New analogues of (E)-β-farnesene with insecticidal activity and binding affinity to aphid odorant-binding proteins

Article abstract of DOI:10.1021/jf104712c

(E)-β-Farnesene is a strong and efficient alarm pheromone in most aphid species. However, applications in agriculture are prevented by its relatively high volatility, its susceptibility to oxidation and its complex and expensive synthesis. To develop novel compounds for aphid control, we have designed and synthesized analogues of (E)-β-farnesene, containing a pyrazole moiety present in several insecticides. Their structures have been confirmed by ¹H NMR, elemental analysis, high-resolution mass spectroscopy and IR. Binding activities to three odorant-binding proteins (OBPs) of the pea aphid Acythosiphon pisum have been evaluated and correlated with their structures with reference to (E)-β-farnesene. Several derivatives were shown both to bind to A. pisum OBPs with a specificity similar to that of (E)-β-farnesene and to have aphicidal activity comparable to that of thiacloprid, a commercial insecticide. The compounds synthesized in this work represent new potential agents for aphid population control and provide guidelines to design analogues of (E)-β-farnesene endowed with both insecticidal and repellent activity for aphids.

Full text of DOI:10.1021/jf104712c

ARTICLE
pubs.acs.org/JAFC
New Analogues of (E)-β-Farnesene with Insecticidal Activity and
Binding Affinity to Aphid Odorant-Binding Proteins
Yufeng Sun,†,‡ Huili Qiao,‡ Yun Ling,† Shaoxiang Yang,† Changhui Rui,§ Paolo Pelosi,*,‡ and Xinling Yang*,†
†Department of Applied Chemistry, College of Science, China Agricultural University, Key Laboratory of Pesticide Chemistry and
Application, Ministry of Agriculture, Beijing 100193, People’s Republic of China
‡Department of Biology of Agricultural Plants, University of Pisa, via S. Michele 4, 56124 Pisa, Italy
§Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China
ABSTRACT: (E)-β-Farnesene is a strong and efficient alarm pheromone in most aphid species. However, applications in
agriculture are prevented by its relatively high volatility, its susceptibility to oxidation and its complex and expensive synthesis. To
develop novel compounds for aphid control, we have designed and synthesized analogues of (E)-β-farnesene, containing a pyrazole
moiety present in several insecticides. Their structures have been confirmed by 1H NMR, elemental analysis, high-resolution mass
spectroscopy and IR. Binding activities to three odorant-binding proteins (OBPs) of the pea aphid Acythosiphon pisum have been
evaluated and correlated with their structures with reference to (E)-β-farnesene. Several derivatives were shown both to bind to A.
pisum OBPs with a specificity similar to that of (E)-β-farnesene and to have aphicidal activity comparable to that of thiacloprid, a
commercial insecticide. The compounds synthesized in this work represent new potential agents for aphid population control and
provide guidelines to design analogues of (E)-β-farnesene endowed with both insecticidal and repellent activity for aphids.
KEYWORDS: (E)-β-farnesene, aphids, insecticides, odorant-binding proteins, OBP, ligand-binding assay
alarm message by the aphids, while modeling studies could also
help designing new semiochemicals for aphid population
control.14
Based on such working hypothesis, we have designed and
synthesized analogues of (E)-β-farnesene endowed with insecti-
cidal activity and investigated their biological activity, as well as
their affinity to aphid OBPs.
1. INTRODUCTION
Aphids are one of the major pests in agriculture, responsible
for spreading plant virus infections, apart from their direct
damage to the plant. Mildew is nourished by their excreted
honeydew that causes fulvia leaf mold. The great number of
aphid species, their fast breeding and their ability to develop
resistance to chemical insecticides make their population control
challenging. The major component of alarm pheromone, (E)-β-
farnesene (Figure 1, EBF), that is released by most aphid species
when disturbed,1-5 has been shown to be endowed with multiple
biological functions. Recently, this compound has been reported
to be involved in pea aphid (Acyrthosiphon pisum) wing
induction,6 while its insecticidal activity at high doses has also
been demonstrated.7 However, the use of this pheromone in
aphid population control presents some disadvantages. In fact,
(E)-β-farnesene is relatively volatile and unstable in the environ-
ment, due to the easy oxidation of its several double bonds.8
Therefore it is necessary to find analogues that are more stable
and efficient.
Recent studies have indicated that odorant-binding proteins
(OBPs), water-soluble polypeptides that mediate olfactory de-
tection in the sensillar lymph of insects,9,10 are required for
correct functioning of the olfactory system and are responsible for
recognition and discrimination of different olfactory messages.11-13
A recent study on the functional characterization of three OBPs
of the pea aphid A. pisum has suggested that OBP3 could be
specifically involved in the detection of the alarm pheromone
(E)-β-farnesene.14 Under such hypothesis, that however still
needs further support, simple and fast binding assays with OBPs
could predict if a synthetic compound might be perceived as an
2. MATERIALS AND METHODS
2.1. Apparatus and Chemicals. Melting points of compounds
were determined on a Yanagimoto microscope, with an uncorrected
1
thermometer. H NMR spectra were recorded on a Bruker Avance
DPX300 spectrometer. Chemical shifts are reported in δ (ppm) relative
to the signal of tetramethylsilane (TMS) as internal standard, using
deuterochloroform as solvent. Coupling constants are given in Hz.
