Avermectins and flea control: Structure-activity relationships and the selection of selamectin for development as an endectocide for companion animals

Article abstract of DOI:10.1016/S0968-0896(00)00120-6

Evaluation of a wide range of avermectin derivatives for flea activity in an in vitro feeding screen using the cat flea, Ctenocephalides felis, revealed a narrow structure-activity relationship (SAR) with activity surprisingly associated with monosaccharides and especially their C-5-oximes. We discovered commercially exploitable flea activity in a single compound, selamectin 33, which also possessed the necessary antiparasitic spectrum and margin of safety for development as a broad-spectrum companion animal endectocide. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00120-6

Bioorganic & Medicinal Chemistry 8 (2000) 2017±2025
Avermectins and Flea Control: Structure±Activity Relationships
and the Selection of Selamectin for Development as an
Endectocide for Companion Animals
B. J. Banks,* B. F. Bishop, N. A. Evans, S. P. Gibson, A. C. Goudie,
K. A. F. Gration, M. S. Pacey, D. A. Perry and M. J. Witty
P®zer Central Research, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
Received 27 January 2000; accepted 4 May 2000
AbstractÐEvaluation of a wide range of avermectin derivatives for ¯ea activity in an in vitro feeding screen using the cat ¯ea,
Ctenocephalides felis, revealed a narrow structure±activity relationship (SAR) with activity surprisingly associated with mono-
saccharides and especially their C-5-oximes. We discovered commercially exploitable ¯ea activity in a single compound, selamectin
33, which also possessed the necessary antiparasitic spectrum and margin of safety for development as a broad-spectrum compa-
nion animal endectocide. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Avermectins and milbemycins have been the subjects of
extensive research and are known to bind to chloride
channels in a wide range of invertebrates and verte-
brates.1 However, their antiparasitic potencies are also
known to be dramatically altered by (sometimes minor)
structural modi®cation.2 We therefore screened a wide
range of compounds in an in vitro feeding screen using
the cat ¯ea, Ctenocephalides felis. Close analogues of the
¯ea active structures discovered were then prepared and
the optimum compound, selamectin 33, was identi®ed.
Extensive evaluation demonstrated that the compound
satis®ed all our antiparasitic spectrum and safety criteria
(including safety in collies) and it was developed as a
safe, broad-spectrum endectocide for cats and dogs.3
Commercial products containing avermectins (e.g. Dec-
tomax1 and Ivomec1) or milbemycins (e.g. Cydectin1)
are well established for the treatment of a wide range of
both endo- and ecto-parasite infections of livestock and
hence are referred to as endectocides. Although safe for
use as an endectocide in livestock, ivermectin 2 pro-
duces idiosyncratic toxicity in collie dogs and cross-
bred collies at doses required to give e€ective control
of gastrointestinal nematodes. Its use in dogs (as
HeartgardTM), at the very low dose required for safety
in all dog breeds, provides only prophylactic control of
heartworm. Milbemycin oxime 20 has also been com-
mercialised (as Interceptor1). The compound's greater
safety permits doses that provide additional control of
gastrointestinal nematodes but it is ine€ective against
¯eas, the major ectoparasite of commercial importance
of cats and dogs. We set ourselves the objective of
®nding a safe, broad-spectrum endectocide for compa-
nion animals from this class of macrolides. Most
importantly we wished to identify a compound which
had systemic in vivo activity against ¯eas. Prior to the
inception of our work, signi®cant ¯ea activity was not
associated with the structural class.
Results
The majority of the compounds evaluated have been
previously reported and are referenced in the Tables.
Syntheses of novel avermectin derivatives, which gen-
erally follow reported strategies,4 are shown in Schemes 1
and 2. C-5-oximes and related compounds were obtained
by selective oxidation employing manganese dioxide fol-
lowed by treatment of the crude ketone so obtained with
the appropriate nitrogen nucleophile (Scheme 1).
C-40-deoxygenation of the sugar was achieved via selec-
tive protection of the C-5-alcohol as its tert-butyldi-
methylsilyl ether and thiocarbonylation of the C-40-
*Corresponding author. Tel.:+44-1304-641106; fax:+ 44-1304-656595;
e-mail: bernard_banks@sandwich.p®zer.com
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00120-6

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B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
Scheme 1. Synthesis of oximes 13, 14, 23 and 39 and semicarbazone 42. Reagents and conditions: i. manganese dioxide, diethyl ether, 25 ꢀC, 3±20 h;
ii. 12.2±27.6 equiv of hydroxylamine hydrochloride, 30 equiv of methoxylamine hydrochloride or 5.43 equiv of semicarbazide hydrochloride,
methanol, dioxan, water, 25±50 ꢀC, 2±48 h, 9±66% for two steps.
Table 1. Avermectins, milbemycins and C-5-oxime derivatives
Compound number
R1
R2
X±Y
Z
1,5,a
2,6,b
3,7,c
4,7,d
58,e
69
ds1
ds1
ds2
ds3
ds1
ds1
ds1
ds1
ds1
ds1
ds1
ds1
ds1
ds1
ds1
ds1
H
sec-Butyl
sec-Butyl
sec-Butyl
CH=CH
CH2-CH2
CH=CH
CH=CH
CH=CH
CH2-CH2
CH( OH)
CH( OH)
CH( OH)
CH( OH)
CH( OH)
CH( OH)
CH( OH)
CH( OH)
CH( OCH3)
CH( OCH3)
C=NOH
C=NOH
C=NOH
C=NOH
C=NOH
C=NOH
CH( OH)
sec-Butyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
sec-Butyl
78
CH2CH( OH)
CH2CH( OCH3)
CH=CH
CH2CH( OH)
CH=CH
CH2-CH2
CH=CH
CH2-CH2
CH2CH( OH)
CH2CH( OCH3)
CH2-CH2
810
98
108
1111
1212
13
sec-Butyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Isopropyl
14
1511
1610
1713
1814
H
H
CH2CH( OH)
CH( OH)
CH( OH)
1915,f
CH2C(=NOCH3)
2016,g
2116
H
H
Methyl/ethyl
Isopropyl
CH2-CH2
CH2-CH2
C=NOH
C=NOH
2217
23
H
H
CH2CH( OH)
C=NOH
C=NOH
CH2C(=NOCH3)
aAbamectin.
bIvermectin.
cEmamectin.
dEprinomectin.
eDoramectin.
fMoxidectin.
gMilbemycin oxime.

B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
2019
Scheme 2. Synthesis of oxime 45. Reagents and conditions: i. 1.43 equiv of tert-butyldimethylsilyl chloride, 2.2 equiv of imidazole, dimethylforma-
mide, 25 ꢀC, 3.5 h, 60%; ii. 2 equiv of para-tolyl chlorothionoformate, pyridine, dichloromethane, 25 ꢀC, 3 h; iii. 89.2 equiv of tributyltin hydride,
0.01 equiv of 2,20-azobis(isobutyronitrile), toluene, 110 ꢀC, 1 h; iv. 1.31 equiv of para-toluenesulphonic acid, methanol, 25 ꢀC, 35% over 3 steps; v.
