Modification of the butenyl-spinosyns utilizing cross-metathesis

Article abstract of DOI:10.1016/j.bmc.2009.02.036

The discovery of a strain of Saccharopolyspora sp. that produced a number of spinosyn analogs that had not before been seen gave an ideal opportunity for extending our knowledge of that SAR of these highly efficacious insecticides. In particular, these co

Full text of DOI:10.1016/j.bmc.2009.02.036

Bioorganic & Medicinal Chemistry 17 (2009) 4197–4205
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modification of the butenyl-spinosyns utilizing cross-metathesis
*
John Daeuble, Thomas C. Sparks, Peter Johnson, Paul R. Graupner
Dow AgroSciences, 9330 Zionsville Road, Indianapolis, IN 46268, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of a strain of Saccharopolyspora sp. that produced a number of spinosyn analogs that had
not before been seen gave an ideal opportunity for extending our knowledge of that SAR of these highly
efficacious insecticides. In particular, these compounds contained a butenyl group connected to C-21
which in the regular spinosyns was substituted with a simple ethyl group. The double bond therefore
gave us a handle to further modify this position allowing us to substitute different groups there. In this
paper we show one of our approaches to this modification using olefin cross-metathesis. Even though the
spinosyns were not highly efficient substrates for metathesis reactions, we were nevertheless successful
in extending their chemistry accordingly.
Received 26 September 2008
Revised 11 February 2009
Accepted 18 February 2009
Available online 23 February 2009
Keywords:
Spinosyns
Natural Products
Semi-synthesis
Olefin cross-metathesis
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
naturally occurring molecules. Some limited success was found
utilizing enzyme approaches both in our labs and others by which
The spinosyns are an important family of natural products that
are highly efficacious against a number of different insect pests.1
The compounds are extracted from a fermentation of the actino-
mycete Saccharopolyspora spinosa—originally isolated from a soil
sample collected in a disused rum distillery on a Caribbean Is-
land—from which over 20 factors were isolated. They are marketed
alcohol groups could be incorporated onto the tail,5 but no pro-
gress was made using classical chemical techniques. Manipulation
of the genome did give access to simple variants including
branched alkyl and cycloalkyl groups through splicing of the star-
ter unit from the avermectins into that of spinosyn allowing some
access to new derivatives. However none of these could be further
manipulated by synthesis.6
TM
under the trade mark Tracer , which is primarily a mixture of the
two major factors, spinosyns A and D.2 During the development
stage, much chemistry was undertaken on the spinosyns in an ef-
fort to discover potentially more active compounds, in particular
by manipulating both glycoside units and, where possible, under-
taking substitutions at other parts of the molecule.3 This effort
was rewarded with the discovery of Spinetoram, a semi-synthetic
product derived from fermentation of a strain of S. spinosa that pro-
duces spinosyn J and L which are both missing one methyl group
on the rhamnose moiety. Ethylation of this remaining hydroxyl
group followed by selective hydrogenation of the isolated double
bond of spinosyn J yields a spinosyn derivative that has a better
activity profile than the parent.4
Amongst the many synthetic manipulations of the spinosyn
skeleton undertaken, it was not possible to make significant
changes to the ethyl group substituted alpha to the lactone oxygen
(the ‘tail’ group on C-21). Early structure activity relationship (SAR)
studies were, therefore limited to the few variations found in the
The tail SAR was significantly advanced with the discovery of a
closely related organism Saccharopolyspora pogona which pro-
duced spinosyns extended with an extra polyketide synthase mod-
ule at the start of the biosynthetic pathway.7 The group at C-21
was predominately but-1-enyl [for example, the major factor
spinosyn
a1 (1)], with minor variants including butadienyl, and
3-hydroxybut-1-enyl which contained an allylic alcohol group.
