Avermectin chemistry and action: Ester- and ether-type candidate photoaffinity probes

Article abstract of DOI:10.1016/S0968-0896(99)00259-X

Avermectin B(1a) (1) is a potent anthelmintic, insecticide, miticide and chloride channel activator on interaction with a specific nerve membrane site analyzed by binding assays with [³H]1. Candidate photoaffinity probes were prepared replacing the dioleandrosyl substituent with potential isosteric esters and ethers approximating the original overall atom length and terminating in a phenyl moiety substituted with azido, iodo or hydroxy. Several of the candidates met the goal of high potency on mouse, housefly and fruit fly brain chloride channels with IC₅₀ values of 7-57 nM in competing for the [³H]1 binding site. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00259-X

Bioorganic & Medicinal Chemistry 8 (2000) 19±26
Avermectin Chemistry and Action: Ester- and Ether-type
Candidate Photoanity Probes
Yoshihisa Tsukamoto,1 Loretta M. Cole and John E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management,
University of California, Berkeley, CA 94720-3112, USA
Received 18 May 1999; accepted 4 August 1999
AbstractÐAvermectin B1a (1) is a potent anthelmintic, insecticide, miticide and chloride channel activator on interaction with a
speci®c nerve membrane site analyzed by binding assays with [3H]1. Candidate photoanity probes were prepared replacing the
dioleandrosyl substituent with potential isosteric esters and ethers approximating the original overall atom length and terminating
in a phenyl moiety substituted with azido, iodo or hydroxy. Several of the candidates met the goal of high potency on mouse,
house¯y and fruit ¯y brain chloride channels with IC50 values of 7±57 nM in competing for the [3H]1 binding site. # 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
53-, 47- and 8-kDa polypeptides from the free-living
nematode Caenorhabditis elegans but does not bind to
rat brain membranes and, therefore, is selective for the
insect and nematode membranes.16±18 Although target
site selectivity is a distinct advantage for safe use of a
pharmaceutical or pesticide, it does not enable a single
probe to be employed for comparing vertebrate and
invertebrate targets and de®ning di€erences in their
binding sites within the chloride channel, as has been
successfully applied to chloride channel blockers.19
The avermectins are potent anthelmintics, insecticides
and miticides used to control pests of humans, veter-
inary animals and crops.1,2 The prototype avermectin
B1a (1) consists of a macrocyclic lactone with a 13a-
dioleandrosyl substituent (R) (Fig. 1). Compound 1 and
many analogues bind to vertebrate and invertebrate
g-aminobutyric acid (GABA)-gated and invertebrate
glutamate-gated chloride channels to increase ion con-
ductance, thereby disrupting inhibitory or excitatory
action. These mechanisms are established by physio-
logical, biochemical and molecular biological investi-
gations3±8 with an important contribution from
radioligand binding studies with [5a-3H]1 and its 22,23-
ditritio derivative [3H]ivermectin.9±13 Photoanity
labeling has been successfully applied by Merck scien-
tists to the action of the avermectins, using radioligand
2 which is 400-amino-400-deoxy-1 with 4-azido-3-125
iodosalicylic acid attached to the amino group through
a b-alanyl-e-aminocaproyl spacer (Fig. 1).14,15 Photo-
anity probe 2 labels a 47-kDa polypeptide from head
membranes of the fruit ¯y Drosophila melanogaster and
The goal of this investigation is to design a candidate
photoanity probe e€ective for both mammals and
insects. The failure of 2 to act in mammalian brain16 is
conceivably due to the far greater length of its R sub-
stituent than that of 1. An alternative is that 2 has
amide bonds versus only ether linkages in the R sub-
stituent of 1. The candidate photoanity probes 3±6
prepared here, therefore, include ester and ether moieties
more nearly isosteric with the dioleandrosyl substituent
(Fig. 1). Potency is evaluated using the [3H]1 binding
assay with membranes from mouse brain and house¯y
(Musca domestica) and fruit ¯y heads.
Keywords: antiparasitics; labelling; neurologically-active compounds;
receptors; substituent e€ects.
Structural modi®cations
Additional or alternative key words; Chloride channel; insecticides;
isosteres.
*Corresponding author. Tel.:+1-510-642-5424; fax:+1-510-642-6497;
e-mail: ectl@nature.berkeley.edu
1Present address: Agroscience Research Laboratories, Sankyo Com-
pany, Ltd., Shiga-ken 520-2342, Japan.
Esters 3a±f of 1-aglycone (Figs 1 and 2). Key inter-
mediate 9 was synthesized by a two-step reaction from
5-O-tert-butyldimethylsilylavermectin B1a aglycone
(7).20 Treatment of 7 with the anhydride prepared in situ
from N-(2,2,2-trichloroethoxycarbonyl)-b-alanine and
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00259-X

20
Y. Tsukamoto et al. / Bioorg. Med. Chem. 8 (2000) 19±26
Figure 1. Structures of avermectin B1a (1) and the corresponding aglycone (1-aglycone) and of an amide (2), esters (3) and ethers (4±6) derived
therefrom.
pivaloyl chloride in the presence of triethylamine gave
ester 8 in high yield, and reductive cleavage of the 2,2,2-
trichloroethoxycarbonyl group of this ester a€orded 9.