Elemental analysis was performed with a ST-Carlo Erba Co. elemental
analyzer. High-resolution mass spectra were recorded under electron
impact (150 eV) condition using a Bruker APEX IV instrument. Liquid
chromatography-mass spectrometry (LC/MS) was performed on a
Waters Alliance 2695/ZQ4000 spectrometer. IR spectra were recorded
on neat samples on a Shimadzu IR-435 spectrophotometer using a KBr
pellets. All reagents and solvents were of analytical reagent and when
necessary were purified and dried before use.
2.2. Synthesis of (E)-β-Farnesene Analogues. 2.2.1. General
Synthetic Procedure for Intermediates 2a-2j. Metal sodium (5.0 g,
Received: September 20, 2010
Accepted: January 25, 2011
Revised:
January 24, 2011
Published: February 22, 2011
r
2011 American Chemical Society
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Scheme 1. General Synthetic Routes for the Target
Compounds
Figure 1. Structure of (E)-β-farnesene (EBF), the alarm pheromone
for most aphid species, thiacloprid, the insecticide used as reference in
the mortality experiments, and the general structures of the two classes
of synthetic compounds described in this work and containing a region
structurally similar to EBF and one reproducing part of an insecticide
molecule.
217 mmol) was slowly added to ethanol (125 mL) in a 250 mL round-
bottom flask equipped with a condenser. After the sodium was com-
pletely dissolved, the mixture was heated at 50 °C under stirring for 1 h
and then cooled to 10 °C. A mixture of the appropriate methyl ketone
(1a-1j, 200 mmol) and diethyl oxalate (29.2 g, 200 mmol) was then
added dropwise. The reaction mixture was stirred for 4 h while the
temperature was kept below 10 °C. A solid precipitate formed, which
was filtered and dissolved in 100 mL of iced water. The solution was then
brought to pH 3.0 with 20% sulfuric acid and extracted with dichlor-
omethane (2 ꢀ 50 mL). The organic layer was dried with anhydrous
Na2SO4, and the solvent was removed under reduced pressure to yield
intermediates 2a-2j (see refs 15-20 and Scheme 1) all as sticky oils of
pale pink color. Yields were as follows: 2a or 2b, 22.8 g (72%); 2c or 2d,
26.0 g, (70%); 2e, 2f or 2g, 28.0 g, (69%); 2h, 2i or 2j, 13.96 g (32%).
2.2.2. General Synthetic Procedure for Intermediates 3a-3j. A
solution containing the intermediate 2a-2j (25 mmol) in methanol
(10 mL) was slowly added to a solution of hydrazine (25 mmol) in
methanol at 0 °C (8.0 mL). The mixture was adjusted to neutrality with a
saturated solution of sodium bicarbonate and stirred at 60 °C for 2 h.
After concentration under reduced pressure, the residue was added into
7 mL of 6 M aq NaOH and stirred at 80 °C for 2 h. After cooling to room
temperature, the resulting mixture was poured into 50 mL of iced water
and adjusted to pH 3.0 with concentrated hydrochloric acid. The solid
thus formed was filtered and recrystallized from methanol to give white
to yellow crystals (see refs 16 and 20 and Scheme 1). Yields were as
follows: 3a, 2.6 g (57%); 3b, 3.5 g (70%); 3c, 4.23 g (74%); 3d, 2.84 g
(49%); 3e, 4.13 g (79%); 3f, 1.73 g (30%); 3g, 5.96 g (91%); 3h, 3.16 g
(52%); 3i, 4.47 g (68%); 3j, 5.53 g (73%).
J = 6.66 Hz, CH2-O), 5.09 (t, 1H, J = 1.41 Hz, C=CH), 5.43-5.48
(m, 1H, C=CH), 6.55 (d, 1H, J = 0.68 Hz, pyrazole-H). Anal. Calcd
for C19H30N2O2: C (71.66), H (9.50), N (8.80); found, C (71.75), H
(9.42), N (8.87). IR (KBr pellet press, cm-1): 2972, 2924, 1718,
1671, 1376, 1217, 1011.