The presence of these new functional groups gave potential for fur-
ther manipulation of the side chain through reduction, substitu-
tion, oxidation, etc. However of particular interest was the
presence of the olefin on the C21-tail which opened up the oppor-
tunity for further modifications using cross-metathesis (CM).8 Clo-
ser examination of the overall structure raised concerns about the
chances of success for this chemistry though; basic nitrogen groups
have been shown to affect metathesis chemistry,9 and the two
ring-olefins present in the spinosyn molecule could undergo
ring-opening metathesis. One example of natural product manipu-
lation by CM which drew our attention was the derivitization of
erythromycin, particularly considering the presence of a 3-amino
glycoside in the starting material.10 This gave hope that the foros-
amine sugar in spinosyn derivatives would not hinder the cross-
metathesis reaction.
* Corresponding author. Tel.: +1 317 337 3496; fax: +1 317 337 3546.
E-mail address: prgraupner@dow.com (P.R. Graupner).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.036

4198
J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
N
N
R
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
O
O
H
H
O
O
H
R
H
O
O
O
O
R = Me Spinosyn J
R = H Spinosyn M
R = H Spinosyn A
R = Me Spinosyn D
N
N
1``
O
O
O
O
O
O
O
O
O
O
21
3`
O
O
H
H
H
H
H
H
1
24
O
O
1`
H
H
O
O
H
H
O
O
O
O
6
1: Spinosyn α1
Spinetoram - main factor
2.1. Choice of starting material
2. Results and discussion
There was substantial interest in spinosyns that contained
incomplete alkylation of the rhamnose glycoside. Since some effort
had been made to produce more demethylated ‘J’ type spinosoids
with alkenyl groups substituted on C-21, selection of precursors
for this study was largely determined by the greater availability
of these butenyl ‘J’ factors. For purposes of biological comparisons,
C21-modified analogs prepared in this study were subsequently
converted into their 30-O-ethylated derivatives and evaluated rela-
tive to both spinosyn A and the corresponding 30-O-alkyl spinosyn
analog. There were some fully methylated derivatives available
though, and initial experiments were undertaken with them.
Unfortunately, reaction of 1 with either generation of Grubbs cat-
alyst (A or B) either ethylene gas or cis-1,4-diacetoxy-2-butene in
a sealed vial at reflux temperature in methylene chloride (CH2Cl2)
returned only starting material. On the other hand, when the ‘J’
There are many catalysts available for metathesis chemistry
covered by a massive body of publications since development
has taken place over recent decades for both CM and ring-clos-
ing metathesis (RCM) chemistry.8 During this work a number of
these catalysts were utilized for example the so called ‘first
generation’ Grubbs catalyst (A), the more recent ‘second gener-
ation’ Grubbs (B) and the phosphine-free Grubbs/Hoyveda cata-
lyst (C). The first two catalysts require displacement of
a
phosphine ligand to allow coordination of an olefin to the metal
center whereas the third one removes that requirement, while
retaining the selectivity and functional group compatibility of
the earlier catalysts. Ability to coordinate to the metal com-
plexes is key to the success of metathesis reactions, and is
dependent on electronic and steric properties of the reactants
and products. These were unknown factors for the spinosyns
prior to this study.
derivative 30-O-demethyl-24-hydroxy spinosyn
a1c (2) was re-
acted with catalyst B in ethylene saturated CH2Cl2, substantial
conversion to the 22,23-dehydro spinosyn J (3) was observed
(Scheme 1). In several instances throughout this investigation, it
was noted that the 24-hydroxy compounds appeared to be more
reactive metathesis partners than the unadorned butenyl groups,
suggesting either a positive influence from the free hydroxyl group
or that allylic alcohols are more reactive than simple internal ole-
fins—an effect that has been noted in RCM reactions.11 In any
event, the incomplete conversion of starting material suggested
that, as with steric or electronic factors, the basic amine group
can serve to make a poor metathesis partner even worse, while
not precluding metathesis occurring altogether. Therefore it was
decided to explore protection procedures for the nitrogen function
that would allow higher-yielding metathesis chemistry to occur
PCy3
Cl
Cl
Rh
N
N
Cy3P
Cl
Cl
Cy3P
Rh
A, 1st generation
Grubb's catalyst
B, 2nd generation
N
N
Cl
Cl
Rh
O+
Naming of the spinosyns follows the conventions described in the literature.