(a). manganese dioxide, diethyl ether, 25 ꢀC, 22 h; (b). 11.7 equiv of hydroxylamine hydrochloride, methanol, dioxan, water, 25±40 ꢀC, 4 h, 76% over
2 steps.
Discussion
alcohol. Tributyltin hydride reduction of the thiocarbo-
nate, desilylation with para-toluenesulphonic acid in
methanol, followed by oxidation and oxime formation
completed the sequence (Scheme 2).
The avermectins, milbemycins and C-5-oxime derivatives
shown in Table 1 were devoid of activity in the ¯ea feeding
assay at the initial screening dose of 1mg/mL. Their MDDs
were not determined as a value of 0.25 mg/mL in this assay
provides an activity threshold; only compounds of at least
this potency were subsequently e€ective against ¯eas on
cats and dogs at doses up to the limit imposed for viability
as a commercial product. Inspection of Table 2 shows that
this threshold level of in vitro activity is only found
amongst monosaccharide C-5-oximes. The most potent in
vitro ¯ea active (MDD 0.1mg/mL) was 25-cyclohexyl-25-
de(1-methylpropyl)-5-deoxy-22,23-dihydro-5-(hydroxy-
amino)-avermectin B1a monosaccharide (selamectin) 33.
Compounds with alternative C-5 substituents, prepared
The insecticidal activity of avermectin derivatives
against ¯eas was determined using an in vitro feeding
assay employing the cat ¯ea Ctenocephalides felis. The
selection of this model, in which ¯eas feed through an
arti®cial membrane upon blood containing the test com-
pound, is consistent with our objective of identifying
systemically active molecules. Potency of a test com-
pound is most conveniently expressed as its minimum
detectable dose (MDD), which is de®ned as the con-
centration which caused at least 30% mortality at 24 h
post-feeding.

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B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
Table 2. Avermectin monosaccharides and C-5-oxime derivatives
Table 4. 25-Cyclohexyl-25-de(1-methylpropyl)-5-deoxy-22,23-dihy-
dro-5-(hydroxyimino)-avermectin B1a monosaccharide derivatives.
Compound
number
R
X±Y
Z
Flea MDD
mg/mL
Compound number
X
Y
Z
Flea MDD mg/mL
2418
2518
2611
2711
2811
2910
3011
3111
3211
3311,a
3411
3510
3611
sec-Butyl
sec-Butyl
Cyclohexyl
Cyclohexyl
CH=CH
CH2-CH2
CH=CH
CH2-CH2
CH( OH)
CH( OH)
CH( OH)
CH( OH)
*
b
4311
4411
45
4611
4711
4811
4922
OH
H
H
CH3
H
NHAc
H
H
OAc
H
OH
NHAc
H
H
0.25
a
*
*
1
*
1
H
H
H
H
H
H
*
*
*
*
*
1
Cyclohexyl CH2CH( OH) CH( OH)
Cyclohexyl CH2CH( OCH3) CH( OH)
sec-Butyl
sec-Butyl
Cyclohexyl
Cyclohexyl
Cyclohexyl CH2CH( OH)
CH=CH
CH2-CH2
CH=CH
CH2-CH2
C=NOH
C=NOH
C=NOH
C=NOH
C=NOH
1
OH
0.25
0.25
0.1
0.25
1
a*Inactive at 1 mg/mL, MDD not determined.
Cyclohexyl CH2CH( OCH3) C=NOH
Cyclohexyl CH2C(=NOCH3) C=NOH
tion of disaccharides which is tolerant to substitution,
with, for example, 400-O-acetyl and 400-epi-acetylamino-400-
deoxy derivatives retaining full potency. The report that
disaccharides epimeric at C-13 possess full anthelmintic
1
aSelamectin.
b*Inactive at 1 mg/mL, MDD not determined.
24
potency coupled with an improved margin of safety
Table 3. C-5-derivatives of 25-cyclohexyl-25-de(1-methylpropyl)-5-
deoxy-22,23-dihydro-avermectin B1a monosaccharide.
prompted the preparation of the ®nal compound 49, the
C-13 epimer of 33. Whilst ¯ea activity was retained with
this modi®cation, potency was substantially reduced.
From this work it was concluded that 33 was uniquely
potent and the compound was selected for development.
Conclusion
Systematic evaluation of avermectin derivatives for ¯ea
activity has revealed a narrow SAR with activity sur-
prisingly associated with monosaccharides and especially
their C-5-oximes. We have discovered commercially
useful levels of activity in one compound, selamectin 33,
which also has the necessary antiparasitic spectrum and
margin of safety for development as a broad-spectrum
companion animal endectocide.3
Compound number
X
Flea MDD mg/mL
3719
3819
39
4020
4121
42
O
CH2
NOMe
*
0.25
a
1
*
*
*
NNH2
NNMe2
NNHCONH2
Experimental
General
a*Inactive at 1 mg/mL, MDD not determined.
in a follow-up series, all possessed inferior activity
(Table 3).
All common reagents and solvents were purchased at
highest commercial quality and used without further
puri®cation. All solutions used in work up procedures
were saturated. All reactions were monitored by thin-
layer chromatography carried out on 0.25 mm E. Merck
silica gel plates (60F-254) using UV light as visualising
agent. E. Merck silica gel (kieselgel 60, 230-400 mesh)
was used for column chromatography. Yields, which are
unoptimised, refer to chromatographically and spectro-
scopically (1H NMR) homogenous materials.
To probe the structure±activity relationship (SAR) of
the sugar moiety, a series of C-5-hydroxyimino-mono-
saccharides, each with a single modi®cation at C-40 was
prepared. Inspection of Table 4 again reveals inferior
activity for these analogues, with only the epimeric alco-
hol 43 retaining moderate potency. This is in contrast to
the antiparasitic SAR reported in the literature23 and
generated in our laboratories for the corresponding posi-

B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
2021
1H NMR spectra were acquired on a Varian Inova 500
spectrometer in CDCl3 at 30ꢀC using 3 mm tubes and
calibrated using residual undeuterated solvent as an
internal reference. The following abbreviations were used
to explain the multiplicities: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet. High resolution mass spectra
(HRMS) were recorded on a Micromass Autospec Q
under electrospray conditions.
25-Cyclohexyl-5-demethoxy-25-de(1-methylpropyl)-22,
23-dihydro-5-(hydroxyimino)-avermectin A1a (14).
A
solution of alcohol 69 (334 mg, 0.37 mmol) in diethyl
ether (20 mL) containing a suspension of activated
manganese dioxide (670 mg) was stirred at 25 ꢀC for
15 h. A further portion of manganese dioxide (334 mg)
was added and stirring continued for a further 2 h after
which the mixture was ®ltered through Celite1 and
evaporated to dryness under reduced pressure. The
residue was dissolved in a mixture of dioxan (10 mL)
and methanol (10 mL). A solution of hydroxylamine
hydrochloride (314 mg, 4.52 mmol) in water (10 mL)
was added and the mixture was stirred at 50 ꢀC for 2 h.
