Synthesis and Insecticidal Activity of Spinosyns with C9- O -Benzyl Bioisosteres in Place of the 2′,3′,4′-Tri- O -methyl Rhamnose

Article abstract of DOI:10.1021/acs.jafc.5b01987

The spinosyns are fermentation-derived natural products active against a wide range of insect pests. They are structurally complex, consisting of two sugars (forosamine and rhamnose) coupled to a macrocyclic tetracycle. Removal of the rhamnose sugar results in a >100-fold reduction in insecticidal activity. C9-O-benzyl analogues of spinosyn D were synthesized to determine if the 2′,3′,4′-tri-O-methyl rhamnose moiety could be replaced with a simpler, synthetic bioisostere. Insecticidal activity was evaluated against larvae of Spodoptera exigua (beet armyworm) and Helicoverpa zea (corn earworm). Whereas most analogues were far less active than spinosyn D, a few of the C9-O-benzyl analogues, such as 4-CN, 4-Cl, 2-isopropyl, and 3,5-diOMe, were within 3-15 times the activity of spinosyn D for larvae of S. exigua and H. zea. Thus, although not yet quite as effective, synthetic bioisosteres can substitute for the naturally occurring 2′,3′,4′-tri-O-methyl rhamnose moiety.

Full text of DOI:10.1021/acs.jafc.5b01987

Article
pubs.acs.org/JAFC
Synthesis and Insecticidal Activity of Spinosyns with C9‑O‑Benzyl
Bioisosteres in Place of the 2′,3′,4′-Tri‑O‑methyl Rhamnose
M. Paige Oliver, Gary D. Crouse, David A. Demeter, and Thomas C. Sparks*
Dow AgroSciences, Discovery Research, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States
*
S Supporting Information
ABSTRACT: The spinosyns are fermentation-derived natural products active against a wide range of insect pests. They are
structurally complex, consisting of two sugars (forosamine and rhamnose) coupled to a macrocyclic tetracycle. Removal of the
rhamnose sugar results in a >100-fold reduction in insecticidal activity. C9-O-benzyl analogues of spinosyn D were synthesized to
determine if the 2′,3′,4′-tri-O-methyl rhamnose moiety could be replaced with a simpler, synthetic bioisostere. Insecticidal
activity was evaluated against larvae of Spodoptera exigua (beet armyworm) and Helicoverpa zea (corn earworm). Whereas most
analogues were far less active than spinosyn D, a few of the C9-O-benzyl analogues, such as 4-CN, 4-Cl, 2-isopropyl, and 3,5-
diOMe, were within 3−15 times the activity of spinosyn D for larvae of S. exigua and H. zea. Thus, although not yet quite as
effective, synthetic bioisosteres can substitute for the naturally occurring 2′,3′,4′-tri-O-methyl rhamnose moiety.
KEYWORDS: macrocyclic lactone insecticides, spinosyns, semisynthetic analogues, natural products, rhamnose sugar bioisostere
INTRODUCTION
milbemycin derivative resulted in a new lepidopteran active
■
21
insecticide, lepimectin.
The unique nature of the spinosyns and the appearance of
The global demand for food continues to expand in response to
a rapidly growing world population. As important com-
petitors for food crops, the control of pest insects remains an
1
,2
24
3
resistance in some insect pests have prompted further
synthetic exploration of the spinosyn structure with the goal
of simplifying the spinosyn tetracycle and producing analogues
that potentially circumvent mechanisms of spinosyn resist-
ongoing, essential component to successful food production.
However, pest insect resistance to existing insecticides
4
−6
continues to increase, limiting the utility of many established
classes of chemicals and creating the need for new pest insect
control options. Simultaneously, regulatory requirements and
costs for developing and registering new pesticides continue to
25,26
ance.
Other simplifications of the spinosyn structure
potentially leading to improvement to insecticidal activity
and/or spectrum could arise by replacement of the naturally
occurring sugars with synthetic moieties. As demonstrated by
the research leading to spinetoram, modifications of the
7
,8
rise, making the discovery and development of new insect
control agents a substantial challenge.
In the quest for new insect control agents, natural products
have been and remain an excellent source of novel chemistry
1
3,17
rhamnose sugar can lead to increased insecticidal efficacy.
27
A previous study examined the insecticidal efficacy of
replacing rhamnose with alternative sugars and a few selected
nonsugar moieties. Although some of the alternative sugars
proved to be insecticidal, spinosyn analogues coupled with 9-O-
9
,10
and inspiration for insecticides.
Although very few natural
products are currently in extensive use as pesticides, based on
1
0
global sales, they have served as templates and inspiration for
9
−12
a wide range of fungicides, herbicides, and fungicides.
Among natural product-based insecticides, the spinosyns are a
unique family of fermentation-derived, large, complex macro-
cyclic lactones possessing a novel mode of action.
exemplified by the first spinosyn-based product, spinosad
(
substituted) benzoyl or other nonsugar moieties as rhamnose
replacement bioisosteres were insecticidally inactive against
27
larvae of Heliothis virescens (tobacco budworm). Spinosyn
1
3,14
As
analogues containing a C9-O-(arylalkyl) oxime group (Figure
2
) have also been reported to be insecticidal, although these
(
Figure 1), the spinosyns are highly effective as insect control
1
3,15,16
analogues also contained either a 17-β-D-desosaminyl sugar or a
agents.