Compounds with
2 or fewer carbon atoms attached to C-21 are described as
derivatives of the ‘classical’ spinosyns (A, D, J, etc.)1 whereas those with three or more
are named according to the nomenclature described in the preceeding paper on the
butenyl compounds.7
C, Grubbs/Hoyveda Catalyst

J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
4199
N
N
O
O
O
O
O
O
O
R2
R2
O
i
O
O
O
H
O
H
H
H
H
H
R1
O
O
H
O
H
H
O
O
H
O
O
O
1; R1 = H, R2 = Me
2; R1 = OH, R2 = H
R2 = Me, 0%
3; R2 = H, 50%
Scheme 1. General scheme for CM of unprotected spinosyns. Reagents and conditions: (i) catalyst A or B (10–20 mol %), ethylene gas, DCM or dichloroethane.
either by protection of the secondary amine, or by formation of
amine salt derivatives.
and their reactivity examined in the metathesis reaction with eth-
ylene. The results of this study are shown in Table 1.
There are a number of observations to be made regarding the
data in Table 1. Interestingly, the metal complex seemed to serve
as an oxidation catalyst in the presence of the N-oxide (2a), deox-
ygenating the N-oxide and oxidizing the allylic alcohol at C-24.
Protecting the amine as the tetrafluoroborate (2b) or acetate salt
(2c) offered no benefit over 2. In all three cases, some conversion
to the vinyl product was observed but starting material remained
even after prolonged reaction time indicating degradation of the
catalyst. Use of hydrochloric acid (HCl) or trifluoroacetic acid
(TFA) to protonate the amine proved optimal. With either salt, 2
was completely consumed giving rise to reasonable yields of 3
which was readily ethylated to give the required Spinetoram deriv-
ative (12). As mentioned previously, there appeared to be some
benefit from using the 24-hydroxy butenyl spinosyn 2 rather than
the non-hydroxylated 1 in metathesis reactions. Thus even as the
HCl or TFA salt, incomplete conversion was observed with 1 as
the substrate (1a and 1b). One caveat to using the HCl or TFA salts
was their sensitivity to moisture and subsequent degradation to
the 17-pseudo aglycone. Addition of ethereal HCl to a solution of
a spinosyn in ether produced a fine white precipitate which was
isolated by filtration or concentration. If the salt was not used
immediately or carefully protected from moisture, noticeable loss
of forosamine occurred, severely affecting yields. Even with fresh
salt samples however, some loss of forosamine was observed with
2.2. Protection of amine
In the first example, the dimethyl amino group of 2 was mono-
demethylated (4)12 and protected as the tert-butyl carbamate (Boc)
(5, Scheme 2). Exposure of 5 to conditions similar to those de-
scribed in Scheme 1, led to complete conversion of the starting
material providing Boc protected 21-vinyl derivative (6) in 65%
yield.
Unfortunately, all attempts to remove the Boc protecting group
from 6 resulted in loss of the forosamine sugar, thus negating the
usefulness of this strategy. A second protecting group strategy in-
volved use of 2-(trimethylsilyl)ethyl carbamate group (Teoc) in
place of Boc (7, Scheme 3). As with the Boc protected substrate,
metathesis with ethylene went smoothly giving rise to the Teoc
protected 21-vinyl compound (9) in 88% yield. After capping the
30-hydroxyl group as an ethyl ether (10), the substrate was ex-
posed to tetrabutylammonium fluoride (TBAF) causing slow con-
version to 21-vinyl-30-O-Et-spinosyn M (11) in moderate yield.
While protecting the amine as a carbamate allowed for clean
conversion in this simple metathesis reaction, the process required
three additional steps. A far simpler approach to removing the
effects of the basic amine was to protect it as a salt prior to the
metathesis reaction.9a Several salts and the N-oxide were prepared
HN
N
O
O
O
O
O
O
O
i
HO
ii
O
OH
OH
O
H
O
H
H
H
H
H
HO
O
O
H
O
H
H
O
O
H
O
O
O
4
2
N
N
O
O
O
O
O
O
O
O
O
O
O
O
iii
OH
OH
O
O
H
H
H
H
HO
O
O
H
H
O
O
H
H
O
O
O
O
H
H
6
5
Scheme 2. Protection of amine with Boc group. Reagents and conditions: (i) NaOAc, I2, MeOH, NaOH (aq); (ii) Boc2O, NaHCO3, EtOH, 70%; (iii) ethylene, B, DCM, reflux, 65%.