The cooled mixture was concentrated under reduced
pressure to remove the methanol and dioxan and then
extracted with diethyl ether (50 mLÂ2). The organic
phases were combined, washed with water (50 mL) and
brine (50 mL), dried (Na2SO4) and evaporated under
reduced pressure to provide a colourless glass (669 mg).
The crude product was puri®ed by column chromato-
graphy on silica gel (25 g, dichloromethane:ethyl acetate,
4:1, v/v) and then reverse phase preparative HPLC
25-Cyclohexyl-5-demethoxy-25-de(1-methylpropyl)-5-
(hydroxyimino)-avermectin A1a (13). A solution of alco-
hol 58 (500 mg, 0.556 mmol) in diethyl ether (50 mL)
containing a suspension of activated manganese dioxide
(1 g) was stirred at 25 ꢀC for 15 h. A further portion of
manganese dioxide (200 mg) was added and stirring
continued for a further 3 h after which the mixture was
®ltered through Celite1 and evaporated to dryness under
reduced pressure. The residue was dissolved in a mixture of
dioxan (20 mL) and methanol (20 mL). A solution of
hydroxylamine hydrochloride (538 mg, 7.74 mmol) in
water (20 mL) was added and the mixture was stirred at
50 ꢀC for 2 h. The cooled mixture was poured into a
mixture of diethyl ether (50 mL) and water (50mL). The
layers were separated and the organic phase was washed
with water (50 mL) and brine (50 mL). The aqueous phase
was extracted with diethyl ether (50mLÂ2). The organic
phases were combined, dried (Na2SO4) and evaporated
under reduced pressure to provide a white solid
(552 mg) which was puri®ed by column chromatography
on silica gel (50g, dichloromethane:ethyl acetate, 2:1, v/v)
to provide oxime 13 (296 mg, 58%) as an amorphous
white solid: Rf=0.24 (dichloromethane:ethyl acetate,
(250Â21.2 mm diameter Zorbax1 8 m ODS column
1
at ambient temperature and eluted at 9 mL min
,
methanol:water, 90:10, v/v) to provide oxime 13 (141 mg,
42%) as an amorphous white solid: Rf=0.25 (dichloro-
1
methane:ethyl acetate, 1:1, v/v); H NMR (500 MHz,
CDCl3) d 8.22 (broad s, 1H, NOH), 5.95 (dt, J=10.4,
2.4 Hz, 1H, C=CHCH= CHCH), 5.83 (m, 1H,
CH=C(CH3)C=N), 5.78 (dd, J=9.4, 15.0 Hz, 1H, C=
CHCH=CHCH), 5.74 (dd, J=10.4, 15.0 Hz, 1H, C=
CHCH=CHCH), 5.46±5.39 (m, 1H, CHOCˆO), 5.40
(d, J=3.2 Hz, 1H, CH(O)(O) CH2), 4.99 (broad d,
J=10.7 Hz, 1H, CH2CH=C(CH3)), 4.79 (d, J=4.2 Hz,
1H, CH(O)(O)CH2), 4.76 (dd, J=14.4, 2.4 Hz, 1H,
OCHH), 4.70 (dd, J=14.4, 2.4 Hz, 1H, OCHH), 4.68 (s,
1H, CHC=NOH), 3.98 (broad s, 1H, COH), 3.95
(broad s, 1H, CH(O)), 3.86±3.81 (m, 1H, CH(O)(CH2)
(CH2)), 3.84 (dq, J=9.0, 6.3 Hz, 1H, CH(O)(CH3)CH),
3.77 (dq, J=9.2, 6.3 Hz, 1H, CH(O) (CH3)CH), 3.63
(ddd, J=13.3, 9.0, 4.8 Hz, 1H, CH (OCH3)), 3.49 (ddd,
J=13.6, 9.2, 4.9 Hz, 1H, CH(OCH3)), 3.42 (s, 6H,
2ÂOCH3) 3.41 (quintet, J=2.3 Hz, 1H, CHCˆO), 3.25
(t, J=9.0 Hz, 1H, CH(O)(CH)(CH)), 3.17 (t, J=9.2 Hz,
1H, CH(OH)(CH)(CH)), 3.08 (broad d, J=10.0 Hz, 1H,
CHC6H13), 2.60 (broad s, CHOH, 1H), 2.56±2.49 (m,
1H, C=CHCH=CHCHCH3), 2.35±2.22 (m, 4H), 1.97
(ddd, J=11.9, 5.2, 1.4 Hz, 1H, CHHC), 1.95±1.94 (m,
3H, CH=C(CH3)C=N), 1.81±1.78 (m, 3H), 1.66±1.43
(m, 12H), 1.50 (broad s, 3H, CH2CH= CCH3), 1.37 (t,
J=11.9 Hz, 1H, CHHC), 1.30±1.14 (m, 4H), 1.28 (d,
J=6.3Hz, 3H, OCHCH3), 1.26 (d, J=6.3Hz, 3H,
OCHCH3), 1.17 (d, J=7.0 Hz, 3H, C= CHCH=CH
CHCH3), 0.85 (q, J=12.0 Hz, 1H, CHHCH OCˆO), 0.80
(broad d, J=5.4 Hz, 3H, CHCH3); HRMS, calcd for
C50H75NO14 (M+Na+) 936.508527, found 936.512494.
1
1:1, v/v); H NMR (500 MHz, CDCl3) d 8.11 (broad s,
1H, NOH), 5.96 (dt, J=10.4, 2.4 Hz, 1H, C=
CHCH=CHCH), 5.83±5.82 (m, 1H, CH=C(CH3)
C=N), 5.84 (dd, J=9.4, 15.4Hz, 1H, C=CHCH=
CHCH), 5.74 (dd, J=9.8, 2.8 Hz, 1H, CH=CHCH), 5.70
(dd, J=10.4, 15.4 Hz, 1H, C= CHCH=CHCH), 5.55 (dd,
J=9.8, 2.5Hz, 1H, CH= CHCH), 5.50±5.44 (m, 1H,
CHOCˆO), 5.40 (d, J= 3.4 Hz, 1H, CH(O)(O)CH2),
5.01 (broad d, J=10.1 Hz, 1H, CH2CH=C(CH3)), 4.79
(d, J=4.0 Hz, 1H, CH(O)(O)CH2), 4.76 (dd, J=14.6,
2.4 Hz, 1H, OCHH), 4.70 (dd, J=14.6, 2.4 Hz, 1H,
OCHH), 4.68 (s, 1H, CHC=NOH), 3.95 (broad s, 1H,
COH), 3.90 (broad s, 1H, CH(O)), 3.91±3.82 (m, 1H,
CH(O)(CH2)(CH2)), 3.83 (dq, J=9.0, 6.3 Hz, 1H, CH(O)
(CH3)CH), 3.77 (dq, J=9.2, 6.3 Hz, 1H, CH(O)(CH3)
CH), 3.63 (ddd, J=13.3, 9.0, 4.8 Hz, 1H, CH(OCH3)),
3.49 (ddd, J=13.7, 9.2, 4.8 Hz, 1H, CH(OCH3)), 3.43 (s,
3H, OCH3), 3.42 (s, 3H, OCH3) 3.42 (quintet, J=2.3Hz,
1H, CHCˆO), 3.32 (d, J=10.0 Hz, 1H, CHC6H13), 3.25
(t, J=9.0Hz, 1H, CH(O)(CH)(CH)), 3.17 (t, J=9.2 Hz,
1H, CH(OH)(CH) (CH)), 2.80 (broad s, CHOH, 1H),
2.57±2.50 (m, 1H, C=CHCH=CHCHCH3), 2.35±2.22
(m, 5H), 2.01 (ddd, J=12.1, 4.8, 2.5 Hz, 1H, CHHC),
1.95±1.94 (m, 3H, CH=C(CH3)C=N), 1.83±1.77 (m,
3H), 1.70±1.48 (m, 8H), 1.50 (broad s, 3H, CH2CH=
CCH3), 1.33±1.19 (m, 4H), 1.28 (d, J=6.3 Hz, 3H,
OCHCH3), 1.26 (d, J=6.3 Hz, 3H, OCHCH3), 1.18 (d,
J=7.0 Hz, 3H, C= CHCH=CHCHCH3), 0.93 (d,
J=7.3 Hz, 3H, CH= CHCHCH3), 0.87 (q, J=12.0 Hz,
1H, CHHCHOCˆO); HRMS, calcd for C50H73NO14
(M+Na+) 934.492877, found 934.489348.