Continued exploration of the spinosyns through
2
8
1
7-hydroxy group instead of a forosamine sugar. Because the
modifications of the rhamnose sugar led to semisynthetic
spinosyn derivatives with greater insecticidal efficacy and
spectrum giving rise to a second commercial insecticide,
nonsugar bioisosteres examined were limited in number, the
objective of the present study is to extend the examination of
nonsugar rhamnose bioisosteres to determine whether simple
O-benzyl substitutions (Figure 2) would be more suitable
bioisosteric replacements for the spinosyn rhamnose sugar.
1
6,17
spinetoram
(Figure 1). Similarly, modifications to the sugar
moieties of the avermectins resulted in significant shifts in
spectrum and efficacy (e.g., emamectin benzoate).
1
8,19
Several
studies have investigated the replacement of one or both of the
oleandrose sugars of the avermectins with nonsugar bioisosteres
Received: January 2, 2015
Revised: May 19, 2015
Accepted: May 20, 2015
Published: May 20, 2015
20−23
resulting in insecticidal analogues.
Similarly, the milbemy-
cins are insecticidal−acaricidal non-sugar-containing analogues
1
8
of avermectin. The addition of a nonsugar bioisostere to a
©
2015 American Chemical Society
5571
DOI: 10.1021/acs.jafc.5b01987
J. Agric. Food Chem. 2015, 63, 5571−5577

Journal of Agricultural and Food Chemistry
Article
Figure 2. Spinosyn analogues containing rhamnose replacements at C-
9
9
: (A) 9-O-benzoyl analogues; (B) 9-(O-aralkyl) oxime analogues; (C)
-O-benzyl analogues.
evaporative light scattering detector (Alltech Associates, Inc., Deer-
field, IL, USA) with impactor on and to a ZQ mass spectrometer
detector (Waters Corp., Milford, MA, USA) or by a second LC-MS
(
Waters Corp.) composed of a 2777 autosampler, 1525 μL binary
pumps with 100 μL pump heads, and a 2996 photodiode array
detector linked to a 50/50 splitter going simultaneously to a 2420
evaporative light scattering detector (Alltech Associates, Inc.) and a
ZQ mass spectrometer detector (Waters Corp.). Masses are detected
by electrospray ionization (ESI) on both systems. The main method
consists of a linear gradient from 5 to 95% organic in 5 min. Solvents
are 94.9% H O with 5% acetonitrile (aqueous) and 99.9% acetonitrile
2
(
4
organic) both spiked with 0.1% acetic acid. The column used was a
.6 mm × 50 mm i.d., 5 μm, Sunfire Prep C18 OBD (Waters Corp.).
Reverse phase high-performance liquid chromatography (RP HPLC)
was conducted on a liquid handler 215 (Gilson) with HPLC grade
1
acetonitrile and water (both with 0.1% acetic acid). H NMR
spectroscopy data were collected using chloroform-d solvent with
tetramethylsilane as internal standard.
Preparation of 3,4,5-Trimethoxybenzyl Bromide. In a 1 dram
vial equipped with a magnetic stir bar were added 0.198 g of 3,4,5-
trimethoxybenzyl alcohol (1.00 mmol), 1.5 mL of 9:1 dichloro-
methane/diethyl ether, and hydrogen bromide (40% aqueous
solution). The reaction mixture was stirred for 4.5 h. The reaction
mixture was poured into a 25 mL vial containing water and ice. This
mixture was extracted three times with ethyl acetate, dried over
magnesium sulfate, filtered, concentrated under reduced pressure and
low heat, and then stored in the freezer.
Figure 1. Structures of spinosad, spinetoram, spinosyns: (A) spinosad
(
primary component spinosyn A (R = H), minor component spinosyn
D (R = Me)); (B) spinetoram (primary component 5,6-dihydro-3′-O-
ethyl spinosyn J (R = H, 5,6 single bond), minor component 3′-O-
ethyl spinosyn L (R = Me, 5,6-double bond)); (C) general structure of
the spinosyns (R = H or Me, boxes highlight the two sugar moieties).
Preparation of 9-O-Benzyl Spinosyn D Analogues. Method A:
Preparation of 9-O-(3,4,5-Tri-O-methylbenzyl)spinosyn D, 6q. In a 1
dram vial equipped with a magnetic stir bar were added 0.144 g of
3
,4,5-trimethoxybenzyl bromide (0.55 mmol), 0.10 g (0.18 mmol) of
MATERIALS AND METHODS
■
spinosyn L C-9 pseudoaglycone, 0.150 g of powdered 10:1 potassium
hydroxide/tertrabutylammonium hydride (excess), and 3.0 mL of
dichloromethane. The reaction mixture was stirred overnight at room
temperature. The reaction mixture was diluted with ether and then
passed through a 10 mL Celite cartridge. The eluent was concentrated
under vacuum. Purification using a Redi Sep 10 g flash column (Isco,
Inc., Lincoln, NE, USA), eluting with ethyl acetate/hexanes/
dichloromethane/methanol (10:10:10:1, v/v/v/v), yielded 0.066 g
(50% yield) of 6q as a solid foam.