Acylation of the deprotected amino group of 9 was
carried out with benzoyl chloride for 3a or with the
previously known azidobenzoic acid derivatives in the
presence of 1,3-dicyclohexylcarbodiimide (DCC) for
3b±d or with the N-hydroxysuccinimidyl ester of 4-
azido-2-hydroxybenzoic acid and 4-hydroxy-2-iodo-
benzoic acid for 3e and 3f, respectively. Finally, desilyla-
tion of the 5-hydroxy group gave the esters 3a±f.14,20,21
Structure±activity relationships
General (Fig. 1 and Table 1). Compound 1 di€ers only
3-fold in potency for the mouse, house¯y and fruit ¯y
brain [3H]1 binding site and 1-aglycone is 18- to 68-fold
less active. Photoanity probe 2 is similar to ivermectin
in its anity for the C. elegans membrane binding site
despite the long spacer group between the macrocyclic
lactone and the terminal photoreactive 4-azido-3-125
iodosalicylate moiety.16 However, 2 does not bind to the
rat brain [3H]ivermectin binding site16 suggesting that
an R substituent more isosteric with that of 1 may be
more appropriate. Several of the candidates in the pre-
sent study met the goal of high potency on mouse,
house¯y and fruit ¯y brain chloride channels with IC50
values of 7±57 nM in competing for the [3H]1 binding
site.
Ethers 4 of 1-aglycone, 5a and 5b of 1-monosaccharide
and 6a and 6b of 1 (Figs 1 and 3). The ether-type deri-
vatives (4±6) were obtained from 7, 5-O-tert-butyl-
dimethylsilylavermectin B1a monosaccharide (12),
and 5-O-tert-butyldimethylsilylavermectin B1a (14)22 by
coupling with phenoxyethoxymethyl chloride or 2-(2-
azido-4-iodophenoxy)ethoxymethyl chloride followed
by deprotection of the 5-hydroxy group.
Esters 3a±f (Fig. 1 and Table 1). The goal was to replace
the dioleandrosyl unit with an ester of approximately

Y. Tsukamoto et al. / Bioorg. Med. Chem. 8 (2000) 19±26
21
Figure 2. 5-O-(tert-Butyldimethylsilyl)-13-O-(2-aminopropionyl)avermectin B1a aglycone (9) and precursors (7 and 8) and derivatives (10) thereof.
Su refers to N-hydroxysuccinimidyl ester of the acid.
10-atom equivalent length including an aryl moiety that
would potentially bear azido, iodo and possibly hy-
droxy substituents. The b-alanyl group was used as a
candidate isosteric spacer unit. All of the esters are
potent in the house¯y receptor assays (IC50 values 7±31
nM) while 3c and 3e are best for the mouse preparation
(IC50 values 57 and 105 nM) and 3d±f for the fruit ¯y
membranes (IC50 values 26±56 nM). On this basis iodo-
containing compounds 3d and 3f are candidates for
radiolabeling, especially for the insect receptor. While a
portion of the activity of the esters may be due to
hydrolysis to 1-aglycone, this is probably not a major
factor since the IC50 values of 3e for mouse brain and
house¯y membrane preparations were not changed by
treating with a potent esterase inhibitor (phenyl sali-
genin cyclic phosphonate or phenylmethanesulfonyl
¯uoride, 10 mM, 30 min preincubation, data not detailed
here) and the ester linkage can be substituted with a
non-hydrolyzable ether moiety with enhanced potency
relative to the aglycone (see below).
candidate photoanity probes. The 13-O-(2-phenoxy-
ethoxymethyl) derivatives as prepared from 1-aglycone
(4), 1-monosaccharide (5a) and 1 (6a) retain a high level
of potency in the house¯y receptor assay (IC50 values
19±97 nM). However, the 2-azido-4-iodophenoxy ana-
logues (5b and 6b) are ꢀ100-fold less active than their
phenoxy counterparts with mouse and 2±5-fold less
active with house¯y. Further optimization is required in
the phenoxy substituents for a general photoanity
probe.
Experimental
Chemistry
General. 1H NMR spectra were recorded for CDCl3
solutions with a Bruker AM-300 spectrometer. Fast
atom bombardment (FABMS) (both low- and high-
resolution, LR and HR) was conducted with the Fisons
ZAB2-EQ spectrometer. Analytical thin-layer chroma-
tography (TLC) was performed on silica gel with ¯uor-
escent indicator using precoated plastic sheets.
Preparative TLC involved 20Â20 cm plates coated with
Ethers 4, 5a, 5b, 6a and 6b (Fig. 1 and Table 1). Ether
linkages were examined as one way to rule out the pos-
sibility of esterases or amidases limiting the potency of

22
Y. Tsukamoto et al. / Bioorg. Med. Chem. 8 (2000) 19±26
Figure 3. 5-O-(tert-Butyldimethylsilyl)-13-O-(2-phenoxyethoxymethyl)avermectin B1a aglycone (11) and related compounds (12±15).
0.25 or 0.5 mm silica gel containing PF250 indicator.
Compounds were visualized with short wavelength UV
light. Column ¯ash chromatography was performed
using silica gel 60. All ®nal products were puri®ed to
mixture was stirred at room temperature for 18 h and
poured into ice-cold water (40 mL). The resulting mix-
ture was extracted with ethyl acetate (40 mL, and then
3Â20 mL) and the combined organic layer was washed
with water (20 mL) and brine (20 mL), then dried over
Na2SO4 and evaporated in vacuo. The residue was pur-
i®ed by silica gel column chromatography (hexane-ethyl
acetate gradient) to give 188 mg (99%) of 8.
