Semi-synthesis and insecticidal activity of spinetoram J and its D-forosamine replacement analogues

Article abstract of DOI:10.3762/bjoc.14.207

Spinetoram, a mixture of spinetoram J (XDE-175-J, major component) and spinetoram L (XDE-175-L), is a new kind of fermentation-derived insecticide with a broad range of action against many insect pests, especially Cydia pomonella, Leaf miner and Thrips. Similar to spinosad, spinetoram is friendly to the environment, and non-toxic to animals and human beings. Therefore, spinetoram has been widely applied in pest control and grain storage. In a previous study, we had reported a semi-synthesis of spinetoram J. However, in that synthesis, there were more experimental steps, and the operations were troublesome. So an improved synthesis based on a self-protection strategy was designed and discussed. In this work, 3-O-ethyl-2,4-di-O-methylrhamnose was used as both the reaction substrate of C9–OH and the protecting group of C17–OH. The number of synthetic steps and costs were significantly reduced. In addition, a variety of D-forosamine replacement analogues of spinetoram J were synthesized based on the improved semi-synthesis, and their insecticidal activities were evaluated against third-instar larvae of Plutella xylostella. Although none of the analogues were as potent as spinetoram, a few of the analogues have only a 20–40 times lower activity than spinetoram. In particular, one of these analogues was approximately as active as spinosad. This study highlights the possibility of developing new insecticidal chemistries by replacing sugars on natural products with other groups, and the improved semi-synthesis will be helpful for further researches on spinetoram.

Full text of DOI:10.3762/bjoc.14.207

Semi-synthesis and insecticidal activity of spinetoram J and
its D-forosamine replacement analogues
Kai Zhang1, Jiarong Li*1, Honglin Liu2, Haiyou Wang2 and Lamusi A*2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 2321–2330.
1School of Chemistry and Chemical Engineering, Beijing Institute of
Technology, 5 South Zhongguancan Street, Haidian District, Beijing,
P. R. China and 2Institute of Grassland Research of CAAS, No. 120
Wulanchabu East Street, Saihan District, Hohhot, P. R. China
doi:10.3762/bjoc.14.207
Received: 10 July 2018
Accepted: 23 August 2018
Published: 04 September 2018
Email:
Jiarong Li* - jrligroup@126.com; Lamusi A* - alms721@163.com
Associate Editor: D. Spring
* Corresponding author
© 2018 Zhang et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
analogues; forosamine replacement; improved semi-synthesis;
insecticide; natural products; spinetoram
Abstract
Spinetoram, a mixture of spinetoram J (XDE-175-J, major component) and spinetoram L (XDE-175-L), is a new kind of fermenta-
tion-derived insecticide with a broad range of action against many insect pests, especially Cydia pomonella, Leaf miner and Thrips.
Similar to spinosad, spinetoram is friendly to the environment, and non-toxic to animals and human beings. Therefore, spinetoram
has been widely applied in pest control and grain storage. In a previous study, we had reported a semi-synthesis of spinetoram J.
However, in that synthesis, there were more experimental steps, and the operations were troublesome. So an improved synthesis
based on a self-protection strategy was designed and discussed. In this work, 3-O-ethyl-2,4-di-O-methylrhamnose was used as both
the reaction substrate of C9–OH and the protecting group of C17–OH. The number of synthetic steps and costs were significantly
reduced. In addition, a variety of D-forosamine replacement analogues of spinetoram J were synthesized based on the improved
semi-synthesis, and their insecticidal activities were evaluated against third-instar larvae of Plutella xylostella. Although none of the
analogues were as potent as spinetoram, a few of the analogues have only a 20–40 times lower activity than spinetoram. In particu-
lar, one of these analogues was approximately as active as spinosad. This study highlights the possibility of developing new insecti-
cidal chemistries by replacing sugars on natural products with other groups, and the improved semi-synthesis will be helpful for
further researches on spinetoram.