Method B: Preparation of 9-O-(2-Isopropylbenzyl)spinosyn D,
6d. In a 1 dram vial equipped with a magnetic stir bar were added
0.050 g of 1-(bromomethyl)-2-isopropylbenzene (0.21 mmol), 0.020 g
(0.036 mmol) of spinosyn L C-9 pseudoaglycone, 0.10 g of powdered
10:1 potassium hydroxide/tertrabutylammonium hydride (excess),
and 1.5 mL of dichloromethane. The reaction mixture was stirred
overnight at room temperature. The supernatant solution was then
Synthesis of Spinosyn Analogues. Unless otherwise indicated,
all commercial reagents were used without purification. Solvents were
of reagent grade. Benzyl alcohol precursors were available
commercially or were prepared using published routes. Purity was
1
established as >95% by H NMR spectroscopy and LC-MS. Mass
spectral data were obtained by electron ionization on one of several
systems including a series 1100 mass selective detector (MSD)
Hewlett-Packard, Palo Alto, CA, USA) or a 5890 series II (Hewlett-
Packard) with a 5890A mass spectrometer (Hewlett-Packard) or a
890 series GC system with a 5973 MSD (Hewlett-Packard). Mass
spectral data were also obtained by LC-MS analysis using a 215 liquid
handler (Gilson, Middleton, WI, USA) for injection coupled to a 1100
chromatography system (Agilent Technologies, Santa Clara, CA,
USA) composed of a quaternary pump system and photodiode array
detector linked to a 50/50 splitter going simultaneously to a 2000
(
6
5
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DOI: 10.1021/acs.jafc.5b01987
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Journal of Agricultural and Food Chemistry
Article
Figure 3. Synthesis of spinosyn D C9 pseudoaglycone and spinosyn D analogues with rhamnose replacements.
added directly to the top of a Redi Sep 2.5 g flash column (Isco, Inc.)
and eluted, first with 25 mL of dichloromethane, then with 25 mL of
spraying both leaf surfaces until runoff. There were four replicates
(plants) for each treatment, whereas controls consisted of eight
replicates treated with a solvent blank. Following treatment, plants
were held at 23 °C and 40% RH with a 24 h photoperiod prior to
grading. Grading occurred at 72 h post-treatment and consisted of live
aphid counts (all nonwinged stages) on each of the replicates
compared to the averaged aphid population on the solvent blank
controls to estimate a percent mortality.
1% methanol in dichloromethane, and finally with 25 mL of 2%
methanol in dichloromethane, which resulted in elution of the desired
9-O-benzyl product. Removal of solvent furnished 0.0205 g (79%) of
6d as a solid foam.
Molecular Modeling Overlays. The spinosyn D and the benzyl
and benzoyl structures were built by hand in Sybyl-X 2.1 (Certata,
Princeton, NJ, USA) starting from the minimized X-ray structure of
spinosyn A. The structures were then minimized and flexibly fit to the
minimized X-ray structure of spinosyn A. All of the molecules were
RESULTS AND DISCUSSION
■
2
9
initially geometry optimized using the Tripos force field and
Synthesis. Rhamnose replacement analogues were made by
first deglycosylating spinosyns J, 1, and L, 2 (Figure 3), to form
the pseudoaglycones 3 and 4, respectively, as described
30,31
Gasteiger−Huckel charges
(Tripos Associates, St. Louis, MO,
USA). The shape-based flexible fit of spinosyn D and the benzyl and
benzoyl structures was done using Surflex-Sim v2.706 distributed with
Sybyl-X 2.1. The top-scoring conformer of each compound from
Surflex-Sim was used in the overlay.
34
previously. A free C-9 hydroxyl group was then available to
prepare benzylated or benzoylated derivatives. Twenty-eight
benzyl bromides were selected on the basis of diverse steric and
electronic properties. Some of these were available commer-
cially, with the remainder prepared as shown in Figure 3.
Commercially available benzyl alcohols were converted into the
bromides using hydrogen bromide in dichloromethane/ether.
When the benzyl alcohols themselves were unavailable, they
were prepared either from the corresponding aldehyde, for 6x,
or acid, for 6l, by reduction with sodium borohydride or lithium
Insecticide Bioassays. Dose−response data for each compound
against two susceptible laboratory strains of the lepidopteran species
Spodoptera exigua or Helicoverpa zea was determined with a diet-based
bioassay using 128-well diet trays (Bio-Serv, Frenchtown, NJ, USA).
Three to five second-instar larvae of either lepidopteran were placed in
each well (3 mL) of the diet tray that had been previously filled with 1
mL of artificial diet. Selected dosages (12.5, 3.125, 0.78, 0.195, 0.049
2
μg/cm ) of the test compound (dissolved in 50 μL of 90:10 acetone/
water mixture, v/v) were then applied to the diet in each of 16 wells,
allowed to dry, and covered with a clear self-adhesive cover. Controls
received solvent only. All treatments were held at 25 °C and 14:10 h
light/dark for 6 days. Each well was recorded as a unit (all alive or all
dead), the activity across the 16 wells (for each dose) was then
averaged. The lethal concentration for 50% of the test population
aluminum hydride (LiAlH ), respectively. The substituted aryl
4
methyl bromides were then reacted with 3 or 4, using individual
1
dram vials with powdered potassium hydroxide/tetra-n-
butylammonium iodide as the basic catalyst to produce the
desired targets 5 and 6 (Figure 3). In addition to exhibiting the
expected mass parent ion by LC-MS, the 9-O-benzyl products
(
LC ) and 95% fiducial limits for each compound, with correction for
50
32
mortality in the controls, were determined using probit analysis.