1
homogeneity as determined by TLC and H NMR and
1
had appropriate H NMR spectra (Table 2) and mole-
cular mass by FABMS.
5-O-(tert-Butyldimethylsilyl)-13-O-[2-(2,2,2-trichloroeth-
oxycarbonylamino)propionyl]-avermectin B1a aglycone
(8). To a solution of N-(2,2,2-trichloroethoxycarbonyl)-
b-alanine23 (530 mg) in CH2Cl2 (7 mL) in an ice bath
were added triethylamine (280 mL) and pivaloyl chloride
(250 mL). The mixture was stirred for 30 min at 0 ꢁC and
a solution of 720 (141 mg) in CH2Cl2 (3 mL), diisopro-
pylethylamine (174 mL), and 4-(N,N-dimethylamino)-
pyridine (DMAP) (123 mg) was added at 0 ꢁC. The
5-O-(tert-Butyldimethylsilyl)-13-O-(2-aminopropionyl)-
avermectin B1a aglycone (9). A mixture of 8 (184 mg),
water (10 mL), acetic acid (100 mL) and zinc dust
(747 mg) in tetrahydrofuran (THF) (8 mL) was stirred
for 4.5 h at room temperature. The reaction mixture was
®ltered through Celite and the solid was washed with
ethyl acetate (80 mL). The ®ltrate and the ethyl acetate
were combined and washed with water (20 mL) and

Y. Tsukamoto et al. / Bioorg. Med. Chem. 8 (2000) 19±26
23
Table 1. Structure±activity relationships of avermectin derivatives as
inhibitors of [3H]avermectin binding in brain membranes of mouse,
house¯y and fruit¯y
5-O-(tert-Butyldimethylsilyl)-13-O-[2-(4-azido-2-hydroxy-
5-iodobenzoyl)-aminopropionyl]avermectin B1a aglycone
(10f). The above procedure with 9 (17.8 mg) and N-
hydroxysuccinimidyl-4-azido-2-hydroxy-5-iodobenzoate27
(14.5 mg) in the dark gave 16.0 mg (66%) of 10f.
Compound
IC50, nM‹SD (n)
House¯y
Mouse
Fruit ¯y
5-O-(tert-Butyldimethylsilyl)-13-O-(2-phenoxyethoxyme-
thyl)avermectin B1a aglycone (11). To a solution of 7
(20 mg) and 2-phenoxyethoxymethyl chloride (106 mg)
in CH2Cl2 (150 mL) was added diisopropylethylamine
(120 mL). The reaction mixture was stirred for 3 days,
poured into ice-cold saturated aqueous NaHCO3
(20 mL), extracted with CH2Cl2 (4Â10 mL) and the
combined organic layer was washed with NaHCO3
solution (2Â10 mL) then dried over Na2SO4 and eva-
porated in vacuo. The residue was puri®ed by pre-
parative TLC (hexane:ethyl acetate, 6:1) to give 16.3 mg
(67%) of 11.
Avermectin series
1
1-aglycone
2.9‹0.9 (10)
196‹46 (3)
1.5‹0.6 (9)
64‹20 (4)
4.2‹1.3 (5)
77‹18 (3)
Esters
3a
3b
3c
3d
653, 424a
170‹98 (4)
57‹34 (3)
185‹72 (3)
105‹28 (3)
130‹2 (3)
13‹5 (3)
15‹10 (3)
15‹9 (3)
31‹12 (5)
6.7‹3.7 (3)
21‹15 (3)
73‹28 (4)
243‹86 (3)
477, 304a
44‹38 (3)
54‹24 (3)
56, 26a
3e
3f
Ethers
4
5a
5b
6a
29‹18 (5)
38‹7 (30)
3400, 506a
33‹13 (3)
3684, 1210a
19‹8 (4)
33‹21 (3)
174, 154a
97, 62a
Ð
Ð
Ð
Ð
Ð
5-O-(tert-Butyldimethylsilyl)avermectin B1a monosac-
charide (12). A solution of avermectin B1a mono-
saccharide28 (326 mg), imidazole (183 mg), and tert-
butyldimethylsilyl chloride (202 mg) in DMF (4 mL)
was stirred for 40 min. Then additional imidazole
(183 mg) and tert-butyldimethylsilyl chloride (202 mg)
were added followed by stirring for 75 min. The reaction
mixture was poured into ice-cold water (50 mL) and
extracted with hexane:ethyl acetate (1/1) (4Â25 mL).
The combined organic layer was washed with water
(2Â25 mL) and brine (25 mL) then dried over Na2SO4
and evaporated in vacuo. The residue was puri®ed by
silica gel column chromatography (hexane±ethyl acetate
gradient) to give 190 mg (50%) of 12.
6b
201, 153a
aValues from separate experiments.
brine (20 mL), then dried over Na2SO4 and evaporated
in vacuo. The residue was puri®ed by preparative TLC
(CH2Cl2:MeOH, 10:1) to give 85 mg (57%) of 9.