5-Demethoxy-23,28-dideoxy-25-(1,3-dimethyl-1-butenyl)-
6,28-epoxy-5-(hydroxyimino)-23-(methoxyimino)-milbe-
mycin B (23). A solution of methoxime 1915 (250 mg,
0.391 mmol) in diethyl ether (5 mL) containing a suspen-
sion of activated manganese dioxide (500mg) was stirred
at 25 ꢀC for 2 h. A further portion of manganese dioxide

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B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
(500 mg) was added and stirring continued for a further
0.5 h after which the mixture was ®ltered through Celite1
and evaporated to dryness under reduced pressure. The
residue was dissolved in a mixture of dioxan (12 mL)
and methanol (6 mL). A solution of hydroxylamine
hydrochloride (750 mg, 10.79 mmol) in water (6 mL)
was added dropwise over 5 min and the mixture was
stirred at 25 ꢀC for 48 h. The mixture was concentrated
under reduced pressure to remove the methanol and
dioxan and then extracted with diethyl ether (50 mLÂ3).
The organic phases were combined, washed with water
(50 mL) and brine (50 mL), dried (MgSO4) and evapo-
rated under reduced pressure. The crude product was
puri®ed by column chromatography on silica gel (5 g,
dichloromethane/diethyl ether 0!5%, v/v) and then
reverse phase preparative HPLC (250Â21.4 mm dia-
carbonate solution and then extracted with diethyl ether
(50 mLÂ3). The organic phases were combined, washed
with water (50 mL) and brine (50 mL), dried (Na2SO4)
and evaporated under reduced pressure. The crude pro-
duct was puri®ed by reverse phase preparative HPLC
(250Â21.4 mm diameter Rainin Dynamax1 Micro-
sorbTM 5 m ODS column maintained at 40 ꢀC and eluted
at 20 mL min 1, methanol:water, 90:10, v/v) to provide
oxime 39 (86 mg, 33%) as an amorphous white solid:
1
Rf=0.39 (dichloromethane:ethyl acetate, 4:1, v/v); H
NMR (500 MHz, CDCl3) d 5.93 (dt, J=10.4, 2.4 Hz, 1H,
C=CHCH=CHCH), 5.79±5.78 (m, 1H, CH=
C(CH3)C=N), 5.76 (dd, J=9.4, 15.0 Hz, 1H, C=
CHCH=CHCH), 5.73 (dd, J=10.4, 15.0 Hz, 1H, C=
CHCH=CHCH), 5.46±5.39 (m, 1H, CHOCˆO), 4.98
(broad d, J=10.9 Hz, 1H, CH2CH=C(CH3)), 4.83 (d,
J=3.6 Hz, 1H, CH(O)(O)CH2), 4.72 (dd, J=14.4, 2.4 Hz,
1H, OCHH), 4.66 (dd, J=14.4, 2.4Hz, 1H, OCHH), 4.56
(s, 1H, CHC=NOH), 4.00 (s, 3H, C=NOCH3), 3.96
(broad s, 1H, COH), 3.91 (broad s, 1H, CH(O)), 3.86 (dq,
J=9.2, 6.2 Hz, 1H, CH(O)(CH3)CH), 3.65 (tdd, J=11.8,
4.6, 1.9 Hz, 1H, CH(OCH3)), 3.49 (ddd, J=13.6, 9.0,
4.8 Hz, 1H, CH(OCH3)), 3.47 (s, 3H, OCH3), 3.38 (quintet,
J=2.4 Hz, 1H, CHCˆO), 3.16 (t, J=9.2 Hz, 1H, CH(O)
(CH)(CH)), 3.07 (broad d, J=7.6 Hz, 1H, CHC6H13),
2.55±2.49 (m, 1H, C=CHCH= CHCHCH3), 2.51
(broad s, CHOH, 1H), 2.34±2.22 (m, 3H), 1.97 (ddd,
J=12.0, 5.1, 1.5 Hz, 1H, CHHC), 1.95±1.94 (m, 3H,
CH=C(CH3)C=N), 1.81±1.77 (m, 3H), 1.66±1.42 (m,
11H), 1.57 (broad s, 3H, CH2CH=CCH3), 1.37 (t,
J=12.0 Hz, 1H, CHHC), 1.30±1.14 (m, 4H), 1.27 (d,
J=6.2Hz, 3H, OCHCH3), 1.16 (d, J=6.9Hz, 3H,
C=CHCH=CHCHCH3), 0.82 (q, J=11.8 Hz, 1H, CHH
CHOCˆO), 0.80 (broad d, J=5.5Hz, 3H, CHCH3);
HRMS, calcd for C44H65NO11 (M+Na+) 806.445532,
found 806.445465.