Introduction
Nowadays, insect pests are one of the primary hazards that insect pests. In the quest for new and efficient insecticides,
affect crop production and storage, and pest control depends natural products have always been considered an excellent
mainly on the use of insecticides. Therefore, it is particularly source of inspiration for insecticides [1,2]. Among natural prod-
important to identify an efficient insecticide for controlling uct-based insecticides, spinosyns are a novel kind of green
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insecticides with broad insecticide spectrum. Spinosyns, deriva- productivity of spinosyn J and spinosyn L is low. So a chemi-
tives of bioactive substances produced by the soil actinomycete cal synthesis of spinetoram would be helpful for the application
Saccharopolyspora spinosa [3], have been widely applied in of spinetoram, as well as for the further research on spinetoram.
pest control and crop protection due to their broad pest spec- In the previous study [13], we had reported a semi-synthesis of
trum, high insecticidal activity, low environmental impact, and spinetoram J with spinosyn A aglycon as initiator (Scheme 1).
low toxicity to non-target species [4]. So far, more than However, in the previous route, C9–OH and C17–OH of the
24 natural spinosyns and 800 semi-synthetic spinosyn ana- aglycone needed to be protected with different protecting
logues have been obtained and characterized, and their relative groups, resulting in cumbersome steps and high costs. To
insecticidal activities have also been reported [5,6]. Since 1990, reduce the synthetic steps and simplify the operations, we de-
there are two generations of commercial products of spinosyns veloped a new semi-synthesis of spinetoram J based on a self-
(spinosad) and spinetoram (Figure 1). Structurally, spinosad and protection strategy (Scheme 2). In this study, 3-O-ethyl-2,4-di-
spinetoram are both composed of D-forosamine and rhamnose O-methylrhamnose was used not only as the glycosylation
coupled to a macrocyclic tetracycle. Spinosad, a mixture of donor of C9–OH, but also the protecting group of C17–OH,
≈85% spinosyn A and ≈15% spinosyn D [7], is considered a greatly reducing the synthetic steps and costs.
highly effective bioinsecticide, and it has been widely used for
the management of various insect pests. Compared with spin- Macrolide compounds are a new kind of insecticides and fungi-
osad, spinetoram is more active and has a longer duration of cides which have also been widely applied in medicine [14,15].
control against many insect pests, such as codling moth (Cydia Currently, research on structural modification of macrolide
pomonella), a major pest of pome fruits, tobacco budworm, compounds, such as modification of the macrolide and the
cotton and vegetable crops [8]. Like spinosad, spinetoram elicits branched chain glycosyl residue, and replacement of the sugar
toxicity in the pest species via a neurotoxic mode of action. Al- group, are in the limelight [16,17]. The unique macrolide struc-
though the exact mechanism of action has yet to be character- ture of spinosyns and the efficient appearance of resistance in
ized, it is hypothesized that the spinosyn molecule interacts with some insect pests have prompted further exploration of spin-
both gamma-aminobutyric acid (GABA) receptor and nicotine osyns via chemical modification [18]. Bioactivities of many
acetylcholine (NACh) receptors [9]. Spinosad and spinetoram microbial secondary metabolites are highly dependent on their
can be degraded via a combination of photolysis and microbial sugar constituents which are transferred as nucleotide-activated
action, ultimately producing CO2, H2O, and nitrogen oxides sugars to an aglycon by glycosyltransferases [19]. Therefore,
[10]. That suggests that along with a broad-pest spectrum and bioactivities of these metabolites could change when the sugar
crop-uses, spinosad and spinetoram are both environment- and constituents are altered. Previous studies have shown that modi-
human-friendly. In addition, spinosad and spinetoram show fication of the spinosyn structure could potentially improve
high specificity to target insects and low toxicity to both insecticidal activity and expand the insect spectrum [20,21].
mammals and beneficial insects [11]. Therefore, spinosad and Indeed, a large number of researchers have altered spinosad,
spinetoram were successively awarded the Presidential Green replacing rhamnose or D-forosamine groups with sugar and
Chemistry Challenge Award in 1999 and 2008 [12].
nonsugar substituents [22-26], and evaluated the insecticidal
efficacy of these new spinosyn products. Although most spin-
At present, spinetoram is obtained from spinosyn J and spin- osyn analogues were not as potent as spinosyns, a few ana-
osyn L by a chemical modification. However, the fermentation logues demonstrated good potential for the use in pest control.
Figure 1: Structures of spinosad and spinetoram.
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Beilstein J. Org. Chem. 2018, 14, 2321–2330.
Scheme 1: Comparison of previous synthesis and self-protection synthesis.
However, up until now, there were no reports about the chemi- against third-instar larvae of Plutella xylostella. This work ex-
cal modification of spinetoram. Therefore, in this study, a series tended the examination of sugar and nonsugar substituents to
of D-forosamine replacement analogues of spinetoram J were determine whether simple sugar or nonsugar substitutions
synthesized based on the improved semi-synthesis (Scheme 3), would be suitable bioisosteric substituents for the D-forosamine
and the insecticidal activities of analogues were evaluated sugar of spinetoram J.