4200
J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
O
O
O
N
SMT
N
O
O
O
8
O
STM
O
O
O
i
4
O
OH
O
H
H
H
HO
N
O
H
O
H
O
O
7
Teoc
O
O
O
ii
iii
O
OH
O
H
H
H
O
H
O
H
O
O
9
HN
N
Teoc
O
O
O
O
O
O
O
iv
O
O
O
O
O
H
H
H
H
H
O
O
H
H
O
O
H
O
O
O
O
H
H
10
11
Scheme 3. Teoc protection. Reagents and conditions: (i) 8 Et3N, DCM, EtOH, 54%; (ii) B, ethylene, DCM, reflux, 88%; (iii) EtBr, KOH/Bu4NI (10:1) 88%; (iv) Bu4NF, THF, 66%.
nearly all these reactions and contributed to the relatively modest
yields.
ity than the cross coupling utilizing the corresponding terminal
olefin.14 Therefore the metathesis of butenyl spinosyn analogs
and various butene analogs was examined (Table 3).
2.3. Extension of the alkenes
The reaction with allyl acetate (entry 4) was perplexing. It was
assumed that this material would homo-dimerize to the 1,4-bu-
tene diol derivative faster than productive cross-metathesis would
occur. However, it is obvious that the dimer itself participated in
the reaction (entries 2 and 3), thus the lack of conversion to the de-
sired product was surprising. This could simply point to a relatively
short half life for the catalyst under these conditions. Perhaps not
unexpectedly, the Boc protected 21-vinyl compound (5) was an
excellent substrate for cross-metathesis, at least when compared
to other spinosyns. This result supported the idea that while pro-
tecting basic amines as salts can be effective, it is not an ideal solu-
tion and the salts themselves may have a detrimental effect on the
longevity of the catalyst or catalyst precursor. The low conversion
observed with the 21-vinyl-30-OEt spinosyn J (12) was also intrigu-
ing. This result may lend further support to the aforementioned
idea that a free hydroxyl group somehow facilitated the metathesis
reaction and that substrates lacking a free hydroxyl were not as
well suited for the reaction.11
2.3.1. Terminal olefins
We first investigated the use of terminal olefins to extend the C-
21 side chain, with 2d or 2e as starting materials (Table 2).
Under the conditions tried, most reactions gave mixtures of
the desired metathesis product and the 21-vinyl analog. Presum-
ably this is due, in part, to the reluctance of the spinosyn-based
olefin to undergo metathesis as well as the facility with which
these simple terminal dienes undergo homocoupling to the sym-
metrical dimer. This was indicated by the substantial amount of
stilbene recovered when using styrene as the coupling partner. It
was further expected that the internal olefin of the homodimer
would not be as reactive as its terminal counterpart. Also, as
the reactions became dark in color and began to precipitate solids,
it appeared that the catalyst was decomposing under the reaction
conditions no matter how much care was taken to exclude air
and moisture, further indicating that spinosyn is a reluctant
metathesis partner at best.13 Lastly, attempts to use vinyl cyclohex-
ane in the reaction gave only trace amounts of the 21-vinyl analog
while allyl cyclohexane coupled readily pointing to the influence of
steric interactions on the outcome of the reaction.