Data for 4b: 1.69 g of yellow oil. Yield: 71.5%. 1H NMR (CDCl3):
1.60 (s, 3H, CH3-C), 1.68 (d, 6H, J = 0.85 Hz, (CH3)2-C), 1.99-2.10
(m, 4H, C-CH2-CH2-C), 2.33 (t, 3H, J = 5.58 Hz, CH3-CdN),
4.68 (d, 2H, J = 7.14 Hz, CH2-O), 5.04-5.09 (m, 1H, C=CH), 5.26-
5.31 (m, 1H, C=CH), 6.81 (s, 1H, pyrazole-H), 7.37-7.47 (m, 5H,
ArH). Anal. Calcd for C21H26N2O2: C (74.52), H (7.74), N (8.28);
found, C (74.04), H (7.74), N (8.17). IR (KBr pellet press, cm-1): 2967,
2924, 1729, 1670, 1598, 1502, 1377, 1222, 1029.
Data for 4c: 1.13 g of yellow oil. Yield: 47.6%. 1H NMR (CDCl3):
1.04 (t, 3H, J = 7.33 Hz, CH3-CH2), 1.60 (s, 3H, CH3-C), 1.66 (s, 3H,
CH3-C), 1.67 (s, 9H, (CH3)3-C), 1.70-1.78 (m, 5H, CH3-C,
CH3-CH2), 2.05-2.11 (m, 4H, C-CH2-CH2-C), 2.77 (t, 2H, J =
7.77 Hz, CH2CH2CH3), 4.82 (d, 2H, J=6.99Hz, CH2-O), 5.09 (t, 1H, J=
1.38 Hz, CdCH), 5.46 (d, 1H, J = 1.20 Hz, CdCH), 6.62 (s, 1H, pyrazole-
H). HRMS: calcd for C21H34N2O2 (M þ H), 347.2693; found, 347.2699.
MS m/z (%): 347 (M þ H, 20), 211 (100), 155 (20), 137 (35). IR (KBr
pellet press, cm-1): 2967, 2932, 1714, 1671, 1371, 1215, 1006.
Data for 4d: 1.37 g of yellow oil. Yield: 53.6%. 1H NMR (CDCl3):
0.99 (t, 3H, J = 7.35 Hz, CH3-CH2), 1.60 (s, 3H, CH3-C), 1.67 (d, 6H,
J = 2.09 Hz, (CH3)2-C), 1.71-1.76 (m, 2H, CH3-CH2), 2.06 (m, 4H,
C-CH2-CH2-C), 2.67 (t, 2H, J = 7.60 Hz, CH2CH2CH3), 4.68 (d,
2H, J = 7.11 Hz, CH2-O), 5.07 (s, 1H, CdCH), 5.29 (d, 1H, J = 1.07
Hz, CdCH), 6.83 (s, 1H, pyrazole-H), 7.38-7.43 (m, 5H, ArH).
HRMS: calcd for C23H30N2O2 (M þ H), 367.2380; found, 367.2385.
MS m/z (%): 367 (M þ H, 100), 265 (2), 232 (9), 231 (44). IR (KBr
pellet press, cm-1): 2962, 2930, 1718, 1671, 1598, 1502, 1378,
1223, 1012.
2.2.3. General Preparation for Geranyl Pyrazole-5-formate Deriva-
tives 4a-4j. A mixture containing intermediate 3a-3j (7 mmol) and
thionyl chloride (11.9 g, 100 mmol) was added into a 50 mL flask
equipped with a condenser and refluxed for 8 h. After cooling to room
temperature, the excess thionyl chloride was distilled under reduced
pressure. The residue was refluxed with geraniol (7 mmol) in 20 mL of
acetonitrile for 8 h in the presence of pyridine as an acid scavenger. After
filtration, the solvent was removed under reduced pressure to give crude
products 4a-4j. The products were purified by column chromatogra-
phy on silica gel using ethyl acetate-petroleum (60-90 °C) at a ratio of
1:4-1:7 as the eluent (see Scheme 1).
Data for 4a: 1.6 g of light yellow oil. Yield: 71.9%. 1H NMR
(CDCl3): 1.60 (s, 3H, CH3-C), 1.66 (s, 3H, CH3-C), 1.67 (s, 9H,
(CH3)3-C), 1.74 (d, 3H, J = 1.06 Hz, CH3-C), 2.04-2.11 (m, 4H, C-
CH2-CH2-C), 2.46 (t, 3H, J = 2.43 Hz, CH3-CdN), 4.82 (d, 2H,
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Data for 4e: 2.0 g of light yellow oil. Yield: 58.3%. 1H NMR (CDCl3):
1.28 (d, 6H, J = 6.78 Hz, (CH3)2CH), 1.60 (s, 3H, CH3-C), 1.66-1.68
(m, 12H, (CH3)3C, CH3-C), 1.74 (s, 3H, CH3-C), 2.03-2.12 (m, 4H,
C-CH2-CH2-C), 3.36 (q, 1H, J=6.78Hz, (CH3)2CH), 4.82(d, 2H, J=
7.35 Hz, CH2-O), 5.07-5.11 (m, 1H, CdCH), 5.44-5.49 (m, 1H,
CdCH), 6.67 (s, 1H, pyrazole-H). Anal. Calcd for C21H34N2O2: C
(72.79), H (9.89), N (8.08); found, C (73.02), H (9.91), N (8.00). IR
(KBr pellet press, cm-1): 2976, 2930, 1715, 1671, 1371, 1214, 1007.