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Beilstein J. Org. Chem. 2018, 14, 2321–2330.
Scheme 2: Synthesis of spinetoram J from spinosyn A based on a self-protection strategy.
Scheme 3: Synthesis of spinetoram J analogues.
Results and Discussion
Semi-synthesis of spinetoram J
more stable than the C17 glycosidic bond under weak sulfuric
acid conditions (Scheme 2). Similar to spinosyn A, both
As reported in the previous study [27], spinosyn A could D-forosamine and 2’,3’,4’-tri-O-methylrhamnose of 5,6-dihy-
be hydrolyzed to 17-pseudoaglycone under weak sulfuric drospinosyn A could be hydrolyzed under strong acidic condi-
acid conditions, indicating that the C9 glycosidic bond was tions to afford the aglycone of spinetoram J (compound 2).
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Beilstein J. Org. Chem. 2018, 14, 2321–2330.
Then compound 2 was glycosylated with donor 3 to provide 4.
Since the C9 glycosidic bond was more stable than the
C17 glycosidic bond under weak sulfuric acid conditions, the
3-O-ethyl-2,4-di-O-methylrhamnose at the C17 position could
be removed selectively under the weak acidic conditions to
afford 5. The structure of compound 5 was also ascertained by
NMR and mass spectrometry. Finally, compound 5 was glyco-
sylated with donor 6 to provide spinetoram J. Compared with
the previous semi-synthesis, selective protection and deprotec-
tion of the C9–OH and the C17–OH were simplified in this
self-protection strategy, resulting in less synthetic steps and
costs.
Table 1: The hydrolysis yields of spinetoram J analogues under weak
acidic conditions. (continued)
7h
7i
61%
59%
7j
57%
During the selective hydrolysis of 3-O-ethyl-2,4-di-O-methyl-
rhamnose at the C17 position, neither the 9-pseudoaglycone of
compound 4 nor the aglycone (compound 2) were observed.
Moreover, the analogues 7 and 8 were also hydrolyzed
under 1.5 N H2SO4 at 90 °C, and the hydrolysis yields of
all analogues were shown in Table 1. During the hydrolysis of
analogues 7, only the 17-pseudoaglycone of spinetoram J
(compound 5) was observed, which was the same
as the hydrolysis of compound 4. However, during the
hydrolysis of analogues 8, none of the 17-pseudoaglycone of
spinetoram J, the 9-pseudoaglycone of compound 4, nor com-
7k
7l
62%
63%
8a
8b
8c
0
0
0
Table 1: The hydrolysis yields of spinetoram J analogues under weak
acidic conditions.
compound C17–O substituent structure hydrolysis yield
7a
7b
56%
54%
8d
8e
0
0
7c
7d
7e
53%
55%
53%
8f
0
0
8g
8h
8i
0
0
7f
57%
56%
7g
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pound 2 were observed. It seemed that the ester bond is more similar to spinetoram. The results prompted a comprehensive
stable than the ether bond at the C17 position under acidic evaluation of spinetoram J analogues, which indicated that
conditions.
spinetoram J analogues had a wide range of insecticidal activity.
Indeed, all of the spinetoram J analogues demonstrated toxicity
to P. xylostella. Although most analogues were far less toxic
Synthesis of spinetoram J analogues
All carbohydrates and alcohols were activated by CNCCl3 with than spinetoram (demonstrating 50−400 times lower toxicity),
DBU as catalyst initially to afford glycoside donors, and then but a few of the analogues, such as 7d, 7e, 7g and 8b, exhibited
the 17-pseudoaglycone of spinetoram J was glycosylated with toxicity to P. xylostella that was 20–40 times lower than that of
donors in the presence of BF3·(C2H5)2O under Ar gas. Due to spinetoram. Particularly, the compound 8b was approximately
the high yields and few byproducts in the synthesis of glyco- as active as spinosad.
side donors, the donors could be used directly in the glycosyla-
tion without purification.
Carbohydrate substituents are considered to be related closely to
many secondary metabolites [19], hence, C17–O glycosyl ana-
logues are regarded as efficient insecticides that are likely to
Insecticidal activity
The insecticidal activities of synthetic spinetoram J and its ana- rival present insecticides. However, as noted in Table 2,
logues were evaluated using third-instar larvae of P. xylostella. C17–O-glycosyl analogues 7j, 7k and 7l did not exhibit excel-
The lethal concentration for 50% of the test population (LC50) lent performance as expected.
and 95% confidence intervals were corrected for mortality in
the controls, and then determined using Probit analysis. The Similar to the C17–O-benzyl analogues of spinosyn A, the
results are shown in Table 2.