5-O-(tert-Butyldimethylsilyl)-13-O-(2-benzoylaminopro-
pionyl)-avermectin B1a aglycone (10a). To a solution of
9 (17.5 mg) in CH2Cl2 (1 mL) were added triethylamine
(8 mL) and benzoyl chloride (3 mL) at 0ꢁC. The reaction
mixture was stirred at room temperature for 24 h and
puri®ed by preparative TLC (hexane:ethyl acetate, 2:1)
to give 17.2 mg (87%) of 10a.
5-O-(tert-Butyldimethylsilyl)-40-O-(2-phenoxyethoxy-
methyl)avermectin B1a monosaccharide (13a). To a
solution of 12 (21.9 mg) and 2-phenoxyethoxymethyl
chloride (from treatment of 2-phenoxyethanol and par-
aformaldehyde in toluene with HCl gas) (260 mg) in
CH2Cl2 (150 mL) was added diisopropylethylamine
(243 mL). The reaction mixture was stirred for 1 day
and poured into ice-cold saturated aqueous NaHCO3
(10 mL), extracted with ethyl acetate (4Â10 mL) and the
combined organic layer was washed with aqueous
NaHCO3 (2Â10 mL) then dried over Na2SO4 and eva-
porated in vacuo. The residue was puri®ed by pre-
parative TLC (CH2Cl2:MeOH, 30:1) to give 12.7 mg
(49%) of 13a.
5-O-(tert-Butyldimethylsilyl)-13-O-[2-(4-azidobenzoyl)-
aminopropionyl]avermectin B1a aglycone (10b). A solu-
tion of 9 (16.9 mg), 4-azidobenzoic acid24 (6.1 mg), and
DCC (10 mg) in CH2Cl2 (1 mL) was stirred in the dark
at room temperature for 15 h. The reaction mixture was
puri®ed by preparative TLC (hexane:ethyl acetate, 2:1)
to give 13.1 mg (65%) of 10b.
5-O-(tert-Butyldimethylsilyl)-13-O-[2-(2-azidobenzoyl)-
aminopropionyl]avermectin B1a aglycone (10c). The
above procedure with 9 (14.3 mg), 2-azidobenzoic acid25
(6.0 mg) and DCC (10 mg) in the dark gave 13.1 mg
(77%) of 10c.
5-O-(tert-Butyldimethylsilyl)-13-O-[2-(2-azido-5-iodobenz-
oyl)aminopropionyl]-avermectin B1a aglycone (10d). The
above procedure with 9 (17.0 mg), 2-azido-5-iodo-
benzoic acid26 (11 mg) and DCC (10 mg) in the dark
gave 16.7 mg (72%) of 10d.
5-O-(tert-Butyldimethylsilyl)-40-O-[2-(2-azido-4-iodophen-
oxy)ethoxymethyl]avermectin B1a monosaccharide (13b).
To a solution of 12 (40 mg) and 2-(2-azido-4-iodophe-
noxy)ethoxymethyl chloride (from 2-(2-azido-4-iodo-
phenoxy)ethanol and paraformaldehyde with HCl gas
as above) (101 mg) in CH2Cl2 (100 mL) was added di-
isopropylethylamine (243 mL). The reaction mixture was
stirred for 3 days with work up as for 13a to give 2.6 mg
(4.7%) of 13b.
5-O-(tert-Butyldimethylsilyl)-13-O-[2-(4-azido-2-hydroxy-
benzoyl)aminopropionyl]-avermectin B1a aglycone (10e).
A solution of 9 (17.8 mg) and N-hydroxysuccinimidyl-
4-azido-2-hydroxybenzoate26 (6.9 mg) in CH2Cl2 (1 mL)
was stirred in the dark at 0ꢁC for 30 min and then at
room temperature for 25 h. The reaction mixture was
puri®ed by preparative TLC (hexane:ethyl acetate, 2:1)
to give 14.2 mg (66%) of 10e.
5-O-(tert-Butyldimethylsilyl)-400-O-(2-phenoxyethoxy-
methyl)avermectin B1a (15a). The procedure for 11 but
with 1422 and a reaction time of 1 day gave 14.7 mg
(57%) of 15a.