Data for 4f: 1.49 g of light yellow oil. Yield: 58.3%. 1H NMR
(CDCl3): 1.18 (d, 6H, J = 6.84 Hz, (CH3)2CH), 1.60 (d, 3H, J = 0.3
Hz, CH3-C), 1.67 (d, 3H, J = 0.93 Hz, CH3-C), 1.75 (d, 3H, J = 0.99
Hz, CH3-C), 2.04-2.11 (m, 4H, C-CH2-CH2-C), 2.99 (t, 1H, J =
6.78 Hz, (CH3)2CH), 4.87 (d, 2H, J = 7.11 Hz, CH2-O), 5.06-5.11
(m, 1H, CdCH), 5.46-5.51 (m, 1H, CdCH), 6.77 (d, 1H, J = 0.45 Hz,
pyrazole-H), 7.40-7.51 (m, 5H, ArH). HRMS: calcd for C23H30N2O2
(M þ H), 367.2380; found, 367.2381. MS m/z (%): 367 (M þ H, 14),
232 (15), 231 (100), 213 (3), 137 (25). IR (KBr pellet press, cm-1):
2968, 2929, 1717, 1670, 1597, 1501, 1381, 1222, 1009.
Data for 4k: 1.876 g of colorless oil. Yield: 85.4%. 1H NMR (CDCl3):
1.33 (d, 9H, J = 1.86 Hz, (CH3)3-C), 1.60 (d, 3H, J = 5.19 Hz, CH3-C),
1.68 (d, 3H, J = 0.90 Hz, CH3-C), 1.76 (d, 3H, J = 0.99 Hz, CH3-C),
2.05-2.13 (m, 4H, C-CH2-CH2-C), 4.83 (d, 2H, J = 7.05 Hz, CH2-
O), 5.07-5.12 (m, 1H, CdCH), 5.44-5.49 (m, 1H, CdCH), 7.42-7.47
(m, 2H, ArH), 7.95-8.00 (m, 2H, ArH). Anal. Calcd for C21H30O2: C
(80.21), H (9.62); found, C (80.01), H (9.60). IR (KBr pellet press, cm-1):
2966, 2927, 1718, 1672, 1610, 1572, 1378, 1272, 1017.
Data for 4l: 1.65 g of colorless oil. Yield: 70.0%. 1H NMR (CDCl3):
1.60 (s, 3H, CH3-C), 1.67 (s, 3H, CH3-C), 1.76 (s, 3H, CH3-C), 2.04-
2.15 (m, 4H, C-CH2-CH2-C), 4.83 (d, 2H, J = 7.08 Hz, CH2-O), 5.08
(d, 1H, J =6.93Hz, CdCH), 5.45 (t, 1H, J= 7.08 Hz, CdCH), 7.58 (t, 2H,
J = 5.28 Hz, ArH), 7.92 (t, 2H, J = 5.33 Hz, ArH). Anal. Calcd for
C17H21BrO2: C (60.54), H (6.28); found, C (60.95), H (6.36). IR (KBr
pellet press, cm-1): 2967, 2922, 1721, 1672, 1591, 1378, 1268, 1012.
Data for 4m: 1.26 g of colorless oil. Yield: 61.2%. 1H NMR
(CDCl3): 1.60 (s, 3H, CH3-C), 1.68 (d, 3H, J = 0.78 Hz, CH3-C),
1.76 (d, 3H, J = 1.02 Hz, CH3-C), 2.04-2.17 (m, 4H, C-CH2-
CH2-C), 4.88 (d, 2H, J = 7.20 Hz, CH2-O), 5.06-5.12 (m, 1H,
CdCH), 5.43-5.49 (m, 1H, CdCH), 6.90-6.98 (m, 2H, ArH), 7.34-
7.44 (m, 2H, ArH). Anal. Calcd for C17H20F2O2: C (69.37), H (6.89);
found, C (69.23), H (6.91). IR (KBr pellet press, cm-1): 2968, 2920,
1735, 1670, 1593, 1470, 1378, 1289, 1014.
2.3. Fluorescence Binding Assays. OBP1, OBP3 and OBP8 of
A. pisum were prepared and purified following the reported
procedures.14 To measure the affinity of the fluorescent probes N-
phenyl-1-naphthylamine (1-NPN) to OBPs, a 2 μM solution of the
protein in 50 mM Tris-HCl was titrated with aliquots of 1 mM ligand in
methanol to final concentrations of 2-16 μM. The probe was excited at
337 nm, and emission spectra were recorded between 380 and 450 nm.