C17–O-benzyl analogues of spinetoram J with electron-with-
drawing substituents demonstrated better insecticidal activity
than the C17–O-benzyl analogue with electron-donating substit-
uents, illustrating that the insecticidal activities of spinetoram J
analogues are closely related to the electrical properties of the
substituents. For compound 7c, the methoxy group was at-
tached to the benzene ring at the meta-position, leading to a de-
crease in the conjugate effect, mainly for the role of electron
absorption. So the compound 7c exhibited much more efficient
insecticidal activity than the C17–O-benzyl analogues with
electron-donating substituents. Therefore, an electron-donating
substituent is more likely to generate a promising analogue with
good insecticidal activity. In addition, the majority of C17–O-
acyl analogues did not show as good insecticidal activity as
other analogues, probably indicating that C17–O-ether ana-
logues were more likely to show more efficient insecticidal ac-
tivity than C17–O-ester analogues.
Table 2: The insecticidal activities of spinetoram J and its analogues.
compound
LC50 (mg/L)
95% CI (mg/L)
spinosad [28]
0.627
0.048
0.052
14.621
8.654
2.151
1.577
1.792
2.557
1.226
10.172
7.342
2.166
2.537
3.824
16.353
0.805
18.097
17.633
16.602
13.895
21.996
9.724
12.792
0.509–0.771
0.036–0.060
0.043–0.061
10.824–23.019
7.538–10.016
1.651–3.061
1.272–2.015
1.428–2.350
2.062–3.376
1.047–1.440
8.136–13.585
6.155–8.964
1.844–2.545
2.164–2.986
3.225–4.516
11.659–28.038
0.742–1.051
14.934–22.689
15.591–20.221
13.464–21.413
11.375–17.604
16.964–31.458
8.049–11.825
10.577–15.810
spinetoram [28]
spinetoram J
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
Spinetoram has excellent insecticidal activity against a broad
array of target pests such as Lepidopteran, Thysanoptera,
Diptera, Isoptera, Coleoptera and Themiptera [29]. So the
spinetoram J analogues may be potent against other pests in ad-
dition to P. xylostella, such as leafroller thrips, leafminer flies,
white flies and so on. Moreover, these analogues may also have
antibiotic, antifungal, anticancer, antiparasitic, and immunosup-
pressive properties.
8a
8b
8c
8d
8e
8f
8g
8h
8i
Conclusion
This study demonstrated a viable approach to synthesize spine-
toram J and its analogues. Compared with the previous semi-
synthesis of spinetoram J, the improved semi-synthesis shown
in this study demonstrated less steps and simpler operations.
As shown in Table 2, the semi-synthetic spinetoram J exhibited
excellent insecticidal activity against P. xylostella, which was
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Meanwhile, a variety of spinetoram J analogues were synthe- 74.57, 71.59, 66.87, 64.02, 59.91, 58.01, 57.99, 56.69, 49.03,
sized based on this improved semi-synthesis, and their insecti- 46.82, 45.54, 42.17, 39.99, 39.51, 38.49, 37.75, 37.00, 33.26,
cidal activities were evaluated against third-instar larvae of P. 31.98, 29.49, 28.94, 27.44, 26.01, 23.49, 20.89, 18.43, 18.39,
xylostella. Although spinetoram J analogues displayed a wide 18.30, 16.80, 15.00, 8.36; MS (MALDI) m/z: [M + H]+ calcd
range of insecticidal activity, none of the analogues were as po- for C41H67NO10, 734.483774; found, 734.483663.
tent as spinetoram, but a few of the analogues exhibited insecti-
Preparation of aglycone of
cidal activity that was close to that of spinetoram. This study
demonstrates a viable method to develop natural products 5,6-dihydrospinosyn A 2
that contain glycosidic bonds or hydroxy groups. It also Compound 1 (2.15 g, 3.64 mmol) was added to a solution of
highlights the potential for developing new insecticidal 60 mL MeOH and 130 mL 8 N H2SO4, then the mixture were
products by replacing sugars on natural products with other heated to reflux for 4 hours. After the mixture was cooled, an
groups.
appropriate amount of NaHCO3 was added to adjust the pH to
5–6. Then the mixture was extracted with CH2Cl2 (3 × 15 mL).