meter Rainin Dynamax1 MicrosorbTM 5 m ODS col-
umn maintained at 40 ꢀC and eluted at 10 mL min
,
1
acetonitrile:methanol:water, 50:33:17, v/v/v) to provide
oxime 23 (24 mg, 9%) as an amorphous white solid :
Rf=0.54 (dichloromethane:ethyl acetate, 4:1, v/v); 1H
NMR (500MHz, CDCl3) d 8.03 (broad s, 1H, NOH), 5.86
(dt, J=11.5, 2.4 Hz, 1H, C=CHCH=CHCH), 5.83± 5.82
(m, 1H, CH=C(CH3)C=N), 5.75 (dd, J=11.5, 14.6Hz,
1H, C=CHCH=CHCH), 5.42±5.35 (m, 1H, CHOCˆO),
5.36 (dd, J=10.3, 14.6 Hz, 1H, C=CHCH=CHCH),
5.18 (broad dd, J=9.0, 1.0 Hz, 1H, CH(CHCH3)2), 4.92
(broad dd, J=9.8, 3.7 Hz, 1H, CH2CH=C(CH3)), 4.76
(dd, J=14.3, 2.4 Hz, 1H, OCHH), 4.71 (dd, J=14.3,
2.4 Hz, 1H, OCHH), 4.66 (s, 1H, CHC=NOH), 3.84 (s,
3H, C=NOCH3), 3.77 (broad s, 1H, COH), 3.62 (d,
J=10.3 Hz, 1H, OCH) 3.50±3.45 (m, 1H, CH(O)(CH2)
(CH2)), 3.40 (quintet, J=2.4 Hz, 1H, CHCˆO), 3.29 (d,
J=14.8 Hz, 1H, CHHC=N), 2.65±2.55 (m, 1H,
CH(CH3)s), 2.47±2.38 (m, 1H, C=CHCH=CHCH
CH3), 2.31 (dq, J=10.3, 6.6 Hz, 1H, CHCH3), 2.26±2.18
(m, 3H), 2.15 (ddd, J=11.9, 5.2, 1.4 Hz, 1H, CHHC), 1.94
(m, 3H, CH=C(CH3)C=N), 1.92 (d, J=14.8 Hz, 1H,
CHHC=N), 1.87 (t, J=12.6, Hz, 1H, CHHC
(CH3)=CH), 1.81 (broad d, J=12.0 Hz, 1H, CHHC
HOCˆO), 1.66 (m, 3H, C(CH3)=CHCH(CH3)2), 1.51 (s,
3H, CH2CH=CCH3), 1.44 (t, J=11.9 Hz, 1H, CHHC),
1.05 (d, J=6.6Hz, 3H, CH(CH3)(CH3)), 1.00 (d,
J=6.6 Hz, 3H, C=CHCH=CHCHCH3), 0.97 (d, J=
6.7 Hz, 3H, CH(CH3)(CH3)), 0.92 (d, J=6.6 Hz, 3H,
CHCH3), 0.88 (q, J=12.0 Hz, 1H, CHHCHOCˆO);
HRMS, calcd for C37H52N2O8 (M+Na+) 675.362137,
found 675.361564.
25-Cyclohexyl-5-demethoxy-25-de(1-methylpropyl)-22,
23-dihydro-5-(semicarbazono)-avermectin A1a monosac-
charide (42). A solution of alcohol 2711 (125 mg,
0.33 mmol) in diethyl ether (20 mL) containing a suspen-
sion of activated manganese dioxide (125 mg) was stir-
red at 25 ꢀC for 18 h. A further portion of manganese
dioxide (125 mg) was added and stirring continued for a
further 2 h after which the mixture was ®ltered through
Celite1 and evaporated to dryness under reduced pres-
sure. The residue was dissolved in a mixture of dioxan
(10 mL) and methanol (10 mL). A solution of semi-
carbazide hydrochloride (200 mg, 1.79mmol) in water
(5 mL) was added and the mixture was stirred at 25 ꢀC for
48h. The mixture was concentrated under reduced pres-
sure to remove the methanol and dioxan. Aqueous sodium
hydrogen carbonate solution (10 mL) was added and the
mixture extracted with diethyl ether (50 mLÂ3). The
organic phases were combined, washed with water (50 mL)
and brine (50mL), dried (MgSO4) and evaporated under
reduced pressure to provide semicarbazone 42 (91 mg,
66%) as an amorphous white solid: Rf=0.14 (ethyl ace-
25-Cyclohexyl-5-demethoxy-25-de(1-methylpropyl)-22,
23-dihydro-5-(methoxyimino)-avermectin A1a monosac-
charide (39). A solution of alcohol 2711 (250 mg,
0.33 mmol) in diethyl ether (20 mL) containing a suspen-
sion of activated manganese dioxide (250 mg) was stirred
at 25 ꢀC for 18 h. A further portion of manganese dioxide
(250 mg) was added and stirring continued for a further
2 h after which the mixture was ®ltered through Celite1
and evaporated to dryness under reduced pressure. The
residue was dissolved in a mixture of dioxan (20 mL)
and methanol (20 mL). A solution of methoxylamine
hydrochloride (835 mg, 10 mmol) in water (20 mL) was
added and the mixture was stirred at 50ꢀC for 2 h. The
mixture was basi®ed with aqueous potassium hydrogen
1
tate); H NMR (500MHz, CDCl3) d 8.50 (broad s, 1H,
NNH), 5.98 (dt, J=11.0, 2.4 Hz, 1H, C=CH
CH=CHCH), 5.87±5.86 (m, 1H, CH=C(CH3)C=N),
5.80 (dd, J=9.7, 15.0 Hz, 1H, C=CHCH=CHCH), 5.72
(dd, J=11.0, 15.0Hz, 1H, C=CHCH=CHCH), 5.49±

B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
2023
5.43 (m, 1H, CHOCˆO), 4.99 (broad d, J=10.8 Hz, 1H,
CH2CH=C(CH3)), 4.83 (d, J=3.5Hz, 1H, CH(O)
(O)CH2), 4.72 (d, J=2.4 Hz, 2H, OCH2), 4.21 (s, 1H,
CHC=N), 4.07 (broad s, 1H, CH(O)), 3.97 (broad s, 1H,
COH), 3.86 (dq, J=9.1, 6.2, 1H, CH(O)(CH3)CH), 3.66
(tdd, J=11.8, 4.3, 1.8 Hz, 1H, CH(OCH3)), 3.56 (ddd,
J=13.5, 9.1, 4.7 Hz, 1H, CH(OCH3)), 3.47 (s, 3H, OCH3),
3.32 (quintet, J=2.4 Hz, 1H, CHCˆO), 3.17 (t, J=9.1Hz,
1H, CH(O)(CH)(CH)), 3.08 (broad d, J=7.8Hz, 1H,
CHC6H13), 2.57±2.51 (m, 2H), 2.34±2.22 (m, 3H), 1.97±
1.96 (m, 3H, CH=C(CH3)C=N), 1.95 (ddd, J=12.0, 4.8,
1.3 Hz, 1H, CHHC), 1.81±1.77 (m, 3H), 1.66±1.42 (m,
11H), 1.51 (broad s, 3H, CH2CH=CCH3), 1.38 (t,
J=12.0 Hz, 1H, CHHC), 1.30±1.14 (m, 4H), 1.28 (d,
J=6.2 Hz, 3H, OCHCH3), 1.17 (d, J=7.0 Hz, 3H,
C=CHCH=CHCHCH3), 0.82 (q, J=11.8 Hz, 1H, CH
HCHOCˆO), 0.80 (broad d, J=5.3 Hz, 3H, CHCH3);
HRMS, calcd for C44H65N3011 (M+Na+) 834.451680,
found 834.455976.
25-Cyclohexyl-5-O-demethyl-25-de(1-methylpropyl)-400-
deoxy-22,23-dihydro-avermectin A1a monosaccharide (27d).