For determining binding constants of 1-NPN, the intensity values
corresponding to the maximum of fluorescence emission (406-409
nm) were plotted against free ligand concentrations. Bound ligand was
then evaluated from the values of fluorescence intensity assuming that
the protein was 100% active, with a stoichiometry of 1:1 protein:ligand at
saturation. The curves were linearized using Scatchard plots. The affinity
of each (E)-β-farnesene analogue was measured in competitive binding
assays, using both 1-NPN and protein at 2 μM concentration and each
analogue as the competitor at 2-16 μM. Dissociation constants of (E)-
β-farnesene analogues were calculated from the corresponding IC50
values, using the equation Ki = [IC50]/(1 þ [1 - NPN]/K1-NPN), with
[1-NPN] being the free concentration of 1-NPN and K1-NPN being the
dissociation constant of the protein/1-NPN complex.
2.4. Molecular Modeling. A three-dimensional model of A. pisum
OBP3 was generated using the online program SWISS MODEL,21-23 using
the crystal structure of the PBP of Leucophaea maderae (PDB:1ORG_A;ref
24) as a template. Amino acid identity between the two proteins is 23%
based on the models was displayed using the SwissPdb Viewer program
“Deep-View”22 (http://www.expasy.org/spdbv/).
2.5. Insecticidal Activity. The insecticidal activity of (E)-β-
farnesene analogues against Aphis gossypii was evaluated using the
following procedure.
The compounds were dissolved in acetone/methanol/water (1:1:18)
at the concentration of 600 μg/mL. Cabbage leaves of about 5 cm
diameter with 3 day old aphids on them were immersed in the solution
for 5 s, air-dried and kept in Petri dishes for 24 h at 24-26 °C, and then
the number of dead aphids was counted. Experiments were performed
three times and the results statistically analyzed.
Data for 4g: 0.98 g, yellow oil. Yield. 35.3%. 1H NMR (CDCl3): 1.23
(d, 6H, J = 6.81 Hz, (CH3)2CH), 1.60 (d, 3H, J = 0.42 Hz, CH3-C), 1.67
(d, 3H, J = 0.96 Hz, CH3-C), 1.76 (d, 3H, J = 1.11 Hz, CH3-C), 2.04-
2.13 (m, 4H, C-CH2-CH2-C), 3.08 (t, 1H, J = 6.77 Hz, (CH3)2CH),
4.89 (d, 2H, J = 6.99 Hz, CH2-O), 5.07-5.11 (m, 1H, CdCH), 5.45-
5.51 (m, 1H, CdCH), 6.83 (d, 1H, J= 0.57 Hz, pyrazole-H), 7.66-7.71(m,
2H, ArH), 8.35-8.40 (m, 2H, ArH). HRMS: calcd for C23H29N3O4 (M þ
H), 412.2230; found, 412.2231. MS m/z (%): 412 (M þ H, 6), 277 (10),
276 (78), 258 (3), 138 (8), 137 (100). IR (KBr pellet press, cm-1): 2969,
2929, 1720, 1671, 1597, 1526, 1501, 1379, 1345, 1224, 1005, 856.
Data for 4h: 1.33 g, yellow oil. Yield. 59.8%. 1H NMR (CDCl3): 1.49
(t, 9H, J = 6.20 Hz, (CH3)3-C), 1.60 (s, 3H, CH3-C), 1.67 (d, 3H, J =
0.87 Hz, CH3-C), 1.75 (d, 3H, J = 0.96 Hz, CH3-C), 2.02-2.12 (m,
4H, C-CH2-CH2-C), 4.85 (d, 2H, J = 7.00 Hz, CH2-O), 5.06-5.11
(m, 1H, CdCH), 5.44-5.49 (m, 1H, CdCH), 6.67 (s, 1H, pyrazole-
H), 7.31-7.42 (m, 5H, ArH). HRMS: calcd for C24H32N2O2 (M þ H),
381.2536; found, 381.2540. MS m/z (%): 381 (M þ H, 10), 245 (100),
187 (90), 137 (50). IR (KBr pellet press, cm-1): 2976, 2930, 1716, 1671,
1606, 1577, 1462, 1397, 1217, 1179, 1004.
1
Data for 4i: 2.10 g of yellow oil. Yield: 75.0%. HNMR(CDCl3):
1.60 (s, 3H, CH3-C), 1.68 (d, 3H, J = 0.84 Hz CH3-C), 1.77 (d, 3H, J
= 0.90 Hz, CH3-C), 2.02-2.13 (m, 4H, C-CH2-CH2-C), 4.92 (d,
2H, J = 6.99 Hz, CH2-O), 5.07-5.11 (m, 1H, CdCH), 5.48-5.53 (m,
1H, CdCH), 7.05 (s, 1H, pyrazole-H), 7.19-7.39 (m, 10H, ArH).
HRMS: calcd for C26H28N2O2 (M þ Na), 423.2043; found, 423.2048.