24
Y. Tsukamoto et al. / Bioorg. Med. Chem. 8 (2000) 19±26
Table 2. Characteristic 300-MHz 1H NMR chemical shifts (CDCl3) of avermectin B1a derivatives
Chemical shift, d (multiplicity, coupling constants)
0
C4 H
00
C4 H
Compound (s)
C5H
C13H
13-Substituent other than saccharide
Avermectin series
1
14
4.28 (brs)
4.43±4.45 (m)
4.29 (brs)
3.93 (s) 3.24 (t, 9) 3.16 (t, 9)
3.94 (s) 3.25 (t, 9) 3.17 (t, 9)
1-monosaccharide
12
1-aglycone
7
3.96 (s) 3.17 (t, 9)
ÐÐ
ÐÐ
ÐÐ
ÐÐ
4.43±4.45 (brs) 3.95 (s) 3.17 (t, 9)
4.00 (s)
4.01 (s)
4.29 (brs)
4.41±4.44 (m)
ÐÐ
ÐÐ
Intermediates
8
4.41±4.44 (m)
4.42±4.43 (m)
5.19 (s)
5.20 (s)
ÐÐ
ÐÐ
ÐÐ
ÐÐ
2.62±2.73 (2H, m, N-C-CH2CO), 3.55 (2H, q, J=6 Hz, NCH2-C-CO),
4.70 (1H, s, Cl3CCHOCO-N), 4.71 (1H, s, Cl3CCHOCO-N)
2.61±2.72 (2H, m, N-C-CH2CO), 2.84 (2H, brs, NH2), 3.08 (2H, t,
J=6 Hz, NCH2-C-CO)
9
Esters
3a
4.29 (brs)
4.41±4.43 (m)
4.28±4.32 (m)
4.41±4.44 (m)
4.29 (brs)
5.20 (s)
5.20 (s)
5.21 (s)
5.21 (s)
5.23 (s)
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
2.63±2.81 (2H, m, N-C-CH2CO), 3.70±3.86 (2H, m, NCH2-C-CO), 6.85
(1H, t, J=6 Hz, NH), 7.38±7.49 (3H, m, PhCO), 7.72±7.75 (2H, m,
PhCO)
2.64±2.81 (2H, m, N-C-CH2CO), 3.70±3.86 (2H, m, NCH2-C-CO), 6.86
(1H, t, J=6 Hz, NH), 7.39±7.51 (3H, m, PhCO), 7.72±7.75 (2H, m,
PhCO)
2.64±2.81 (2H, m, N-C-CH2CO), 3.71±3.86 (2H, m, NCH2-C-CO), 6.84
(1H, t, J=6 Hz, NH), 7.06 (2H, dd, J=7 and 2 Hz, PhCO), 7.75 (2H, dd,
J=7 and 2 Hz, PhCO)
2.64±2.81 (2H, m, N-C-CH2CO), 3.71±3.85 (2H, m, NCH2-C-CO), 6.84
(1H, t, J=6 Hz, NH), 7.06 (2H, dd, J=7 and 2 Hz, PhCO), 7.75 (2H, dd,
J=7 and 2 Hz, PhCO)
10a
3b
10b
3c
2.65±2.82 (2H, m, N-C-CH2CO), 3.76±3.86 (2H, m, NCH2-C-CO), 7.17
(1H, dd, J=8 and 1 Hz, PhCO), 7.22 (1H, td, J=8 and 1 Hz, PhCO),
7.48 (1H, td, J=8 and 2 Hz, PhCO), 7.92 (1H, t, J=5 Hz, NH), 8.11 (1H,
dd, J=8 and 2 Hz, PhCO)
10c
4.42±4.44 (m)
5.23 (s)
ÐÐ
ÐÐ
2.65±2.82 (2H, m, N-C-CH2CO), 3.76±3.85 (2H, m, NCH2-C-CO), 7.18
(1H, d, J=9 Hz, PhCO), 7.22 (1H, td, J=8 and 1 Hz, PhCO), 7.48 (1H,
td, J=8 and 2 Hz, PhCO), 7.93 (1H, t, J=5 Hz, NH), 8.11 (1H, dd, J=8
and 2 Hz, PhCO)
3d
4.27±4.31 (m)
4.41±4.31 (m)
4.27±4.31 (m)
4.41±4.44 (m)
4.27±4.31 (m)
4.42±4.44 (m)
5.23 (s)
5.23 (s)
5.21 (s)
5.21 (s)
5.23 (s)
5.23 (s)
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
2.63±2.80 (2H, m, N-C-CH2CO), 3.73±3.86 (2H, m, NCH2-C-CO), 6.92
(1H, d, J=8 Hz, PhCO), 7.76 (1H, dd, J=8 and 2 Hz, PhCO), 7.90 (1H,
t, J=6 Hz, NH), 8.41 (1H, d, J=2 Hz, PhCO)
2.65±2.80 (2H, m, N-C-CH2CO), 3.75±3.86 (2H, m, NCH2-C-CO), 6.92
(1H, d, J=9 Hz, PhCO), 7.76 (1H, dd, J=9 and 2 Hz, PhCO), 7.90 (1H,
t, J=6 Hz, NH), 8.41 (1H, d, J=2 Hz, PhCO)
2.67±2.80 (2H, m, N-C-CH2CO), 3.75±3.86 (2H, m, NCH2-C-CO), 6.47
(1H, dd, J=9 and 2 Hz, PhCO), 6.62 (1H, d, J=2 Hz, PhCO), 7.02±7.06
(1H, m, NH), 7.28 (1H, d, J=9 Hz, PhCO), 12.30 (1H, s, HOPhCO)
2.64±2.85 (2H, m, N-C-CH2CO), 3.75±3.85 (2H, m, NCH2-C-CO), 6.47
(1H, dd, J=9 and 2 Hz, PhCO), 6.62 (1H, d, J=2 Hz, PhCO), 7.03 (1H,
t, J=6 Hz, NH), 7.28 (1H, d, J=9 Hz, PhCO), 12.30 (1H, s, HOPhCO)