The CH2Cl2 extracts were combined, dried with Na2SO4 and
evaporated under reduced pressure. The residue was purified by
Experimental
Insecticide bioassays
The insecticidal activities of spinetoram J analogues were eval- column chromatography on silica gel (200–300 mesh) to afford
uated against third-instar larvae of P. xylostella. Stock solu- pure 2 (1.04 g, yield 71%). TLC (ethyl acetate/petroleum ether
tions were prepared by weighting manufactured analogues and 2:1, v:v); 1H NMR (400 MHz, CDCl3) δ 6.84 (s, 1H, C13-H),
dissolving them in acetone. Dosing solutions were then pre- 4.60 (m, 1H, C21-H), 4.27 (m, 1H, C9-H), 3.58 (m, 1H, C17-H),
pared by diluting stock solutions to different concentrations 3.16–3.06 (m, 2H, C16-H, one of C2-H), 2.89 (m, 1H, C3-H),
with 0.01% Triton X-100 aqueous solution. Cabbage leaves 2.75 (m, 1H, C12-H), 2.51 (m, 1H, one of C2-H), 2.27–2.22 (m,
were then soaked in the dosing solutions for 10 s, dried, and 2H, one of C8-H, C7-H), 1.62–1.19 (m, 13H, C18-H, C20-H,
placed in Petri dishes. Approximately twenty P. xylostella were C22-H, one of C10-H, C5-H, C6-H, one of C8-H, one of C19-H),
added to each dish. After 48 h, the number of P. xylostella 1.14 (d, J = 3.4 Hz, 3H, C16-CH3), 1.10 (m, 1H, C4-H), 0.94
deaths was recorded. Three replicates were used for each com- (m, 1H, C11-H), 0.75 (t, J = 7.4 Hz, 3H, C23-H); 13C NMR
pound of exposure.
(101 MHz, CDCl3) δ 202.53, 171.70, 148.75, 144.38, 71.25,
70.50, 52.47, 49.11, 47.28, 46.21, 42.04, 41.23, 40.26, 40.06,
38.50, 33.95, 31.75, 28.83, 27.46, 26.05, 23.45, 21.13, 14.64,
Preparation of 5,6-dihydrospinosyn A (1)
Spinosyn A (3.96 g, 5.43 mmol) was added to 110 mL metha- 8.39; MS (MALDI) m/z: [M + Na]+ calcd for C24H36O5,
nol, and then 10% Pd/C (0.36 g, 0.33 mmol) was added. The 427.245495; found, 427.245611.
mixture was stirred under hydrogen at room temperature for
Preparation of 3-ethoxy-2,4-
48 h. The mixture was then filtered. The filtrate was evaporated
under reduced pressure, and the residue was purified by column dimethoxyrhamnosyltrichoroacetimidate (3)
chromatography on silica gel (200–300 mesh) to afford pure 1 3-O-Ethyl-2,4-di-O-methylrhamnose (0.91 g, 4.09 mmol) was
(3.70 g, yield 93%). TLC (methanol/dichloromethane 1:9, v:v); dissolved in 20 mL CH2Cl2, then CNCCl3 (0.81 g, 5.61 mmol)
1H NMR (400 MHz, CDCl3) δ 6.79 (s, 1H, C13-H), 4.75 (s,1H, and 0.15 mL DBU was added. The mixture was stirred at room
C1’-H), 4.58 (m, 1H, C21-H), 4.39 (d, J = 7.6 Hz, 1H, C1”-H), temperature for 20 min, then diluted with CH2Cl2 and washed
4.13 (m, 1H, C9-H), 3.57 (m, 1H, C2’-H), 3.48–3.41 (m, 14H, with saturated sodium bicarbonate solution (3 × 10 mL). The
C5”-H, C17-H, C5’-H, C4-H, C4’-OCH3, C2’-OCH3, C3’-OCH3, combined organic layers were dried with Na2SO4 and evaporat-
C3’-H), 3.20 (m, 1H, C16-H), 3.21–3.09 (m, 2H, one of C2-H, ed under reduced pressure. The residue can be used directly in
C3-H), 2.89–2.67 (m, 3H, C4’-H, C12-H, one of C2-H), the next reaction step.