To a stirred solution of silyl ether 27a (435.5mg,
0.5 mmol) in dichloromethane (5mL) at 25ꢀC was added
pyridine (2 mL) and then para-tolyl chlorothionoformate
(187mg, 1 mmol, added dropwise over 1 min). After 2 h
para-tolyl chlorothionoformate (187mg, 1 mmol) was
added and stirring continued for 1 h. The reaction mixture
was then evaporated under reduced pressure and the resi-
due puri®ed by column chromatography on silica gel
(20 g, dichloromethane:hexane, 1:1, v/v) to provide
thionocarbonate 27b (420 mg) which was taken up in
toluene (80 mL). To this solution was added tributyltin
hydride (12 mL, 44.6 mmol) and 2,20-azobis(isobutyr-
onitrile) (0.8 mg, 0.005 mmol). The mixture was heated
at 110 ꢀC for 1 h, then cooled and evaporated under
reduced pressure. The residue was puri®ed by column
chromatography on silica gel (20 g, hexane and then di-
chloromethane) to provide silyl ether 27c (271 mg) which
was taken up in methanol (50 mL). To this solution was
added para-toluenesulphonic acid monohydrate (125mg,
0.657 mmol) and the mixture was stirred at 25 ꢀC for
0.75h and then poured into aqueous potassium hydrogen
carbonate solution (25 mL). Water (25 mL) was added
and the mixture extracted with diethyl ether (50 mLÂ3).
The combined organic phases were washed with water
(20 mLÂ3) and brine (20 mLÂ2), then dried (Na2SO4)
and evaporated under reduced pressure. The crude pro-
duct was puri®ed by column chromatography on silica gel
(10 g, dichloromethane:ethyl acetate, 9:1, v/v) and then
reverse phase preparative HPLC (250Â21.4 mm diameter
Rainin Dynamax1 MicrosorbTM 5 m ODS column main-
tained at 40ꢀC and eluted at 20mL min 1, methanol:-
water, 85:15, v/v/v) to provide alcohol 27d (131mg, 35%)
as an amorphous white solid: Rf=0.33 (dichlor-
25-Cyclohexyl-5-O-demethyl-5-O-[(1,1-dimethylethyl)di-
methylsilyl]-25-de(1-methylpropyl)-22,23-dihydro-aver-
mectin A1a monosaccharide (27a). To a stirred solution
of alcohol 2711 (3.03 g, 4 mmol) and imidazole (600mg,
8.8 mmol) in dimethylformamide (10 mL) at 25ꢀC was
added tert-butyldimethylsilyl chloride (663mg, 4.4 mmol).
After 3 h tert-butyldimethylsilyl chloride (200mg,
1.33mmol) was added and stirring continued for 0.5 h.
Water (100mL) was then added and the mixture extracted
with dichloromethane (50 mLÂ2). The organic phases
were combined, dried (Na2SO4) and evaporated under
reduced pressure to provide a yellow oil which was pur-
i®ed by column chromatography on silica gel (100g,
dichloromethane:ethyl acetate, 9:1, v/v) to provide silyl
ether 27a (2.1g, 60%) as a pale yellow amorphous solid:
Rf=0.57 (dichloromethane:ethyl acetate, 4:1, v/v); 1H
NMR (500MHz, CDCl3) d 5.84 (dt, J=10.3, 2.4 Hz, 1H,
C=CHCH=CHCH), 5.74 (dd, J=9.2, 14.8Hz, 1H, C=
CHCH=CHCH), 5.71 (dd, J=10.3, 14.8Hz, 1H, C=
CHCH=CHCH), 5.39±5.32 (m, 1H, CHOCˆO), 5.34±
5.33 (m, 1H, CH=C(CH3)CHOTBDMS), 4.99 (broad d,
J=10.9 Hz, 1H, CH2CH=C(CH3)), 4.83 (d, J=3.4 Hz,
1H, CH(O)(O)CH2), 4.67 (dd, J=14.6, 2.4 Hz, 1H,
OCHH), 4.58 (d, J=14.6, 2.2 Hz, 1H, OCHH), 4.44
(broad d, J=5.6 Hz, 1H, CHOTBDMS), 4.17 (broad s,
1H, CH(O)), 3.95 (broad s, 1H, COH), 3.86 (dq, J=9.2,
6.3 Hz, 1H, CH(O)(CH3)CH), 3.82 (d, J=5.6 Hz, 1H,
CH(O)CHOTBDMS, 3.65 (tdd, J=11.8, 4.6, 2.0 Hz, 1H,
CH(OCH3)), 3.56 (ddd, J=13.5, 9.2, 4.6 Hz, 1H,
CH(OCH3)), 3.47 (s, 3H, OCH3), 3.39 (sextet, J=2.4Hz,
1H, CHCˆO), 3.16 (t, J=9.2Hz, 1H, CH(O)(CH)(CH)),
3.07 (broad d, J=7.2 Hz, 1H, CHC6H13), 2.59 (broad s,
1H, CHOH), 2.54±2.48 (m, 1H, C=CHCH=
CHCHCH3), 2.34±2.22 (m, 3H), 1.98 (ddd, J=11.9, 5.0,
1.3 Hz, 1H, CHHC), 1.79 (broad s, 3H, CH=C
(CH3)C=N), 1.80±1.75 (m, 3H), 1.68±1.40 (m, 11H),
1.51 (broad s, 3H, CH2CH=CCH3), 1.34 (t, J=11.9 Hz,
1H, CHHC), 1.30±1.13 (m, 4H), 1.27 (d, J=6.3Hz, 3H,
OCHCH3), 1.15 (d, J=6.8Hz, 3H, C=CHCH=
CHCHCH3), 0.93 (s, 9H, t-Bu), 0.82 (q, J=11.8 Hz, 1H,
CHHCHOCˆO), 0.79 (broad d, J=5.2 Hz, 3H, CHCH3),
0.13 (s, 6H, Si(CH3)2); HRMS, calcd for C49H78O11Si
(M+Na+) 893.521112, found 893.522751.