Bioassays against a susceptible laboratory strain of Aphis gossypii
(
were also characterized by the presence of a −OCH − signal in
2
the proton NMR between 4.4 and 4.56 ppm, in addition to the
expected aromatic signals. This methylene peak was usually a
singlet; however, due to its diastereotopic nature, it was
sometimes observed as a doublet of doublets. Isolated yields
were usually in the range of 50−85%, except when highly
electron-rich benzyl bromides such as 2,4-dimethoxy, 2-
methoxy, and 4-methoxy were used; in these cases,
quaternization of the tertiary amine predominated under the
conditions used for this reaction. Structures and selected
3
3
cotton aphid) were conducted as described previously. Briefly, 1-
week-old summer crookneck squash seedlings, Cucurbita pepo, were
pruned to a single cotyledon. A mixed population (immatures and
adults) of A. gossypii was transferred to the cotyledons 16−24 h prior
to the application of test materials. Test compounds were dissolved in
90:10 acetone/ethanol, v/v, to form a stock solution, from which an
appropriate amount was diluted in water containing 0.05% Tween 20
to form the spray solution of 50 and 200 ppm. Application was made
using a hand-held DeVilbiss airbrush sprayer on aphid-infested plants
5
573
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Journal of Agricultural and Food Chemistry
Article
physical property data of all final targets are provided in Table
Molecular Modeling Overlay. The 3-methoxybenzoyl, 6a,
and 3-methoxybenzyl, 6b, derivatives of spinosyn D were
overlaid on the structure of spinosyn D. As shown in Figure 4,
1.
Table 1. Structure of 9-O-Benzyl or -O-Benzoyl Spinosyn
Analogues
MS (ESI)
analogue
linker
phenyl substitution
3-OCH3
[M + H]+
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
a
b
c
d
e
f
−CO−
678
664
692
678
648
690
722
676
676
736
712
691
696
676
732
706
746
708
738
682
673
662
716
690
724
−CH − 3-OCH3
2
−CO−
3-OCH3
−CH − 3-OCH3
2
−CH − unsubstituted
2
−CH − 2-iPr
2
Figure 4. Overlays of C9 3-O-methylbenzyl, 6a (green), and 3-O-
methylbenzoyl, 6b (light gray), spinosyn D derivatives, with spinosyn
D (orange): (A) side view; (B) end-on view with the C9-benzyl and
benzoyl bioisosteres in the foreground.
−CH − 2-OCH CH OCH
2
2
2
3
−CH − 2,3-(CH3)2
2
g
h
i
−CH − 2,4-(CH3)
2
2
-CH2-
2-OCH , 4-CO CH
3 2 3
−CH − 2-OCH , 4-Cl
2
3
the benzyl derivative, 6b, fits into the space of the rhamnose
sugar better than the benzoyl derivative, 6a. The better fit of the
benzyl derivatives into the space of the rhamnose (Figure 4)
may partially explain their improved insecticidal activity
compared to the corresponding benzoyl derivatives (Table 2).
The above results prompted a more comprehensive
evaluation of C9-O-benzyl analogues. For this study, spinosyn
D analogues were prepared due to the availability of gram
quantities of the corresponding 9-OH precursor. As noted in
Table 2, the relative potencies of A and D analogues are similar.
Although the C9-O-benzyl analogues displayed a wide range of
insecticidal activity (Table 3), all of the analogues were less
potent than spinosyn D. A few of the substitutions exhibited
insecticidal activity against S. exigua that was within 5−15 times
that of spinosyn D, whereas most were 20−200-fold less active
than spinosyn D. The 2-isopropyl analogue, 6d, was the most
potent against S. exigua followed closely by the 3-O-methyl-
ethoxy, 6n, 4-methyl, 6t, 3,5-dimethoxy, 6p, and benzofuran-2-
yl, 6x, analogues. For larvae of H. zea, the most active
substitution was only about 3-fold less active than spinosyn D,
with the majority being 20− 100-fold less active against H. zea
than spinosyn D. The most effective substitution against larvae
of H. zea was the 2-isopropyl, 6d, followed closely by the 3,5-
dimethoxy, 6p, 4-Cl, 6r, and 4-CN, 6s (Table 3). All of the C9-
O-benzyl analogues were also evaluated against A. gossypii.