2.3.3. Incorporation of polar functionalities onto the C-21
spinosyn side chain
Methyl acrylate and acrylic acid were two additional cross-
metathesis substrates of interest as we envisioned a dramatic
change in physical properties that would accompany introduction
of an acidic functional group on the C21 side chain. A variety of
conditions and catalysts were screened using methyl acrylate as
the metathesis partner. As with the butene-1,4-diol derivative dis-
2.3.2. Symmetrical olefins
Derivatives of cis-2-butene-1,4-diol and certain other symmet-
ric internal olefins are reported to be excellent cross-metathesis
partners—often providing higher yields and greater trans selectiv-

J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
4201
Table 1
Comparison of amine salts of butenyl spinosyns in cross-metathesis with ethylene
N+
N
H
X
O
O
O
O
O
O
O
O
i, ii
OH
OH
O
O
H
H
H
H
H
H
R
O
O
H
H
O
O
H
H
O
O
O
O
3
1a, 1b, 2a-2e
N
O
O
O
O
iii
O
O
H
H
O
H
O
H
O
O
H
12
Reagents and conditions: i. Cross metathesis (see Table for conditions); ii NaHCO3; iii EtBr, KOH/Bu4NI
(10:1).
R
X
Catalyst
Solvent
Time (h)
Yield of 3
Comments
2
OH
OH
OH
OH
OH
H
Free base
(N-Oxide)
BF4
Acetate
Cl
Cl
TFA
TFA
20 mol % B
10 mol % B
20 mol % B
20 mol % B
10 mol % B or C
20 mol % B
20 mol % B
20 mol % B
CH2Cl2
CH2Cl2
CH2Cl2
ClCH2CH2Cl
CH2Cl2
CH2Cl2
ClCH2CH2Cl
CH2Cl2
14
16
3
2
4
14
2
4
ND
ND
ND
ND
65%
ND
83%
ND
Incomplete conversion
2a
2b
2c
2d
1a
2e
1b
50% Conversion to the 24-keto derivative
Incomplete conversion
Incomplete conversion
Complete conversion
Incomplete conversion
Complete conversion
OH
H
Incomplete conversion
ND = Not determined.
cussed above, all reactions employing methyl acrylate led to a mix-
ture of E/Z isomers of the desired compound (26) and the 21-vinyl
spinosyn J (3). The best result was obtained using the Grubbs/
Hoyveda catalyst (C) (Scheme 4).
Several attempts were made to saponify the methyl ester of 26
and the 30-O-alkylated derivative 27 but these were unsuccessful,
leading to degradation of the substrate. Unfortunately, metathesis
reactions using acrylic acid itself met with no success.
A second approach to modify the physical properties of the
spinosyn template through metathesis chemistry involved incor-
poration of a glycine residue into the reaction. There are reports
of suitably protected allyl glycine derivatives participating in olefin
cross-metathesis reactions.15
Cross-metathesis was attempted on Fmoc protected allyl gly-
cine (28)16 with several different C21-butenyl and vinyl spinosyns.
Success was achieved with the HCl salt of 12 using catalyst C
(Scheme 5) to give 29. As with many other metathesis reactions
discussed here, the reaction did not proceed to completion and
approximately 50% of the Fmoc protected allyl glycine ethyl ester
was isolated as the homodimer. Exposure of this homodimer to a
separate cross-metathesis using styrene indicated that it is a viable
metathesis partner thus again suggesting that catalyst decomposi-
tion may contribute to incomplete conversion in metathesis reac-
tions using spinosyns. Exposure of the product mixture of the
Fmoc derivative (29) and unreacted 21-vinyl starting material to
morpholine in tetrahydrofuran (THF) led to clean removal of the
protecting group and allowed for facile separation of the deprotec-
ted glycine analog (30), from the 21-vinyl species.
One compound that could not be made under any conditions we
tried was from reaction with acrylonitrile. This starting material is
known to be recalcitrant, and subsequently much work has gone
into devising procedures for successful CM with it.17 Application
of a variety of these procedures to the cross-metathesis of acrylo-
nitrile and various spinosyns failed to yield any of the required
product. A possible explanation is that while butenyl and vinyl
spinosyns can enter into productive cross-metathesis reactions,
they are not necessarily good alkylidene donors or very nucleo-
philic and when matched with other olefins that are not particu-
larly well suited for cross-metathesis, no reaction is observed.13
Also, the structural complexity of the spinosyns, including the
presence of two additional double bonds that can enter into the
metathesis cycle, may lead to non-productive metathesis events
that contribute to catalyst degradation. Other factors including
the presence of the amine salts and free hydroxyl groups may also
play a role in the success or lack thereof in these reactions.