2.54–2.42 (m, 3H, one of C10-H, C7-H, C4”-H), 2.37 (m, 6H,
N(CH3)2), 2.28–2.16 (m, 2H, one of C8-H, one of C2”-H), Preparation of spinetoram J analogue 4
1.96–1.93 (m, 3H, one of C3”-H, one of C8-H, one of C19-H), Compound 2 (0.32 g, 0.79 mmol) and compound 3 (0.68 g,
1.59–1.40 (m, 10H, C18-H, one of C20-H, C22-H, one of C2”-H, 1.87 mmol) were dissolved in 10 mL dry CH2Cl2 with some
one of C3”-H, one of C5-H, one of C6-H, one of C10-H), 1.31 (d, molecular sieve under Ar. Then BF3·(C2H5)2O (0.12 mL,
J = 7.6 Hz, 3H, C6’-H), 1.21–1.18 (m, 6H, C16-CH3, C6”-H), 1.01 mmol) was added at room temperature. The mixture was
1.11–1.09 (m, 4H, one of C19-H, one of C20-H, one of C5-H, stirred for 14 h, then diluted with CH2Cl2 (15 mL) and washed
one of C6-H), 0.96 (m, 1H, C11-H), 0.74 (t, J = 7.6 Hz, 3H, with saturated sodium bicarbonate solution (3 × 10 mL). The
C23-H); 13C NMR (101 MHz, CDCl3) δ 202.21, 171.51, combined organic layers were dried with Na2SO4 and evaporat-
148.53, 144.13, 102.03, 94.45, 81.29, 80.05, 79.45, 76.74, ed under reduced pressure. The residue was purified by column
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chromatography on silica gel (200–300 mesh) to afford pure 4 Preparation of spinetoram J
(0.42 g, yield 65%). TLC (ethyl acetate/petroleum ether 1:2, Compound 5 (0.15 g, 0.25 mmol) and compound 6 (0.11 g,
v:v); 1H NMR (400 MHz, CDCl3) δ 6.79 (s, 1H, C13-H), 0.36 mmol) were dissolved in 10 mL dry CH2Cl2 with some
4.80–4.73 (m, 2H, C1’-H, C1”-H), 4.57 (m, 1H, C21-H), 4.20 molecular sieve under Ar. Then BF3·(C2H5)2O (0.13 mL,
(m, 1H, C9-H), 3.73–3.52 (m, 16H, C2’-H, C2”-H, C17-H, 1.02 mmol) was added at room temperature. The mixture was
C3’-OCH2-, C3”-OCH2-, C4’-OCH3, C4”-OCH3, C5’-H, C5”-H, stirred for 16 h. The mixture was then diluted with CH2Cl2
C4-H), 3.48–3.44 (m, 8H, C2’-OCH3, C2”-OCH3, C4’-H, (15 mL) and washed with saturated sodium bicarbonate solu-
C4”-H), 3.22 (m, 1H, C10-H), 3.11–2.99 (m, 5H, one of C2-H, tion (3 × 10 mL). The combined organic layers were dried with
C3-H, C3’-H, C3”-H, C12-H), 2.36–2.26 (m, 2H, one of C2-H, Na2SO4 and evaporated under reduced pressure. The residue
C7-H), 2.18 (m, 2H, C10-H), 1.88 (m, 1H, one of C8-H), was purified by column chromatography on silica gel
1.61–1.37 (m, 11H, C5-H, C6-H, one of C8-H, C18-H, one of (200–300 mesh) to afford pure spinetoram J (0.15 g,
C19-H, one of C20-H, C22-H), 1.28–1.22 (m, 7H, one of C20-H, yield 78%). TLC (methanol/dichloromethane 1:8, v/v);
C6’-H, C6”-H), 1.18–1.12 (m, 10H, one of C19-H, C16-CH3, MS (MALDI) m/z: [M + Na]+ calcd for C42H69NO10,
C3’-OC-CH3, C3”-OC-CH3), 0.95 (m, 1H, C11-H), 0.75 (t, 770.481368; found, 770.481016.
J = 7.4 Hz, 3H, C23-H); 13C NMR (176 MHz, CDCl3) δ 202.76,
171.18, 149.17, 144.80, 99.43, 95.79, 82.19, 82.01, 79.68, Preparation of 7a
79.54, 78.51, 78.39, 75.70, 72.70, 68.68, 67.96, 65.68, 61.02, Compound 5 (0.32 g, 0.53 mmol) and 4-methylbenzyl-2,2,2-tri-
60.41, 59.23, 59.02, 50.26, 48.19, 47.07, 46.37, 43.52, 40.79, chloroacetimidate (0.28 g, 0.45 mmol) were dissolved in 15 mL
39.60, 38.80, 38.14, 34.86, 34.22, 31.59, 30.62, 28.05, 26.94, dry CH2Cl2 with some molecular sieve under Ar. Then
24.68, 22.66, 17.85, 17.72, 15.76, 15.69, 14.20, 9.41; BF3·(C2H5)2O (0.15 mL, 1.03 mmol) was added at room tem-
MS (MALDI) m/z: [M + Na]+ calcd for C44H72O13, perature. The mixture was stirred for 24 h, then diluted with
831.486513; found, 831.486515.
