tecnología (CONACyT) through the Fondo Institucional de
́
Fomento Regional para el Desarrollo Científico, Tecnologico
́
y de Innovacion (FORDECyT), Grant 292399. We also
thank to Dr. Juan Luis Monribot Villanueva for technical
support in the HRMS-QTOF analysis.
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