However, no insecticidal activity was observed against A.
gossypii for any of the analogues at either the 50 or 200 ppm
dose (data not shown).
j
−CH − 2-F, 4-CN
2
k
l
−CH − 2-Cl, 6-CH3
2
−CH − 2,6-(CH3)2
2
m
n
o
p
q
r
−CH − 3-OCF3
2
−CH − 3-CH OC H
2
2
2
5
−CH − 3-CF , 4-OCH
3
2
3
−CH − 3,5-(OCH3)2
2
−CH − 3,4,5-(OCH3)3
2
−CH − 4-Cl
2
s
−CH − 4-CN
2
t
−CH − 4-CH3
2
u
v
w
−CH − 4-CF3
2
−CH − 4-iPr
2
−CH − 6-fluoro-4H-benzo[1,3]dioxin-8-
2
yl
6
x
−CH − benzofuran-2-yl
688
2
Insecticidal Activity. Earlier work using C9-benzoyl
derivatives had evaluated 2-, 3-, and 4-methoxy benzoyl
analogues, as well as a C9-O-methyl. Because of the high
reactivity of o- and p-methoxy-substituted benzyl bromides,
which led to the formation of quaternary salts under our
reaction conditions, the initial study focused on 3-methoxy
analogues of spinosyns A and D, 5b and 6b, respectively (Table
2
7
2
). These compounds were evaluated for insecticidal efficacy
against two lepidopteran species, S. exigua and H. zea. As shown
in Table 2, the C9-O-(3-methoxybenzyl) derivatives of
spinosyns A and D, 5b and 6b, were more active than the
corresponding C9 pseudoaglycones or the respective C9-O-
benzoyl derivatives, 5a and 6a. However, neither 5b nor 6b was
as potent as spinosyn A or D (Table 2).
Modifications of the spinosyn structure can greatly alter the
insecticidal activity with significant improvements possible over
1
3,17,35,36
the naturally occurring spinosyns.
As such, there has
Table 2. Insecticidal Activity of 9-O-Benzyl and 9-O-Benzoyl Spinosyns
2
LC , μg/cm (95% FL)
50
compound
C9-O substitution
S. exigua
H. zea
spinosyn A
(2′,3′,4′-(CH O) β-D-rhamnosyl)
0.20 (0.11−0.36)
0.044 (0.029−0.049)
>12.5
0.20 (0.18−0.21)
0.11 (0.09−0.14)
>12.5
3
3
spinosyn D
(2′,3′,4′-(CH O) β-D-rhamnosyl)
3
3
spinA C9-PsA
spinD C9-PsA
H
H
7.9 (6.1−12.2)
4.8 (3.7−9.4)
2.0 (1.0−2.7)
12.5 (8.6−107)
6.3 (3.6−10.8)
4.2 (3.1−8.1)
5a
5b
6a
6b
3-methoxybenzoyl A
3-methoxybenzyl A
3-methoxybenzoyl D
3-methoxybenzyl D
>12.5
>12.5
1.4 (0.97−2.0)
5.3 (3.9−10.0)
5
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Article
Table 3. Insecticidal Activity of 9-O-Benzyl Spinosyns
2
LC , μg/cm (95% FL)
50
compound
C9- phenyl substitution
3-OCH3
S. exigua
H. zea
spinosyn D
0.044 (0.029−0.049)
1.4 (0.97−2.0)
1.6 (1.1−2.3)
0.2 (0.0003−0.4)
1.7 (1.2−2.4)
1.3 (0.9−1.9)
1.6 (0.9−1.7)
3.7 (2.7−7.1)
0.9 (0.5−1.5)
0.9 (0.6−2.8)
8.5 (4.3−10.7)
0.9 (0.5−1.5)
13.0 (9.4−20.7)
0.3 (0.01−1.2)
1.2 (0.8−1.8)
0.5 (0.3−0.7)
8.2 (5.5−12.4)
0.7 (0.5−1.1)
0.7 (0.5−1.2)
0.4 (0.3−0.6)
0.8 (0.4−1.7)
5.3 (2.7−8.0)
0.6 (0.3−0.8)
0.4 (0.2−12.2)
1.4 (0.7−1.7)
0.11 (0.09−0.14)
5.3 (3.9−10.0)
1.8 (1.2−2.6)
0.3 (0.2−0.6)
10.7 (5.8−16.3)
8.2 (8.6−11.2)
3.9 (2.8−7.6)
5.9 (2.09−851)
0.9 (0.5−1.5)
1.6 (0.6−3.9)
6.3 (4.5−9.3)
1.2 (0.7−3.4)
9.6 (5.1−12.2)
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
b
c
d
e
f
unsubstituted
2-isopropyl
2-OCH CH OCH
2
2
3
2,3-(CH3)2
g
h
i
2,4-(CH3)2
2-OCH , 4-CO CH
3
2
3
2-OCH , 4-Cl
3
j
2-F, 4-CN
2-Cl, 6-CH3
2,6-(CH3)2
3-OCF3
k
l
m
n
o
p
q
r
3-OCH OC H
5
∼17
2
2
3-CF , 4-OCH
1.4 (0.8−2.1)
0.5 (0.4−0.7)
6.3 (3.6−12.8)
0.6 (0.4−0.9)
0.6 (0.4−0.8)
4.3 (2.3−10.0)
2.4 (0.8−3.9)
11.1 (7.2−15.5)
3.9 (1.8−10.3)
2.0 (1.4−2.9)
1.4 (1.0−2.0)
3
3
3,5-(OCH3)2
3,4,5-(OCH3)3
4-Cl
s
4-CN
t
4-CH3
u
v
w
x
y
4-CF3
4-isopropyl
6-fluoro-4H-benzo[1,3]dioxin-8-yl
benzofuran-2-yl
7-(OCH )-2-naphthyl
3
2
7,28,33,34,37−41
been interest in exploring further alterations
and
of the C9-O-benzyl analogues exhibited any activity against this
aphid at the highest dose tested (200 ppm). Thus, the
rhamnose bioisosteres were unable to improve on the activity
of the spinosyns toward aphids relative to spinosyn D.