2.67±2.82 (2H, m, N-C-CH2CO), 3.74±3.83 (2H, m, NCH2-C-CO), 6.75
(1H, s, PhCO), 7.00 (1H, t, J=6 Hz, NH), 7.67 (1H, s, PhCO), 12.32 (1H,
s, HOPhCO)
10d
3e
10e
3f
10f
2.66±2.85 (2H, m, N-C-CH2CO), 3.72±3.83 (2H, m, NCH2-C-CO), 6.75
(1H, s, PhCO), 7.00 (1H, t, J=6 Hz, NH), 7.67 (1H, s, PhCO), 12.32 (1H,
s, HOPhCO)
Ethers
4
4.27±4.31 (m)
4.41±4.44 (m)
4.30 (brs)
3.96 (s)
3.96 (s)
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
ÐÐ
3.80±4.18 (4H, m, OCH2CH2O), 4.65±4.68 (1H, obsc, OCH2O), 4.71
(1H, d, J=7 Hz, OCH2O), 6.90±6.96 (2H, m, PhO), 7.25±7.31 (3H, m,
PhO)
3.82±4.16 (4H, m, OCH2CH2O), 4.71 (1H, d, J=7 Hz, OCH2O), 4.77
(1H, d, J=7 Hz, OCH2O), 6.90±6.97 (2H, m, PhO), 7.26±7.30 (3H, m,
PhO)
3.83±4.20 (4H, m, OCH2CH2O), 4.89 (1H, d, J=7 Hz, OCH2O), 5.08
(1H, d, J=7 Hz, OCH2O), 6.91±6.97 (2H, m, PhO), 7.25±7.31 (3H, m,
PhO)
3.82±4.16 (4H, m, OCH2CH2O), 4.89 (1H, d, J=7 Hz, OCH2O), 5.08
(1H, d, J=7 Hz, OCH2O), 6.91±6.97 (2H, m, PhO), 7.24±7.31 (3H, m,
PhO)
11
5a
3.94 (s) 3.23 (t, 9)
3.94 (s) 3.23 (t, 9)
3.94 (s) 3.20 (t, 9)
13a
5b
4.43±4.45 (m)
4.28±4.32 (m)
3.92±4.02 (2H, m, OCH2CH2O), 4.17 (2H, t, J=5 Hz, OCH2CH2O), 4.87
(1H, d, J=7 Hz, OCH2O), 5.06 (1H, d, J=7 Hz, OCH2O), 6.67 (1H, d,
J=9 Hz, PhO), 7.25 (1H, d, J=2 Hz, PhO), 7.35 (1H, dd, J=9 and 2 Hz,
PhO)
(continued)

Y. Tsukamoto et al. / Bioorg. Med. Chem. 8 (2000) 19±26
25
Table 2 (continued)
13b
4.43±4.45 (m)
3.94 (s) 3.20 (t, 9)
ÐÐ
3.92±4.02 (2H, m, OCH2CH2O), 4.17 (2H, t, J=5 Hz, OCH2CH2O), 4.87
(1H, d, J=7 Hz, OCH2O), 5.06 (1H, d, J=7 Hz, OCH2O), 6.67 (1H, d,
J=9 Hz, PhO), 7.25 (1H, d, J=2 Hz, PhO), 7.35 (1H, dd, J=9 and 2 Hz,
PhO)
6a
4.30 (brs)
4.43±4.45 (m)
4.31 (brs)
3.94 (s) 3,24 (t, 9) 3.22 (t, 9) 3.92±4.00 (2H, m, OCH2CH2O), 4.15 (2H, d, J=5 Hz, OCH2CH2O),
4.88 (1H, d, J=7 Hz, OCH2O), 5.06 (1H, d, J=7 Hz, OCH2O), 6.91±6.97
(3H, m, PhO), 7.25±7.30 (2H, m, PhO)
3.93 (s) 3.24 (t, 9) 3.22 (t, 9) 3.80±4.00 (2H, m, OCH2CH2O), 4.15 (2H, t, J=5 Hz, OCH2CH2O), 4.88
(1H, d, J=7 Hz, OCH2O), 5.06 (1H, d, J=7 Hz, OCH2O), 6.91±6.97
(3H, m, PhO), 7.25±7.30 (2H, m, PhO)
3.94 (s) 3.23 (t, 9) 3.20 (t, 9) 3.90±4.05 (2H, m, OCH2CH2O), 4.16 (2H, t, J=5 Hz, OCH2CH2O), 4.87
(1H, d, J=7 Hz, OCH2O), 5.05 (1H, d, J=7 Hz, OCH2O), 6.67 (1H, d,
J=9 Hz, PhO), 7.25 (1H, d, J=2 Hz, PhO), 7.36 (1H, dd, J=9 and 2 Hz,
PhO)
15a
6b
15b
4.42±4.44 (m)
3.93 (s) 3.23 (t, 9) 3.20 (t, 9) 3.80±3.98 (2H, m, OCHCH2O), 4.16 (2H, t, J=5 Hz, OCH2CH2O), 4.85
(1H, d, J=7 Hz, OCH2O), 5.03 (1H, d, J=7 Hz, OCH2O), 6.66 (1H, d,
J=9 Hz, PhO), 7.24 (1H, d, J=2 Hz, PhO), 7.34 (1H, dd, J=9 and 2 Hz,
PhO)
5-O-(tert-Butyldimethylsilyl)-400-O-[2-(2-azido-4-iodophen-
oxy)ethoxymethyl]avermectin B1a (15b). The procedure
of 13b but with 14 (40 mg) in the dark gave 3.6 mg
(6.8%) of 15b.
13-O-(2-Phenoxyethoxymethyl)avermectin B1a aglycone
(4). The procedure for 3a but with 11 (16.3 mg) gave
10.0 mg (71%) of 4. MS-LR: C43H58O10, (MH+) 735.
40-O-(2-Phenoxyethoxymethyl)avermectin B1a monosac-
charide (5a). The procedure for 3a but with 13a
(12.7 mg) gave 9.1 mg (81%) of 5a. MS-LR: C50H70O13,
(MH+) 879.