CH2Cl2 (15 mL) and washed with saturated sodium bicar-
bonate solution (3 × 10 mL). The combined organic layers were
dried with Na2SO4 and evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel
Preparation of spinetoram J
17-pseudoaglycone 5
Compound 4 (0.23 g, 0.28 mmol) was dissolved in 10 mL 1.5 N (200–300 mesh) to afford pure 7a (0.27 g, yield 72%). TLC
H2SO4, then the mixture was heated to 90 °C. After 3 h of stir- (ethyl acetate/petroleum ether 1:2, v/v). 1H NMR (400 MHz,
ring, the mixture was cooled to room temperature, and then CDCl3) δ 7.25–7.13 (m, 4H, C3”-H, C5”-H, C6”-H, C7”-H,),
extracted with CH2Cl2 (3 × 15 mL). The CH2Cl2 extracts were 6.86 (s, 1H, C13-H), 4.81 (s, 1H, C1’-H), 4.65 (m, 1H, C21-H),
combined, dried with Na2SO4 and evaporated under reduced 4.52 (m, 2H, C1”-H), 4.22 (m, 1H, C9-H), 3.73 (m, 1H, C2’-H),
pressure. The residue was purified by column chromatography 3.64 (m, 1H, C17-H), 3.57–3.54 (m, 7H, C3’-OCH2-, C4’-OCH3,
on silica gel (200–300 mesh) to afford pure 5 (0.13 g, yield C5’-H, C4-H), 3.50 (s, 3H, C2’-OCH3), 3.45 (m, 1H, C3’-H),
76%). TLC (ethyl acetate/petroleum ether 1:1, v/v); 1H NMR 3.17–3.09 (m, 3H, C16-H, one of C2-H, C3-H), 2.94 (m, 1H,
(400 MHz, CDCl3) δ 6.87 (s, 1H, C13-H), 4.80 (s, 1H, C1’-H), C4’-H), 2.82 (m, 1H, C12-H), 2.59 (t, 3H, C4”-H), 2.35 (dd, 1H,
4.68 (m, 1H, C21-H), 4.21 (m, 1H, C9-H), 3.72 (m, 1H, C2’-H), one of C2-H), 2.31 (m, 1H, C7-H), 2.27 (m, 2H, C10-H), 1.94
3.62 (m, 1H, C17-H), 3.57–3.56 (m, 5H, C3’-OCH2-, (m, 1H, one of C8-H), 1.73–1.47 (m, 11H, C5-H, C6-H, one of
C4’-OCH3), 3.54 (m, 1H, C5’-H), 3.51 (m, 1H, C4-H), 3.49 (s, C8-H, C18-H, one of C19-H, one of C20-H, C22-H), 1.31–1.25
3H, C2’-OCH3), 3.44 (m, 1H, C4’-H), 3.19–3.11 (m, 3H, C16-H, (m, 11H, one of C19-H, one of C20-H, C6’-H, C16-CH3,
one of C2-H, C3-H), 2.95 (m, 1H, C3’-H), 2.82 (m, 1H, C12-H), C3’-OC-CH3), 1.01 (m, 1H, C11-H), 0.83 (t , 3H, C23-H);
2.36 (dd, J1 = 13.2 Hz, J2 = 2.8 Hz, 1H, one of C2-H), 2.32 (m, 13C NMR (101 MHz, CDCl3) δ 203.52, 172.59, 163.78, 149.16,
1H, C7-H), 2.25 (m, 2H, C10-H), 1.93 (m, 1H, one of C8-H), 144.89, 137.32, 135.47, 129.05, 128.60, 127.99, 95.73, 82.22,
1.73–1.47 (m, 11H, C5-H, C6-H, one of C8-H, C18-H, one of 79.62, 78.61, 75.70, 71.94, 70.99, 67.97, 65.62, 60.92, 59.15,
C19-H, one of C20-H, C22-H), 1.28–1.21 (m, 11H, one of 50.28, 47.06, 46.33, 43.62, 40.78, 39.57, 38.98, 38.77, 38.14,
C19-H, one of C20-H, C6’-H, C16-CH3, C3’-OC-CH3), 1.01 (m, 33.47, 31.93, 30.56, 28.05, 26.94, 24.71, 21.14, 20.84, 17.84,
1H, C11-H), 0.82 (t , J = 7.4 Hz, 3H, C23-H); 13C NMR 15.73, 15.54, 9.36; MS (MALDI) m/z: [M + Na]+ calcd for
(101 MHz, CDCl3) δ 203.24, 172.71, 149.44, 145.42, 95.80, C42H62O9, 733.428604; found, 733.428914.