Although none of the rhamnose bioisostere-based spinosyns
in the present study were as insecticidally active as spinosyn D,
several analogues were within 3−15 times, depending on the
insect species. Thus, the analogues exhibiting insecticidal
activity close to that of spinosyn D demonstrate that an O-
benzyl moiety can be a reasonable bioisostere for the 2′,3′,4′-
tri-O-methyl rhamnose. Therefore, as demonstrated by the
present and previous studies, the replacement of the sugars on
macrolides such as the avermectins, and potentially the
spinosyns, with nonsugar bioisoteres is further demonstrated
to be a viable approach to developing new insecticidal
chemistries.
especially simplification of some components or the core
macrolides tetracycle as a potential means for further
exploration of these novel, complex, macrolides.
1
3,38,41,42
As
noted in prior studies, deviation from the 2′,3′,4′-tri-O-methyl-
β-L-rhamnose, whether by removing it, altering the stereo-
chemistry, removing one or more O-alkyl groups, or replacing it
1
3,27,35,36
with D- sugars, furanose sugars, or nonsugars,
all result
in a large reduction (usually >100-fold) in insecticidal activity.
Interestingly, the C9-O-benzyl ethers evaluated in the present
study retained a significant level of insecticidal potency,
demonstrating that, at least to some degree, a substituted
benzylic group is better able to mimic the size and placement of
the tri-O-rhamnosyl group. However, efforts to more closely
mimic the shape or electronics of the rhamnose through
incorporation of methoxy substituents met with limited success.
For example, although the 3,5-dimethoxybenzyl substitution,
6
p, was among the most potent analogues against S. exigua
larvae, several other simple substitutions, including 4-CN, 6s, 4-
Me, 6t, 2-isopropyl, 6d, 4-Cl, 6r, and 3-methoxyethoxy, 6n,
were equally or more active. Two of the larger moieties, the
benzofuran-2-yl, 6x, and 6-fluoro-4H-benzo[1,3]dioxin-8-yl,
ASSOCIATED CONTENT
■
*
S
Supporting Information
Detailed information on the synthesis of the new spinosyn
analogues in this study and supporting NMR data. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jafc.5b01987.
6
w, were also insecticidal against S. exigua larvae (Table 3),
suggesting that to some degree larger moieties can be tolerated
as replacements for the 2′,3′,4′-tri-O-methyl rhamnose. In
general, the analogues showing the greatest potency against S.
exigua were also among the most active against H. zea larvae,
with the exception of the larger analogues, 6x and 6w.
In contrast to their excellent lepidopteran insecticidal activity,
the spinosyns typically are weak against many sap-feeding
insects such as aphids.
AUTHOR INFORMATION
■
*
Corresponding Author
(T.C.S.) Phone: (317) 337-3064. Fax: (317) 337-3205. E-
mail: tcsparks@dow.com.
1
3,16
For example, spinosyn D is only
Notes
weakly active (LC = 50 ppm) against A. gossypii compared to
commercial aphicides such as imidacloprid (0.06 ppm). None
5
0
14
The authors declare no competing financial interest.
5
575
DOI: 10.1021/acs.jafc.5b01987
J. Agric. Food Chem. 2015, 63, 5571−5577

Journal of Agricultural and Food Chemistry
Article
discovery of spinetoram. J. Comput.-Aided Mol. Des. 2008, 22, 393−
ACKNOWLEDGMENTS
■
4
01.
We thank Cathy Young and James Gifford (Dow Agro-
Sciences) for assistance with the bioassays, Ricky Hunter (Dow
AgroSciences) for the insect photograph, and the journal’s
anonymous reviewers for their very helpful suggestions.
(18) Kornis, G. I. Avermectins and milbemycins. In Agrochemicals
from Natural Products; Godfery, C. R. A., Ed.; Dekker: New York,
1995; pp 215−255.
(19) Fischer, M. H. Structure-activity relationships of the avermectins
and milbemycins. In Phytochemicals for Pest Control; Hedin, P. A.,
Hollingworth, R. M., Masler, E. P., Miyamoto, J., Thompson, D. G.,
Eds.; American Chemical Society: Washington, DC, USA, 1997; pp
220−238.
(20) Mrozik, H.; Linn, B. O.; Eakola, P.; Lusi, A.; Matzuk, A.; Preiser,
F. A.; Ostlind, D. A.; Schaeffer, J. M.; Fisher, M. H. Synthesis and
biological activities of 13-substituted avermectin aglycones. J. Med.
Chem. 1989, 32, 375−381.
(21) Pitterna, T. Chloride channel activators/new natural products:
avermectins and milbemycins. In Modern Crop Protection Compounds,
2nd ed.; Kramer, W., Schirmer, U., Jeschke, P., Witschel, M., Eds.;
Wiley-VCH: New York, 2012; Vol. 3, pp 1305−1326, DOI: 10.1002/
9783527644179.ch32.