13-O-(2-Benzoylaminopropionyl)avermectin B1a aglycone
(3a). To a solution of 10a (17.2 mg) in acetonitrile
(1 mL) was added HF-pyridine (HF=68%, 0.1 mL) and
the mixture was stirred at room temperature. After 3 h,
additional HF-pyridine (0.1 mL) was added and the
mixture was stirred for 1 h. The reaction mixture was
poured into ice-cold saturated aqueous NaHCO3
(15 mL) and extracted with ethyl acetate (4Â5 mL). The
combined organic layer was washed with water (5 mL)
and brine (5 mL) then dried over Na2SO4 and evapo-
rated in vacuo. The residue was puri®ed by preparative
TLC (hexane:ethyl acetate, 2:3) to give 12.9 mg (86%)
of 3a. MS-LR: C44O57O10N, (MH+) 760.
40-O-[2-(2-Azido-4-iodophenoxy)ethoxymethyl]avermectin
B1a monosaccharide (5b). The procedure for 3a but with
13b (2.6 mg) in the dark gave 2.2 mg (94%) of 5b. MS-
LR HR: C50H68O13IN3, (MLi+) 1052.
400-O-(2-Phenoxyethoxymethyl)avermectin B1a (6a). The
procedure for 3a but with 15a (14.7 mg) gave 9.0 mg
(68%) of 6a. MS-LR: C57H82O16, (MH+) 1023.
400-O-[2-(2-Azido-4-iodophenoxy)ethoxymethyl]avermectin
B1a (6b). The procedure for 3a but with 15b (3.3 mg) in
the dark gave 1.4 mg (46%) of 6b. MS-LR: C57H80
O16IN3, (MLi+) 1196.
13-O-[2-(4-Azidobenzoyl)aminopropionyl]avermectin B1a
aglycone (3b). The above procedure with 10b (13.1 mg)
but in the dark gave 11.5 mg (100%) of 3b. MS-LR:
C44H56O10N4, (MH+) 801.
Mouse and insect brain [3H]1 binding assays
13-O-[2-(2-Azidobenzoyl)aminopropionyl]avermectin B1a
aglycone (3c). The above procedure with 10c (13.1 mg)
in the dark gave 11.4 mg (99%) of 3c. MS-LR:
C44H56O10N4, (MH+) 801.
Brain and head preparations. The studies used whole
brain from male mice (25±30 g) and frozen ( 70 ꢁC)
heads from male and female house¯ies (susceptible
strain cultured in this laboratory, 4±7 days after emer-
gence as adults) and male and female adult fruit ¯ies
(provided by Gerald Rubin of the Department of
Molecular and Cell Biology on this campus). The frozen
insects at dry ice temperature were shaken in a ¯ask to
break them into their body parts and the heads recov-
ered by passing through a 2-mm sieve and recovery on a
1-mm sieve for house¯ies or a 1-mm and 0.5-mm sieve,
respectively, for fruit ¯ies. Dry ice temperature was
maintained for glassware and sieves and the frozen
heads were held at 80 ꢁC until used for membrane
preparation. Subsequent steps were carried out at 4 ꢁC.
13-O-[2-(2-Azido-5-iodobenzoyl)aminopropionyl]avermectin
B1a aglycone (3d). The above procedure with 10d
(16.7 mg) in the dark gave 11.7 mg (79%) of 3d. MS-LR:
C44H55O10N4I, (MH+) 927; HR (MH+) calcd 927.3041,
found 927.3020.
13-O-[2-(4-Azido-2-hydroxybenzoyl)aminopropionyl]aver-
mectin B1a aglycone (3e). The above procedure with 10e
(14.2 mg) in the dark gave 8.6 mg (69%) of 3e. MS-LR:
C44H56O11N4, (MH+) 817.
13-O-[2-(4-Azido-2-hydroxy-5-iodobenzoyl)aminopropio-
nyl]avermectin B1a aglycone (3f). The above procedure
with 10f (16.0 mg) in the dark gave 13.0 mg (91%) of 3f
MS-LR: C44H55O11N4I, (MH+) 943; HR (MH+) calcd
943.2990, found 943.3015.
Membrane preparations. Mouse brain homogenate at
10% (w/v) in 0.32 M sucrose was centrifuged at 1000Âg
for 10 min and the supernatant at 10,000Âg for 20 min.
The pelleted membranes were suspended in 1 mM

26
Y. Tsukamoto et al. / Bioorg. Med. Chem. 8 (2000) 19±26
EDTA and dialyzed versus distilled water for 5±6 h at
4 ꢁC. Centrifugation (25,000Âg, 30 min) gave the ®nal
brain membrane fraction. Insect heads were homo-
genized in 0.25 M sucrose-10 mM Tris±HCl bu€er, pH
7.5, and the homogenate was centrifuged at 1000Âg for
10 min and the supenatant thereof at 130,000Âg for
60 min. The pelleted mouse brain and insect head mem-
branes were suspended in assay bu€er (see below) and
stored at 80 ꢁC until used.
2. Fisher, M. H. In Recent Advances in the Chemistry of Insect
Control II; Crombie, L., Ed.; Royal Society of Chemistry:
Cambridge, 1990; pp 52±68.
3. Fritz, L. C.; Wang, C. C.; Gorio, A. Proc. Natl. Acad. Sci.
USA 1979, 76, 2062.