MS m/z (%): 401 (M þ H, 20), 265 (100), 137 (31). IR (KBr pellet
press, cm-1): 2966, 2922, 1720, 1670, 1597, 1498, 1372, 1222, 1001.
Data for 4j: 0.42 g of yellow crystal. Yield: 13.5%. mp 60-61 °C. 1H
NMR (CDCl3): 1.60 (d, 3H, J = 5.66 Hz, CH3-C), 1.08 (s, 3H, CH3-
C), 1.78 (d, 3H, J = 0.89 Hz, CH3-C), 2.04-2.15 (m, 4H, C-CH2-
CH2-C), 4.93 (d, 2H, J = 7.16 Hz, CH2-O), 5.07-5.12 (m, 1H,
CdCH), 5.48-5.53 (m, 1H, CdCH), 7.06 (s, 1H, pyrazole-H), 7.21-
7.42 (m, 5H, ArH), 7.52-7.56 (m, 2H, ArH), 8.18-8.22 (m, 2H, ArH).
Calcd for C26H27N3O4: C (70.09), H (6.11), N (9.43); found, C
(69.73), H (6.16), N (9.28). IR (KBr pellet press, cm-1): 2966, 2918,
1732, 1660, 1596, 1518, 1495, 1368, 1339, 1221, 1002.
2.2.4. General Preparation for Geranyl Benzoate 4k-4m. A mix-
ture of 3k-3m (7 mmol) and thionyl chloride (11.9 g, 100 mmol) was
refluxed in a 50 mL flask for 8 h. The excess thionyl chloride removed
under reduced pressure. The residue was dissolved in 20 mL of
acetonitrile. Then geraniol (7 mmol) was added batchwise and the
mixture was refluxed for 8 h in the presence of pyridine as an acid
scavenger. After filtration, the solvent was evaporated and the residues
(4k-4m) were purified by column chromatography on silica gel with
ethyl acetate and petroleum (60-90 °C) at a ratio of 1:4-1:10 as the
eluent (see Scheme 1).
3. RESULTS AND DISCUSSION
3.1. Aim of the Work. It is known that the efficacy of an
insecticide increases if insects move around, thus coming in
contact with higher amounts of the chemical than if they remain
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Figure 2. Competitive binding of the compounds prepared to OBP3 of A. pisum. (A, B) Displacement curves of the fluorescent probe 1-NPN from the
complex with OBP3 by each synthetic compound. A solution of 2 μM protein and 2 μM 1-NPN in Tris was titrated with aliquots of 1 μM solution of the
ligand up to the final concentration of 16 μM. Fluorescence intensities are reported as percent of the values in the absence of competitor. Parallel
experiments were also performed with OBP1 and OBP8 (data not shown). (C) Comparison of the affinity of each ligand to the three OBPs used.
Reciprocal of dissociation constants (divided by 106) are reported, as calculated from the concentration values of each ligand halving the initial
fluorescence value. EBF: (E)-β-farnesene. TC: thiacloprid. The structures of all the ligands are reported in Figure 1.
in the same place.25 Aphids usually stay on a leaf for a long time
feeding on the same spot. However, in response to their specific
alarm pheromone, they become very active, eventually abandon-
ing the place.26,27 For most aphid species, the alarm pheromone
is (E)-β-farnesene, either alone or in combination with minor
components.5 We therefore decided to combine the insecticidal
effect and the alarm activity in the same molecule, designing
structural analogues of (E)-β-farnesene, that also contained a
pyrazole moiety based on the structures of several insecticides.28
Accordingly, the synthesized compounds contain a geranyl
group, mimicking the terpene structure of (E)-β-farnesene,
linked to heteroaromatic rings, that have been employed in some
commercial insecticides. In particular, the ethylidene group of
(E)-β-farnesene is replaced by the carbonyl of the ester group,
while the vinyl group is part of the aromatic ring in the analogues.
The reduced number of the double bonds in the designed
analogues should also increase the stability of the parent (E)-β-
farnesene.
problems. The compounds were easily purified and were found
to be stable at room temperature. In particular, we checked the
stability of compounds 4a and 4e by mass spectrometry after
leaving them at room temperature and in contact with air for
periods up to 4 days. In such conditions we could not detect any
degradation products of the compounds examined.
3.3. Binding Studies. A previous study showed that (E)-β-
farnesene and several other compounds bound selectively to A.
pisum OBP3, but bound at lower or undetectable levels to OBP1
and OBP8 of the same species, suggesting that OBP3 may be
involved in the detection of the alarm pheromone. Therefore, we
decided to measure the binding specificity of our new ligands to
these three recombinant A. pisum OBPs, as a first step to study
their olfactory properties with reference to (E)-β-farnesene.