1
omethane:ethyl acetate, 4:1, v/v); H NMR (500MHz,
CDCl3) d 5.88 (dt, J=10.6, 2.4 Hz, 1H, C=CHCH=
CHCH), 5.77 (dd, J=9.3, 15.0 Hz, 1H, C=CHCH=
CHCH), 5.71 (dd, J=10.6, 15.0Hz, 1H, C=CHCH=
CHCH), 5.44 (broad s, 1H, CH=C(CH3)CHOH), 5.42±
5.36 (m, 1H, CHOCˆO), 5.03 (broad d, J=11.8 Hz, 1H,
CH2CH=C(CH3)), 4.89 (d, J=3.4 Hz, 1H, CH(O)
(O)CH2), 4.69 (dd, J=14.2, 2.4 Hz, 1H, OCHH), 4.66
(dd, J=14.2, 2.4 Hz, 1H, OCHH), 4.30 (broad t,
J=6.9 Hz, 1H, CHOH), 4.12 (s, 1H, COH), 4.03 (ddq,
J=11.5, 1.8, 6.2 Hz, 1H, CH(O)(CH3)CH2), 3.97 (d,
J=6.9 Hz, 1H, CH(O)CHOH), 3.96 (broad s, 1H,
CH(O)), 3.65 (tt, J=11.3, 4.2 Hz, 1H, CH(OCH3)), 3.56
(tdd, J=11.8, 4.6, 2.1 Hz, 1H, CH(O)CH2)2), 3.40 (s, 3H,
OCH3), 3.29 (sextet, J=2.2 Hz, 1H, CHCˆO), 3.07
(broad d, J=7.9Hz, 1H, CHC6H13), 2.51 (dsextets,
J=2.4, 7.0 Hz, 1H, C=CHCH=CHCHCH3), 2.34 (d,
J=6.9Hz, 1H, CHOH), 2.32 (broad d, J=13.5 Hz, 1H,
(CH3)C=CHCHH), 2.25 (dt, J=13.5, 11.8Hz, 1H, (CH3)
C=CHCHH), 2.14 (broad d, J=12.4 Hz, 1H, CH(O)
(O)CHH), 2.05 (broad d, J=12.4 Hz, 1H, CHHCH
(OCH3)), 1.97 (ddd, J=12.0, 4.9, 1.5 Hz, 1H, CHHC),
1.88 (broad s, 3H, CH=C(CH3)CHOH), 1.83±1.76 (m,
3H), 1.68±1.40 (m, 11H), 1.55 (broad s, 3H,
CH2CH=CCH3), 1.35 (t, J=12.0 Hz, 1H, CHHC),
1.30±1.17 (m, 5H), 1.19 (d, J=6.2 Hz, 3H, OCHCH3),
1.14 (d, J=7.0 Hz, 3H, C=CHCH=CHCHCH3), 0.80 (q,
J=11.8Hz, 1H, CHHCHOCˆO), 0.79 (broad d,

2024
B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
J=5.4 Hz, 3H, CHCH3); HRMS, calcd for C43H64010
(M+Na+) 763.439719, found 763.441418.
feeding apparatus.25 Culture cats were used to provide
regular supplies of an established laboratory strain of
¯eas (Ctenocephalides felis) not more than 48 h post-
eclosion. Twenty-four hours prior to testing, up to 30
adult ¯eas of mixed sex were collected and aspirated
into specially designed feeding chambers and held at
25 ꢀC and 75% relative humidity (RH). Immediately
prior to testing, the number of live ¯eas in each feeding
chamber was recorded and test solutions (blood plus
compound) prepared. Test compounds were made up as
stock solutions in dimethylsulfoxide (DMSO) and 50 ml
of the stock added to 9.95 mL of blood.
25-Cyclohexyl-5-demethoxy-25-de(1-methylpropyl)-400-
deoxy-22, 23-dihydro-5-hydroxyimino-avermectin A1a
monosaccharide (45). A solution of alcohol 27d (91 mg,
0.123 mmol) in diethyl ether (30 mL) containing a sus-
pension of activated manganese dioxide (90 mg) was
stirred at 25 ꢀC for 2 h. A further portion of manganese
dioxide (90 mg) was added and stirring continued for a
further 18 h. A third portion of manganese dioxide
(90 mg) and stirring continued for a further 2 h after
which the mixture was ®ltered through Celite1 and
evaporated to dryness under reduced pressure. The
residue was dissolved in a mixture of dioxan (10 mL)
and methanol (10 mL). A solution of hydroxylamine
hydrochloride (100 mg, 1.44 mmol) in water (5 mL) was
added and the mixture was stirred at 25 ꢀC for 2 h and
then heated at 40 ꢀC for 2 h. The mixture was poured
into aqueous potassium hydrogen carbonate solution
(25 mL) and water (50 mL) and then extracted with die-
thyl ether (50 mLÂ3). The organic phases were com-
bined, washed with water (20 mLÂ3) and brine
(10 mLÂ2), dried (Na2SO4) and evaporated under
reduced pressure. The crude product (117 mg) was pur-
i®ed by column chromatography on silica gel (5g,
dichloromethane:ethyl acetate, 9:1, v/v) and then reverse
phase preparative HPLC (250Â21.4 mm diameter Rainin
Dynamax1 MicrosorbTM 5 m ODS column maintained at
40ꢀC and eluted at 20mL min 1, methanol/water, 85/15,
v/v) to provide oxime 45 (70 mg, 76%) as an amorphous
white solid : Rf=0.43 (dichloromethane:ethyl acetate, 4:1,
Fleas were fed through a 300 mm mesh for 6 h. Knock-
down and mortality were monitored during feeding and
for 24 h post feeding. On removal from the feeding
chamber, ¯eas were maintained at 25 ꢀC and 75% RH.
Mortality was recorded and ecacy calculated as a per-
centage of the control ¯eas. Control ¯eas were fed on
blood containing 0.5% v/v DMSO. The minimum
detectable dose (MDD) was de®ned as the dose produ-
cing >30% mortality of ¯eas after 24 h post feeding.
Acknowledgements
We gratefully acknowledge Mr. G. B. T. Goodwin, Mr.
K. Hall, Mr. G. C. Smith and Mr. V. F. Voss for com-
pound preparation, Dr. T. M. Peakman for the acqui-
sition and assignment of NMR spectra and Mr. B.
Austin, Mr. N. Leach-Bing, Mr. C. J. Spanton and Mrs.
C. Gillot for ¯ea feeding assay data.
1
v/v); H NMR (500MHz, CDCl3) d 8.04 (broad s, 1H,
NOH), 5.95 (dt, J=10.5, 2.4 Hz, 1H, C=CHCH=
CHCH), 5.84±5.83 (m, 1H, CH=C(CH3)CHOH), 5.79
(dd, J=9.3, 15.1 Hz, 1H, C=CHCH=CHCH), 5.73
(dd, J=10.5, 15.1 Hz, 1H, C=CHCH=CHCH), 5.47±
5.40 (m, 1H, CHOCˆO), 5.03 (broad d, J=11.5 Hz,
1H, CH2CH=C(CH3)), 4.89 (d, J=3.4 Hz, 1H,
CH(O)(O)CH2), 4.76 (dd, J=14.4, 2.4 Hz, 1H, OCHH),
4.70 (dd, J=14.4, 2.4 Hz, 1H, OCHH), 4.68 (s, 1H,
CHC=NOH), 4.04 (ddq, J=11.5, 1.8, 6.3 Hz, 1H,
CH(O)(CH3)CH2), 3.99 (s, 1H, CH(O)), 3.97 (broad s,
1H, COH), 3.70 (tt, J=12.3, 4.6 Hz, 1H, CH(OCH3)),
3.66 (tdd, J=11.5, 4.5, 2.0 Hz, 1H, CH(O)(CH2)2), 3.41 (s,
3H, OCH3), 3.40 (quintet, J=2.3 Hz, 1H, CHCˆO), 3.07
(broad d, J=8.0 Hz, 1H, CHC6H13), 2.52 (dsextets,
J=2.5, 6.9 Hz, 1H, C=CHCH=CHCHCH3), 2.34
(broad d, J=13.6 Hz, 1H, (CH3)C=CHCHH), 2.25 (dt,
J=13.6, 11.5 Hz, 1H, (CH3)C=CHCHH), 2.14 (broad d,
J=12.6 Hz, CH(O)(O)CHH), 2.06 (broad d, J=12.3 Hz,
CHHCH(OCH3)), 1.97 (ddd, J=12.1, 4.9, 1.3 Hz, 1H,
CHHC), 1.94 (broad s, 3H, CH=C(CH3)C=NOH),
1.83±1.76 (m, 3H), 1.50 (broad s, 3H, CH2CH=CCH3),
1.68±1.40 (m, 11H), 1.37 (t, J=12.1 Hz, 1H, CHHC),
1.30±1.17 (m, 5H), 1.19 (d, J=6.3 Hz, 3H, OCHCH3),
1.14 (d, J=6.9 Hz, 3H, C=CHCH=CHCHCH3), 0.82
(q, J=11.5 Hz, 1H, CHHCHOCˆO), 0.80 (broad d,
J=5.5 Hz, 3H, CHCH3); HRMS, calcd for C43H63NO10
(M+Na+) 776.434968, found 776.435166.