ABBREVIATIONS USED
■
FL, fiducial limits; RH, relative humidity; TBAI, tetra-n-
butylammonium iodide
REFERENCES
■
(
1) Godfray, H. C. J.; Beddington, J. R.; Crute, I. R.; Haddad, L.;
Lawrence, D.; Muir, J. F.; Pretty, J.; Robinson, S.; Thomas, S. M.;
Toulmin, C. Food security: the challenge of feeding 9 billion people.
Science 2010, 327, 812−818.
(
2) Tilman, D.; Balzer, C.; Hill, J.; Befort, B. L. Global food demand
and the sustainable intensification of agriculture. Proc. Natl. Acad. Sci.
U. S. A. 2011, 108, 20260−20264.
(22) Zhao, J.-H.; Xu, X.-J.; Ji, M.-H.; Cheng, J.-L.; Zhu, G.-N. Design,
synthesis, and biological activities of milbemycin analogues. J. Agric.
Food Chem. 2011, 59, 4836−4850.
(
3) Popp, J.; Peto, K.; Nagy, J. Pesticide productivity and food
security, a review. Agron. Sustainable Dev. 2013, 33, 243−255.
(23) Xu, X.; Lai, S.; Ji, M.; Zhu, G.; Zhao, J. Synthesis,
characterization and insecticidal activity of milbemycin analogues.
Youji Huaxue 2012, 32, 1084−1092.
(
4) Whalon, M. E.; Mota-Sanchez, D.; Hollingworth, R. M. Analysis
of global pesticide resistance in arthropods. In Global Pesticide
Resistance in Arthropods; Whalon, M. E., Mota-Sanchez, D.,
Hollingworth, R. M., Eds.; CAB International: Cambridge, MA,
USA, 2008; pp 5−31.
(24) Sparks, T. C.; Dripps, J. E.; Watson, G. B.; Paroonagian, D.
Resistance and cross-resistance to the spinosyns − a review and
analysis. Pestic. Biochem. Physiol. 2012, 102, 1−10.
(25) Tietze, L. F.; Schutzenmeister, N.; Grube, A.; Scheffer, T.; Baag,
M. M.; Granitzka, M.; Stalke, D. Synthesis of spinosyn analogues for
modern crop protection. Eur. J. Org. Chem. 2012, 29, 5748−5756.
(26) Tietze, L. F.; Sietz, S.; Schutzenmeister, N.; Biller, S.; Hierold, J.;
Scheffer, T.; Baag, M. M. Selective glycosylation with the amino sugar
D-forosamine for synthesis of spinosyns and its analogues. Eur. J. Org.
Chem. 2013, 2013, 7305−7312.
(27) Anzeveno, P. B.; Green, F. R., III. Rhamnose replacement
analogs of spinosyn A. In Synthesis and Chemistry of Agrochemicals VI;
Baker, D. R., Fenyes, J. G., Lahm, G. P., Selby, T. P., Stevenson, T. M.,
Eds.; American Chemical Society: Washington, DC, USA, 2002; pp
262−276.
(28) Jeschke, P.; Velten, R.; Ebbinghaus-Kintscher, U.; Beck, M. E.;
Losel, P.; Malsam, O.; Eberz, G.; Mohrle, V.; Frode, R. Synthesis of 9-
keto-spinosyn derivatives for use in agriculture as pesticides. Bayer
CropScience, Germany, Patent, DE 2003-10301519, Jan 17, 2003; 85
pp.
(
5) Tabashnik, B. E.; Mota-Sanchez, D.; Whalon, M. E.;
Hollingworth, R. M.; Carriere, Y. Defining terms for proactive
management of resistance to Bt crops and pesticides. J. Econ. Entomol.
2
(
014, 107, 496−507.
6) Sparks, T. C.; Nauen, R. IRAC: Mode of action classification and
insecticide resistance management. Pestic. Biochem. Physiol. 2015, 121,
22−128.
7) Lamberth, C.; Jeanmart, S.; Luksch, T.; Plant, A. Current
challenges and trends in the discovery of agrochemicals. Science 2013,
41, 742−746.
8) Sparks, T. C. Insecticide discovery: an evaluation and analysis.
Pestic. Biochem. Physiol. 2013, 107, 8−17.
9) Cantrell, C. L.; Dayan, F. E.; Duke, S. O. Natural products as
sources for new pesticides. J. Nat. Prod. 2012, 75, 1231−1242.
10) Gerwick, B. C.; Sparks, T. C. Natural products for pest control:
an analysis of their role, value and future. Pest Manage. Sci. 2014, 70,
169−1185.
11) Godfrey, C. R. A., Ed. Agrochemicals from Natural Products;
Dekker: New York, 1995.
12) Rimando, A. M.; Duke, S. O. Natural products for pest
1
(
3
(
(
(
1
(
(29) Clark, M.; Cramer, R. D., III; von Opdenbosch, N. Validation of
the general purpose Tripos 5.2 force field. J. Comput. Chem. 1989, 10,
982−1012.
(
management. In Natural Products for Pest Management; American
Chemical Society: Washington, DC, USA, 2006; pp 2−21.
(30) Gasteiger, J.; Marsili, M. Iterative partial equalization of orbital
electronegativity: a rapid access to atomic charges. Tetrahedron 1980,
36, 3219−3222.
(
13) Salgado, V. L.; Sparks, T. C. The spinosyns: chemistry,
biochemistry, mode of action, and resistance. In Insect Control:
Biological and Synthetic Agents; Gilbert, L. I., Gill, S. S., Eds.; Academic
Press: New York, 2010; pp 207−243.