Following the approach previously described,14 we evaluated
the affinity of the ligands to the protein in competitive binding
assays, where the strength of interaction between ligand and
protein is measured by the capacity of the ligand to displace the
fluorescent probe N-phenyl-1-naphthylamine (1-NPN) from its
complex with the protein. The three proteins used, OBP1, OBP3
and OBP8, bind 1-NPN with dissociation constants of 6.9 μM,
5.9 μM and 5.5 μM respectively, as previously reported.14
Figures 2A and 2B show the results of the competitive binding
assays obtained with OBP3. Parallel experiments were performed
with the other two proteins. The calculated binding constants of
the ligands for the three proteins are compared in Figure 2C. We
also included in our binding assay thiacloprid, a commercial
insecticide, that we have used as a positive control when
evaluating the aphicidal activity of our compounds. Thiacloprid
did not show affinity to any of the three OBPs tested. On the
3.2. Preparation and Characterization of the Ligands.
The key intermediates 3a-3j were prepared by the Claisen
condensation reaction (Scheme 1). The target compounds 4a-
4j and 4k-4m were synthesized from geraniol and the corre-
sponding acyl chloride in dry acetonitrile under refluxing with
pyridine as an acid scavenger. Pyridine HCl was found to be the
3
main byproduct, and it was easily removed by column chroma-
tography. The identity of the final compounds was confirmed by
1H NMR, elemental analysis, high-resolution mass spectrometry
and IR. Purity was evaluated by LC/MS.
The syntheses followed well-described routes, starting from
inexpensive commercial chemicals, and did not present particular
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Journal of Agricultural and Food Chemistry
ARTICLE
Figure 4. Aphicidal activity of (E)-β-farnesene analogues. The aphicidal
activity of the synthesized compounds with reference to that of
thiacloprid was assessed using solutions of 0.6 mg/mL, as described in
Materials and Methods. Some derivatives showed insecticidal activity
comparable to or even better than that of thiacloprid. The statistical
analysis was performed with “Statistical Program for Social Sciences”.
allowing an efficient interaction of the aromatic ring with Tyr84
and of the terminal isopropylidene group with Ile43.
Once the role of OBP3 in the detection of (E)-β-farnesene by
the aphids is established, molecular modeling studies could
provide a more detailed understanding of the interactions of
OBP3 with (E)-β-farnesene and its analogues. Such information
would then help in designing new compounds mimicking alarm
pheromones to be used as alteratives to or in combination with
traditional insecticides to control aphid populations in the field.
3.4. Insecticidal Activity. We then evaluated the insecticidal
activity of the synthesized compounds against the aphids in vivo,
using the pesticide standard operation practice (SOP). In such a
test, the solvent, a mixture of acetone, methanol and water
(1:1:18), was used as a negative control, while a commercial
aphicide, thiacloprid (TC), was used as a positive reference. The
results are summarized in Figure 4. Some derivatives, such as 4h,
4i and 4l, showed comparable or even better aphicidal activity
than the commercial insecticide, when applied at the same
concentration of 600 μg/mL.
This work demonstrates that the structure of (E)-β-farnesene
can be modified with the addition of a pyrazole moiety without
losing the affinity of (E)-β-farnesene to aphids OBPs and the
specificity to OBP3, supposed to be responsible for perceiving
the alarm message. On the other hand, the molecules of some
insecticides can be modified by adding a region structurally
similar to (E)-β-farnesene without losing their aphicidal proper-
ties. These results provide guidelines for designing and synthe-
sizing compounds endowed with both repellent properties and
aphicidal activities. Such compounds would represent more
efficient insecticides, as the aphids, stimulated by the alarm
message, will become more active, thus increasing their chances
of coming in contact with the compounds. Therefore, we expect
that in the field the same level of efficacy could be reached with
lower doses of these new insecticides. In the end, such com-
pounds, or others designed following the same strategy, could
prove more environmentally friendly than thiacloprid and other
classical pesticides.
Figure 3. Molecular model of A. pisum OBP3. The model was
generated on line using the Swiss-Model software,21-23 using the PBP
of L. maderae24 as a template. The binding cavity is lined with branched
aliphatic residues (Ile43, Leu48, Leu64, Val104, Leu108), while a
tyrosine (Tyr84) is found at the entrance of the pocket. Binding of
several of the synthesized compounds can be rationalized assuming that
the aromatic part of the ligand establishes p-p bonds with the benzene
ring of Tyr84, while the branched hydrocarbon chain can efficiently
interact with the branched chains of the other amino acids. The distance
between Tyr84 and Ile43 is 9.2 Å, compatible with the length of the
aliphatic chain in the synthesized compounds. N-terminus (Nt) and
C-terminus (Ct) are marked.
contrary, the competition binding curves exhibit an increasing
trend with the concentration of thiacloprid, due to nonspecific
effects. The displacement curve of thiacloprid has not been
included, but only the lack of binding is reported in the bar
graph of Figure 2C.