2.4. Purification of spinosyn c1 (32)
Olefin cross-metathesis found an additional use within the
spinosyn project, enabling the chemical transformation of a mixture
of factors to allow for their chromatographic separation. During the
course of the purification of a large scale butenyl spinosyn fermen-
tation, several grams of a mixture comprised mainly of spinosyn c1
(32) and spinosyn d1 (31) along with several minor factors was set
aside as no chromatographic method, normal or reverse phase,
was found that allowed for their separation (Scheme 6).7

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J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
Table 2
Olefin cross-metathesis with butenyl spinosyns and terminal olefins
H
N+
N
O
O
O
O
O
O
Cl
O
O
i, ii
OH
OH
O
O
H
H
H
H
H
H
HO
O
O
H
H
R
O
O
H
H
O
O
O
O
13-17
2d or 2e
N
O
O
O
O
iii
O
O
O
O
H
H
H
O
H
R
H
O
18-22
Reagents and conditions: i Olefin (see Table), B, DCM; ii NaHCO3; iii, EtBr, KOH/Bu4NI (10:1).
Salt
Olefin
R (E/Z)
Time (h)
Producta
Yieldb (%)
Comments
TFA (2e)
HCl (2d)
TFA (2e)
HCl (2d)
1-Dodecene
1-Octene
1-Octene
1-Hexene
CH3(CH2)9 (10:1)
CH3(CH2)5 (3:1)
CH3(CH2)5 (6:1)
CH3(CH2)3 (8:1)
7
14
2
13 (18)
14 (19)
14 (19)
15 (20)
68
76
68
44
Complete conversion
3:1 Mixture 21-octenyl:21-vinyl
Complete conversion
9:1 Mixture of 21-hexenyl:21-vinyl. (22% 21-hexenyl-17-pseudoaglycone
also isolated)
2
TFA (2e)
TFA (2e)
TFA (2e)
TFA (2e)
Styrene
Phenyl (1:0)
4-O-Et-Phenyl (1:0)
PhCH2
Cyclohexyl-CH2-
(5:1)
3
6
7.5
5.5
16 (21)
17 (22)
61
1:1.5 Mixture of 21-styryl:21-vinyl. (60% stilbene recovered)
1:1 Mixture of 21-styryl:21-vinyl
3:1 Mixture 21-allylbenzyl:21-vinyl
Complete conversion
4-O-Et-Styrene
Allyl benzene
Allyl
ND
ND
61
cyclohexane
ND = not determined.
a
Figure in brackets corresponds to the final (ethylated) product.
Yield corresponds to that of the metathesis product.
b
Exposure of this mixture of spinosyns to our standard metathe-
sis protocol using ethylene gas and catalyst C, led to conversion of
the 31 into the 21-vinyl-6-methyl spinosyn J (33) while spinosyn
1 (32) remained unaffected. This subtle change allowed for facile
separation of the two major factors by reverse phase HPLC.
comparable activity to the standards (Table 4) except 10, which
was much less active. Testing of some of the other analogs in sev-
eral other bioassays all pointed to the styrene derivative (21) as
being the most active—about as active as the 30-O-ethyl analog of
spinosyn J.
c
2.5. Incorporation of 13C label
4. Concluding remarks
Increasing the size of the alkyl moiety at the 30-position of the
rhamnose significantly increases the activity of the spinosyns com-
pared to spinosyn A.18 In contrast, extending the size of the alkyl
moiety at the C21-position of the macrocycle produces little
improvement in activity relative to spinosyn A against larvae of
the tobacco budworm. In the present study, the effect of further
modifications to the butenyl moiety at C21 position was investi-
gated via cross-metathesis. The spinosyn derivatives resulting
from the cross-metathesis allowed further exploration of the space
around the C21 position of the spinosyn macrocycle. With the
exception of the large modification to the forosamine nitrogen
[Teoc] in compound 10, all of the resultant analogs exhibited high
levels of biological activity. Several analogs possessed activity sim-
ilar to that of spinosyn J, while a few were similar in activity to the
30-O-ethyl analog of spinosyn A. Because almost all of the cross-
metathesis derived analogs had an O-ethyl moiety in the 30-posi-
tion of the rhamnose, comparison to the 30-O-ethyl analog of spino-
syn J or its C21-2-butenyl homolog was most appropriate. As such
Further utility for the cross-metathesis reaction was found in
the preparation of a stable isotope derivative of the spinosyns
(Scheme 7). Exposure of 24-hydroxy-butenyl spinosyn J (2) to 13C
labeled ethylene in the presence of catalyst C led to modest conver-
sion to 3 containing a 13C label at the terminal olefin carbon atom.