82.22, 79.69, 78.55, 75.55, 72.48, 67.98, 65.60, 61.02, 59.22,
50.03, 48.21, 46.59, 45.90, 43.17, 41.06, 39.51, 38.80, 38.01, Preparation of 8a
34.96, 32.84, 29.90, 28.47, 27.04, 24.50, 22.03, 17.85, 15.76, Compound 5 (0.33 g, 0.54 mmol), cyclopropanecarbonyl chlo-
15.60, 9.39; MS (MALDI) m/z: [M + Na]+ calcd for C34H54O9, ride (0.06 g, 0.58 mmol) and 4-dimethylaminopyridine (DMAP,
629.366004; found, 629.366428.
0.15 g, 1.23 mmol) were added to 15 mL CH2Cl2 under Ar,
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Beilstein J. Org. Chem. 2018, 14, 2321–2330.
4. Salgado, V. L. Pestic. Biochem. Physiol. 1998, 60, 91–102.
then the mixture was heated to reflux. After about 6 h, the mix-
ture was diluted with CH2Cl2 (15 mL) and washed with satu-
rated sodium bicarbonate solution (3 × 10 mL). The combined
organic layers were dried with Na2SO4 and evaporated under
reduced pressure. The residue was purified by column chroma-
tography on silica gel (200–300 mesh) to afford pure 8a (0.31 g,
yield 85%). TLC (ethyl acetate/petroleum ether 1:3, v/v).
1H NMR (400 MHz, CDCl3) δ 6.75 (s, 1H, C13-H), 4.82 (s, 1H,
C1’-H), 4.67 (m, 1H, C21-H), 4.50 (m, 1H, C9-H), 4.06 (m, 1H,
C2’-H), 3.57 (m, 1H, C17-H), 3.46–3.31 (m, 12H, C5’-H, C4-H,
C3’-OCH2-, C2’-OCH3, C4’-OCH3, C3’-H, C16-H), 3.23 (m, 1H,
one of C2-H), 2.99–2.95 (m, 2H, C3-H, C4’-H), 2.80 (m, 1H,
C12-H), 2.67 (m, 1H, one of C2-H), 2.43 (m, 2H, C10-H), 2.16
(m, 1H, C7-H), 1.94 (m, 1H, one of C8-H), 1.67 (m, 2H, one of
C5-H, one of C6-H), 1.58 (m, 1H, C2”-H), 1.47–1.42 (m, 7H,
one of C8-H, C18-H, one of C19-H, one of C20-H, C22-H),
1.34–1.32 (m, 5H, one of C19-H, one of C20-H, C6’-H),
1.20–1.10 (m, 8H, one of C5-H, one of C6-H, C16-CH3,
C3’-OC-CH3), 0.97–0.90 (m, 5H, C3”-H, C4”-H, C11-H), 0.77
(m, 3H, C23-H); 13C NMR (101 MHz, CDCl3) δ 201.48,
174.28, 172.41, 149.15, 144.99, 95.71, 82.08, 79.48, 78.36,
76.27, 75.70, 74.66, 67.80, 65.39, 60.69, 58.90, 50.03, 49.04,
47.75, 46.34, 45.60, 43.20, 40.84, 39.44, 38.65, 37.94, 32.79,
32.34, 29.94, 28.04, 27.35, 26.83, 21.32, 17.57, 15.50, 12.57,
8.15, 8.01; MS (MALDI) m/z: [M + Na]+ calcd for C38H58O10,
697.392219; found, 697.392189.
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Supporting Information
Supporting Information File 1
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reduction of spinosyn factors Et-J and Et-L to spinetoram. US Patent
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Details for the synthesis of spinetoram J analogues,
analytical data of all compounds, NMR spectra and MS
data of all synthesized compounds.
18.Sparks, T. C.; Dripps, J. E.; Watson, G. B.; Paroonagian, D.
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Acknowledgements
We thank the National Natural Science Foundation of China
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Vousden, W. A.; Kaja, A. L.; Crouse, G. D.; Gifford, J.; Graupner, P. R.;
Karr, L.; Lewer, P.; Sparks, T. C.; Leadlay, P. F.; Waldron, C.;
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(No. 41471198) for financial support.
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