Some of the chemicals tested exhibited good affinity to all
three proteins, whereas others were more selective. In particular,
4a, 4c and 4e exhibited marked selectivity to OBP3. These three
chemicals all bear aliphatic chains on the pyrazole ring. The size
and branching of such groups does not seem to affect the
selectivity of binding, but the affinity is highest for the derivative
with shortest chains (4a). An additional aromatic ring, on the
contrary, seems to produce compounds with no selectivity and
variable affinities for the three proteins. The presence of electron-
withdrawing groups, such as halogens or a nitro group (as in 4g,
4j, 4l and 4m), increases, as expected, the affinity for the three
proteins, without any selectivity to each of them. It has been
suggested that (E)-β-farnesene might bind to A. pisum OBP3 by
establishing hydrophobic interactions between the terpene
branched chain and the side groups of several hydrophobic
amino acids lining the binding pocket (Ile43, Leu48, Leu64,
Val104 and Leu108), while the two conjugated double bonds
could establish π interactions with the benzene ring of Tyr84,
located at the entrance of the binding cavity. In the model of
OBP3, reported in Figure 3, the positions of these key residues
are indicated. Accordingly, substituting the vinyl group with an
aromatic ring would still allow the same type of interactions
between ligand and protein. We can also observe that the
distance between the benzene ring (at the carbon bearing the
phenolic group) of Tyr84 and the CH of the side chain of Ile43 is
9.2 Å, as measured in the model. This value is very close to the
length of the branched chain in the derivatives here described,
Moreover, the region of the molecule structurally similar to
(E)-β-farnesene results as more stable to oxidation when coupled
to the aromatic moiety, and all the molecules are less volatile than
(E)-β-farnesene. In fact, in our compounds the two terminal
double bonds of (E)-β-farnesene, which greatly contribute to the
instability of this molecule,8 are replaced by a carbonyl group and
an aromatic ring. The synthesized derivatives are also much less
volatile than (E)-β-farnesene, being both larger and more polar.
Therefore, our compounds are likely to remain in the aphids'
environment for longer times than (E)-β-farnesene, thus increas-
ing their efficacy.
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Journal of Agricultural and Food Chemistry
ARTICLE
’ AUTHOR INFORMATION
Design and synthesis of peptidomimetic severe acute respiratory syn-
drome chymotrypsin-like protease Inhibitors. J. Med. Chem. 2005,
48, 6767–6771.
(16) Xiong, Z. H.; Fei, X. N.; Chang, K. New process for the
synthesis of 1-methyl-3-propylpyrazole-5-carboxlic acid. Chin. J. Synth.
Chem. 2001, 9, 553–556.
(17) Yuan, J.; Gulianello, M.; De Lombaert, S.; Brodbeck, R.;
Kieltyka, A.; Hodgetts, K. J. 3-Aryl pyrazolo[4,3-d]pyrimidine deriva-
tives: non-peptide CRF-1 antagonists. Bioorg. Med. Chem. Lett. 2002,
12, 2133–2136.
(18) McClure, K.; Hack, M.; Huang, L. M.; Sehon, C.; Morton, M.;
Li, L. N.; Barrett, T. D.; Shankley, N.; Breitenbucher, J. G. Pyrazole
CCK1 receptor antagonists. Part 1: Solution-phase library synthesis and
determination of Free-Wilson additivity. Bioorg. Med. Chem. Lett. 2006,
16, 72–76.
Corresponding Author
*Tel: þ86-10-62732223. Fax: 86-10-62732223. E-mail:
yangxl@cau.edu.cn, paolo_pelosi@yahoo.it.
Funding Sources
This work was supported by the National Key Project for Basic
Research (2010CB126104), the National Natural Science Foun-
dation of China (31071717) and the Research Fund for the
Doctoral Program of Higher Education of China (20090008110014).
Y. F. Sun is indebted to the University of Pisa for a scholarship
supporting her visit to Pisa in 2009-2010.
(19) Wei, F.; Zhao, B. X.; Huang, B.; Zhang, L.; Sun, C. H.; Dong,
W. L.; Shin, D. S.; Miao, J. Y Design, synthesis, and preliminary
biological evaluation of novel ethyl 1-(20-hydroxy-30-aroxypropyl)-3-
aryl-1H-pyrazole-5-carboxylate. Bioorg. Med. Chem. Lett. 2006,
16, 6342–6347.
’ ACKNOWLEDGMENT
Y. F. Sun is grateful to Immacolata Iovinella of Pisa University
for helping in the biochemistry experiments.
(20) Su, T. D.; Zhang, L. J.; Liu, H. Research on the synthesis of
1-methyl-3-n-propyl-pyrazole-5-carboxylic acid. Appl. Chem. Ind. 2004,
33, 48–50.
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MODEL Workspace: A web-based environment for protein structure
homology modelling. Bioinformatics 2006, 22, 195–201.
(22) Guex, N.; Peitsch, M. C. SWISS-MODEL and the Swiss-
PdbViewer: An environment for comparative protein modelling. Elec-
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