The reaction did not go to completion and the yield was further
lowered by some loss of the forosamine to give the 17-pseudoagly-
cone. Nevertheless, this labeled material could be further manipu-
lated to furnish a stable isotope standard for studies of spinosyn
(via methylation of the rhamnose alcohol and selective reduction
of the terminal olefin) or Spinetoram (via ethylation of the alcohol
and reduction of the terminal and the 5,6 olefins).
3. Biological considerations
The activity of a selection of these new spinosyn derivatives was
compared with spinosyn A and 30-O-ethyl spinosyn J. In the neo-
nate tobacco budworm (TBW) bioassay all compounds tested had

J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
4203
Table 3
Conversion of 21-butenyl and 21-vinyl spinosyns to 24-acetoxy-propenyl spinosyns
R2
R2
N
H
N+
O
O
O
O
O
O
Cl
O
O
R3
R3
O
i, ii
O
O
O
O
H
H
H
H
H
H
O
O
H
H
R1
O
O
H
H
O
O
O
O
Ac
23 R2 = Me, R3 = H
24 R2 = Boc, R3 = H
25 R2 = Me, R3 = Et
Reagents and conditions: i Olefin and catalyst (see Table), DCM; ii NaHCO3
Starting Material
R1
Et
R2
R3
H
X
Olefin
AcO
Catalyst
A
Product
Yield (E/Z)
Comments
1
Me
Free base
ND
No reaction (85% recovered 1)
OAc
OAc
2d
CH(OH)CH3
Me
H
Cl
B
23
23
20% (2:1)
Trace 21-vinyl (50% recovered 2d)
25% Recovered 2e
AcO
2e
2e
5
CH(OH)CH3
CH(OH)CH3
Me
Me
H
H
TFA
TFA
B
B
60% (4:1)
ND
AcO
AcO
OAc
45% Conversion to 21-vinyl spinosyn
J (51% recovered 2e)
H
H
Boc
Me
H
NA
B
B
24
25
92% (all Z)
Complete conversion
50% Recovered 12
AcO
AcO
OAc
OAc
12
Et
TFA
40% (1:1)
ND = not determined.
H
N+
N
O
O
O
O
O
Cl
O
O
i
O
OH
OH
O
O
O
H
H
H
H
H
HO
O
O
O
H
H
O
O
H
H
O
O
O
O
+
3
H
2d
26 (3:1 E/Z)
ii
N
O
O
O
O
O
O
H
H
H
O
O
H
O
H
O
O
O
27
Scheme 4. CM with methyl acylate. Reagents and conditions: (i) methyl acrylate, 20 mol % C, DCM, 48 h; (ii) EtBr, KOH/Bu4NI (10:1).
it is interesting that further substitution at the terminus of the C21
most often only resulted in a modest reduction in activity with sev-
eral of the analogs having activity near that of the reference com-
pounds (i.e., 19, 20 and 21). Of particular interest is the styryl
derivative (21) the activity for which was as good as the 30-O-ethyl
analog of spinosyn J, and which is a significant new finding for SAR

4204
J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
N+
N
H
O
O
O
O
O
Cl
O
O
i
O
O
O
O
O
H
H
H
H
H
H
O
O
H
O
H
O
H
ii
O
O
H
O
O
29
O
Fmoc
N
O
12 (HCl salt)
+
N
O
O
O
O
Fmoc
N
H
O
O
O
O
O
H
H
H
O
H
O
H
O
O
28
30
NH2
O
Scheme 5. Reagents and conditions: (i) 20 mol % C, DCM, 48 h; (ii) morpholine, THF, 70%.