4. Nicholson, R. A.; Robinson, P. S.; Palmer, C. L.; Casida, J.
E. Pestic. Sci. 1988, 24, 185.
5. Turner, M. J.; Schae€er, J. M. In Ivermectin and Abamectin,
Campbell, W. C. Ed.; Springer±Verlag: New York, 1989; pp
73±88.
6. Fisher, M. H.; Mrozik, H. Annu. Rev. Pharmacol. Toxicol.
1992, 32, 537.
7. Cully, D. F.; Paress, P. S.; Liu, K. K.; Schae€er, J. M.;
Arena, J. P. J. Biol. Chem. 1996, 271, 20187.
8. Huang, J.; Casida, J. E. J. Pharmacol. Exp. Ther. 1997, 281,
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9. Pong, S.-S.; Wang, C. C. Neuropharmacol. 1980, 19, 311.
10. Drexler, G.; Sieghart, W. Eur. J. Pharmacol. 1984, 99, 269.
11. Schae€er, J. M.; Haines, H. Biochem. Pharmacol. 1989, 38,
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12. Arena, J. P.; Liu, K. K.; Paress, P. S.; Frazier, E. G.;
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81, 286.
13. Deng, Y.; Casida, J. E. Pestic. Biochem. Physiol. 1992, 43,
116.
14. Meinke, P. T.; Rohrer, S. P.; Hayes, E. C.; Schae€er,
J. M.; Fisher, M. H.; Mrozik, H. J. Med. Chem. 1992, 35,
3879.
15. Mrozik, H. In Natural and Engineered Pest Management
Agents, Hedin, P. A.; Menn, J. J.; Hollingworth, R. M., Eds.;
ACS Symposium Series, No. 551, American Chemical Society,
Washington, DC, 1994; pp 54±73.
16. Rohrer, S. P.; Meinke, P. T.; Hayes, E. C.; Mrozik, H.;
Schae€er, J. M. Proc. Natl. Acad. Sci. USA 1992, 89, 4168.
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[3H]1 binding assays. Procedures for the [3H]1 binding
assays were based on earlier reports for mammalian
brain9±12 and house¯ies.13 The assay bu€er was 10 mM
phosphate, pH 7.5, containing 200 mM NaCl for mouse
and 300 mM NaCl for insect membranes. The incuba-
tion mixtures were prepared by adding, in sequence,
assay bu€er (870 mL), the test compound in dimethyl
sulfoxide (DMSO) (10 mL), [3H]1 in DMSO:assay bu€er
(1:3, 20 mL) and ®nally the membrane preparation
(100 mL) with mixing. The protein level29 was 50, 200
and 400 mg/assay for mouse, house¯y and fruit ¯y,
respectively. The ®nal [3H]1 concentration was 2.9 nM
(45,000 dpm) for mouse and 1.9 nM (30,000 dpm) for
the insects. The mixtures were incubated for 90 min
(mouse) or 70 min (insect) at 22 ꢁC and then ®ltered on
Whatman GF/C (mouse) or GF/B (insects) glass-®ber
®lters followed by three 5 mL rinses with ice-cold NaCl
(0.9% w/v) solution containing ethanol (2% v/v). Spe-
ci®c binding was considered to be the di€erence between
total 3H bound with 1.9 or 2.9 nM [3H]1 and nonspeci®c
3H bound on addition of 5 mM unlabeled 1. The speci-
®cally bound [3H]1 with mouse and house¯y membranes
was directly proportional to protein level. Typical
values for percentage and dpm speci®c binding were
67% and 7700 dpm for mouse, 67% and 7500 dpm for
house¯y and 86% and 12,000 dpm for fruit ¯y.
19. Cole, L. M.; Casida, J. E. Pestic. Biochem. Physiol. 1992,
44, 1.
20. Blizzard, T. A.; Margiatto, G. M.; Mrozik, H.; Shoop, W.
L.; Frankshum, R. A.; Fisher, M. H. J. Med. Chem. 1992, 35,
3873.
Acknowledgements
This study was supported by Sankyo AgroSciences
Research Laboratories and by Grant R01 ES08419
from the National Institute of Environmental Health
Sciences (NIEHS), NIH, and its contents are solely the
responsibility of the authors and do not necessarily
represent the ocial views of the NIEHS, NIH. We
thank our laboratory colleagues Gary Quistad, Moto-
hiro Tomizawa, Charlemagne Lacza and Graham Ash-
lock for valuable advice and assistance.
21. Nicolaou, K. C.; Webber, S. E. Synthesis 1986, 453.
22. Mrozik, H.; Eskola, R; Fisher, M. H.; Egerton, J. R.;
Cifelli, S.; Ostlind, D. A. J Med. Chem. 1982, 25, 658.
23. Lapatsanis L.; Milias G.; Froussios K.; Kolovos M.
Synthesis 1983, 671.
24. Pinney, K. G.; Katzenellenbogen, J. A. J. Org. Chem.
1991, 47, 3125.
25. Okawa, T.; Sugimori, T.; Eguchi, S.; Kakehi, A. Hetero-
cycles 1998, 47, 375.
26. Latli, B.; Tomizawa, M.; Casida, J. E. Bioconjugate Chem.
1997, 8, 7.
27. Kometani, T.; Watt, D. S.; Ji, T. Tetrahedron Lett. 1985,
26, 2043.
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