References
1. (a) Fisher, M. H.; Mrozik, H. Annu. Rev. Pharmacol. Tox-
icol. 1992, 32, 537 and references therein. (b) Deng, Y.;
Casida, J. E. Pestic. Biochem. Physiol. 1992, 43, 116. (c)
Rohrer, S.P.; Arena, J. P. ACS Symp. Ser. 1995, 591, 264. (d)
Martin, R. J. Parasitology 1996, 112, 247. (e) Vassilatis, D. K.;
Arena, J. P.; Plasterk, R. H. A.; Wilkinson, H. A.; Schae€er, J.
M.; Cully, D. F.; Aan der Ploeg, L. H. T. J. Biol. Chem. 1997,
272, 33167.
2. Fisher, M. H. ACS Symp. Ser. 1997, 658, 220.
3. Bishop, B. F.; Bruce, C. I.; Evans, N. A.; Goudie, A.C.;
Gration, K. A. F.; Gibson, S.P.; Perry, D. A.; Walshe, N. D.
A.; Witty, M. J. Vet. Parasitol. manuscript submitted.
4. (a) Davies, H. G.; Green, R. H. Chem. Soc. Rev. 1991, 20, 211.
(b) Davies, H. G.; Green, R. H. Chem. Soc. Rev. 1991, 20, 271.
5. Miller, T. W.; Chaiet, L.; Cole, D. J.; Cole, L. J.; Flor, J.
E.; Goegelman, R. T.; Gullo, V. P.; Joshua, H.; Kempf, A. J.;
Krellwitz, W. R.; Monaghan, R. L.; Ormond, R. E.; Wilson,
K. E.; Albers-Schonberg, G.; Putter, I. Antimicrob. Agents
Chemother. 1979, 15, 368.
6. Chabala, J. C.; Mrozik, H.; Tolman, R. L.; Eskola, P.;
Lusi, A.; Peterson, L. H.; Woods, M. F.; Fisher, M. H.;
Campbell, W. C.; Egerton, J. R.; Ostlind, D. A. J. Med. Chem.
1980, 23, 1134.
7. Cvetovich, R. J.; Kelly, D. H.; DiMichele, L. M.; Shuman,
R. F.; Grabowoski, E. J. J. J. Org. Chem. 1994, 59, 7704.
8. Dutton, C. J.; Gibson, S. P.; Goudie, A. C.; Holdom, K. S.;
Pacey, M. S.; Ruddock, J. C.; Bu'Lock, J. D.; Richards, M. K.
J. Antibiot. 1991, 44, 357.
Determination of ¯ea feeding activity. In vitro systemic
activity was determined at 38 ꢀC using a membrane
9. Gibson, S. P.; Goudie, A. C.; Holdom, K. S.; Bu'Lock, J.
D. United States Patent, US-5089480-A, 1992.

B.J. Banks et al. / Bioorg. Med. Chem. 8 (2000) 2017±2025
2025
10. Banks, B. J. World Patent Application, WO-93/15099-A1,
1993.
19. Dutton, C. J.; Gibson, S. P.; Witty, M. J. World Patent
Application, WO-95/04746-A1, 1995
11. Bishop, B. F.; Pacey, M. S.; Perry, D. A. World Patent
Application, WO-94/15944-A1, 1994.
20. Walshe, N. D. A. World Patent Application, WO-94/
29328-A1, 1994
12. Fisher, M. H.; Mrozik, H. European Patent Application,
EP-0379341-A1, 1990.
21. Walshe, N. D. A. World Patent Application, WO-95/
22552-A1, 1995
13. Takiguchi, Y.; Ono, M.; Muramatsu, S.; Ide, J.; Mishima,
H.; Terao, M. J. Antibiot. 1983, 36, 502.
14. Carter, G. T.; Nietsche, J. A.; Hertz, M. R.; Williams, D.
R.; Siegel, M. M.; Morton, G. O.; James, J. C.; Borders, D. B.
J. Antibiot. 1988, 41, 519.
15. Beddall, N. E.; Howes, P. D.; Ramsay, M. V. J.; Roberts,
S. M.; Slawin, A. M. Z.; Sutherland, D. R.; Tiley, E. P.; Wil-
liams, D. J. Tetrahedron Lett. 1988, 29, 2959.
16. Tsukamoto, Y.; Sato, K.; Mio, S.; Sugai, S.; Yanai, T.;
Kitano, N.; Muramatsu, S.; Nakada, Y.; Ide, J. Agric. Biol.
Chem. 1991, 55, 2615.
22. Pacey, M. S.; Dutton, C. J.; Monday, R. A.; Ruddock, J.
C.; Smith, G. C. J. Antibiot. manuscript submitted
23. (a) Meinke, P. T.; O'Connor, S. P.; Ostlind, D. A.; Shoop,
W. L.; Fisher, M. H.; Mrozik, H. Bioorg. Med. Chem. Lett.
1993, 3, 2675. (b) Mrozik, H.; Eskola, P.; Arison, B. H.; Linn,
B. O.; Lusi, A.; Matzuk, A.; Shih, T. L.; Tischler, M.; Waks-
munski, F. S.; Wyvratt, M. J.; Blizzard, T. A.; Margiatto, G.
M.; Fisher, M. H.; Shoop, W. L.; Egerton, J. R. Bioorg. Med.
Chem. Lett. 1995, 5, 2435. (c) Mrozik, H.; Eskola, P.; Fisher,
M. H.; Egerton, J. R.; Cifelli, S.; Ostlind, D. A. J. Med. Chem.
1982, 25, 658.
17. Sutherland, D. R.; Porter, N.; Bain, B. M.; Ramsay, M. V.
J.; Noble, H. M.; Tiley, E. P.; Fletton, R. A.; Ward, J. B.; Noble,
D. European Patent Application, EP-0238258-A1, 1987
18. Chabala, J. C.; Mrozik, H.; Tolman, R. L.; Eskola, P.;
Lusi, A.; Peterson, L. H. J. Med. Chem. 1980, 23, 1134.
24. Blizzard, T. A.; Margiatto, G. M.; Mrozik, H.; Shoop, W.
L.; Frankshun, R. A.; Fisher, M. H. J. Med. Chem. 1992, 35,
3873.
25. Wade, S. E.; Georgi, J. R. J. Med. Entomol. 1988, 25,
186.