(31) Purcel, W. P.; Singer, J. A. A brief review and table of
̈
semiempirical parameters used in the Huckel molecular orbital
method. J. Chem. Eng. Data 1967, 12, 235−246.
(
14) Geng, C.; Watson, G. B.; Sparks, T. C. Nicotinic acetylcholine
(32) Finney, D. J. Probit Analysis, 3rd ed; Cambridge University
Press: Cambridge, UK, 1971.
receptors as spinosyn targets for insect pest management. In Target
Receptors in the Control of Insect Pests: Part I, Advances in Insect
Physiology; Cohen, E., Ed.; Academic Press: New York, 2013; Vol. 44,
pp 101−210.
(33) Sheehan, L. S.; Lill, R. E.; Wilkinson, B.; Sheridan, R. M.;
Vousden, W. A.; Kaja, A. L.; Crouse, G. D.; Gifford, J.; Graupner, P.;
Karr, L.; Lewer, L.; Sparks, T. C.; Leadlay, P. F.; Waldron, C.; Martin,
C. J. Engineering of the spinosyn PKS: directing starter unit
incorporation. J. Nat. Prod. 2006, 69, 1702−1710.
(34) Creemer, L. C.; Kirst, H. A.; Paschal, J. W. Conversion of
spinosyn A and spinosyn D to their respective 9- and 17-
pseudoaglycones and their aglycones. J. Antibiot. 1998, 51, 795−800.
(35) Crouse, G. D.; Sparks, T. C.; Schoonover, J.; Gifford, J. M.;
Bruce, T.; Worden, T. V.; Martynow, J. G. Recent advances in the
chemistry of the spinosyns. Pest Manage. Sci. 2001, 57, 177−185.
(36) Sparks, T. C.; Crouse, G. D.; Durst, G. Natural products as
insecticides: the biology, biochemistry and quantitative structure
(
15) Thompson, G. D.; Dutton, R.; Sparks, T. C. Spinosad − a case
study: an example from a natural products discovery program. Pest
Manage. Sci. 2000, 56, 696−702.
(
16) Dripps, J. E.; Boucher, R. E.; Chloridis, A.; Cleveland, C. B.;
DeAmicis, C. V.; Gomez, L. E.; Paroonagian, D. L.; Pavan, L. A.;
Sparks, T. C.; Watson, G. B. The spinosyn insecticides. In Green
Trends in Insect Control; Lopez, O., Fernadez-Bolanos, J. G., Eds.; RSC
Publishing: Cambridge, UK, 2011; pp 163−212.
(
17) Sparks, T. C.; Crouse, G. D.; Dripps, J. E.; Anzeveno, P.;
Martynow, J.; Gifford, J. Artificial neural network-based QSAR and the
5
576
DOI: 10.1021/acs.jafc.5b01987
J. Agric. Food Chem. 2015, 63, 5571−5577

Journal of Agricultural and Food Chemistry
Article
activity relationships of spinosyns and spinosoids. Pest Manage. Sci.
2
(
001, 57, 896−905.
37) Crouse, G. D.; Sparks, T. C.; DeAmicis, C. V.; Kirst, H. A.;
Martynow, J. G.; Creemer, L. L.; Worden, T. V.; Anzeveno, P. B.
Chemistry and insecticidal activity of the spinosyns. In Pesticide
Chemistry and Bioscience: The Food-Environment Challenge; Brook, G.
T., Roberts, T. R., Eds.; Royal Society of Chemistry: Cambridge, UK,
1
(
999; pp155−166.
38) Kirst, H. A.; Creemer, L. C.; Broughton, M. C; Huber, M. L. B.;
Turner, J. R. Chemical and microbial modifications of spinosyn:
exploring synergies between fermentation microbiology and organic
chemistry. In Synthesis and Chemistry of Agrochemicals VI; Baker, D. R.,
Fenyes, J. G., Lahm, G. P., Selby, T. P., Stevenson, T. M., Eds.;
American Chemical Society: Washington, DC, USA, 2002; pp 251−
261.
(
39) Daeuble, J.; Sparks, T. C.; Johnson, P.; Graupner, P. R.
Modification of the butenyl-spinosyns utilizing cross-metathesis.
Bioorg. Med. Chem. 2009, 17, 4197−4205.
(
40) Chen, X.; Li, L.; Zhang, X.; Zhao, L. Chemical synthesis of
spinosad derivatives. Wuhan University of Light Industry, People;s
Republic of China, Patent CN 2013-10613847, Nov 26, 2013; 10 pp.
(
41) Shi, D.; Feng, X.; Zhung, X.; Chai, H.; Liu, T.; Zhang, Q.; Li, J.
Study of synthesis and biological activities of novel macrolides
derivatives. Youji Huaxue 2014, 34, 2543−2550.
(
42) Tietze, L. F.; Brasche, G.; Grube, A.; Boknke, N.; Stradler, C.
Synthesis of novel spinosyn analogues by Pd-mediated trans-
formations. Chem. - Eur. J. 2007, 13, 8543−8563.
5
577
DOI: 10.1021/acs.jafc.5b01987
J. Agric. Food Chem. 2015, 63, 5571−5577