N
N
O
O
O
O
O
O
O
i, ii
O
OH
OH
O
H
H
H
O
H
+
H
H
O
O
H
O
H
H
O
O
O
H
O
O
33
Spinosyn δ1 31
+
N
O
O
O
OH
O
O
O
32 (unreacted)
H
H
O
H
H
O
O
Spinosyn γ1 32
Scheme 6. CM of 31 in presence of 32. Reagents and conditions: (i) HCl, Et2O; (ii) 20 mol % C, C2H4, DCM.
N
N
O
O
O
O
O
O
O
O
i, ii
OH
OH
O
O
H
H
H
H
H
H
HO
*
O
O
H
H
O
O
H
H
O
O
O
O
[23-13C] 3
2
Scheme 7. Preparation of 13C labeled spinosyn J. Reagents and conditions: (i) HCl, Et2O; (ii) 20 mol % C, 13C2H4, DCM.

J. Daeuble et al. / Bioorg. Med. Chem. 17 (2009) 4197–4205
4205
Table 4
mined five days after treatment. LC50s were again calculated using
the method of Finney.20
Biological activity of selected novel spinosyns
Compound
Neonatea TBW
LC50 (ppm)
BAWb oral
TBWc leaf
LC50 (ppm)
CLd leaf, LC50
(ppm)
LC50
(l
g/cm2)
Acknowledgments
11
12
19
20
21
25
27
10
0.2
0.5
—
—
—
2.0
0.2
47.2
—
0.018
0.022
<0.012
0.013
—
—
—
0.22
1.46
0.37
0.17
—
0.13
0.12
0.13
0.05
—
The authors thank D’Lee McCormick for assistance with the
bioasays, and Paul Lewer and Carl DeAmicis for isolating the start-
ing materials used in this study.
—
—
—
—
—
—
Supplementary data
30-OEt-J
Spin A
0.28
0.42
0.012
0.079
0.15
0.43
0.048
0.10
Supplementary data (Details of synthetic methods) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.02.036.
a
Neonate; TBW—tobacco budworm larvae—neonate drench bioassay.
BAW oral—beet armyworm—diet feeding assay.
TBW leaf—TBW leaf disk bioassay.
b
c
d
References and notes
CL leaf—Cabbage Looper leaf disk bioassay.
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development. These observations suggest that there is some rea-
sonable latitude in the size and substitution extending out from
the C21 position that does not readily interfere with the biological
activity.
5. Experimental
5.1. Spectroscopic methods
NMR spectra were recorded on a Varian Gemini 300 MHz spec-
trometer operating at 299.7 MHz or a Bruker DRX600 spectrometer
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tions were achieved using a Hewlett–Packard 1100 LC system. LC/
MS was performed on a Micromass Platform single-quadrupole
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Neonate tobacco budworm (TBW, Heliothis virescens) larvae
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the test compounds (dissolved in 50
lL of 90:10 acetone–water).
Each dose had eight replicates, and a range of doses were used to
estimate the LC50s. Controls were treated with solvent only. Trea-
ted trays were covered with a clear self-adhesive cover and held
at 25 °C under a 14:10 light:dark regimen in a light chamber.
The average percent mortality for the eight wells for each dose
was determined six days after treatment. LC50s were calculated
using the method of Finney.20 Second instar tobacco budworm or
cabbage looper (CL, Trichoplusia ni) larvae were placed on leaf disks
(six per dose, squash) that had been treated (compound in 250 lL
of 90:10 acetone–water) and allowed to dry. Controls were treated
with solvent only. Infested leaf disks were held in 6-well microtiter
plates covered with a plastic lid at 25 °C under a 14:10 light:dark
regimen as above. Percent mortality of the six replicates was deter-
20. Finney, D. J. Probit Analysis, 3rd ed.; Cambridge University Press: New York,
1971.