Fipronil-based photoaffinity probe for Drosophila and human β3 GABA receptors

Article abstract of DOI:10.1016/S0960-894X(01)00604-7

Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC_50<

Full text of DOI:10.1016/S0960-894X(01)00604-7

Bioorganic & Medicinal ChemistryLetters 11 (2001) 2979–2981
Fipronil-based Photoaffinity Probe for Drosophila and Human
ꢀ3 GABA Receptors
Nilantha S. Sirisoma, Gurpreet S. Ratra, Motohiro Tomizawa 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 6 July2001; accepted 5 September 2001
Abstract—Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and
amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency(IC 50 2–28
nM) in blocking the chloride channel of Drosophila and human b3 GABA receptors. # 2001 Elsevier Science Ltd. All rights
reserved.
g-Aminobutyric acid (GABA) receptors are ligand-
gated chloride ion channels and the target for important
drugs such as benzodiazepines and toxicants including
insecticides.1,2 The GABA-recognition and benzodiaze-
Introduction of the photoreactive substituent
(Scheme 1)13,14 involved conversion of the bromopyr-
azole (4),15 via the trifluoromethylketone (5),16 oxime
tosylate (6),17 and diaziridine (7)18 to the (tri-
fluoromethyl)diazirine (3)19 in an overall yield of 18%.
Compounds were isolated bychromatographyon a
silica gel column with hexane–ethyl acetate and char-
pine binding sites have been labeled and characterized
3,4
with appropriate photoaffinityprobes.
The most
important chloride channel blockers are the botanical
toxicant picrotoxinin and three major insecticides, two
of which are polychlorocycloalkanes (i.e., a-endosulfan
and lindane) and the third is an arylpyrazole (fipronil)
(1) (Fig. 1).5ꢀ7 The localization of the ‘insecticide bind-
ing site’ (also known as the picrotoxinin or non-
competitive blocker site) has onlybeen determined
indirectlyfor lack of a suitable high-affinityphoto-
reactive ligand.5
1
acterized by H and 13C NMR20 and HRMS and UV
for the end product 3.21
The mammalian GABAA receptor consists of hetero-
oligomeric assemblies of several different subunits (a1-
a6, b1-b4, g1-g3, and others), which can be studied in
various recombinant combinations.1,2,22ꢀ24 There are
several lines of evidence that the insecticide binding site
is in the lumen of the chloride channel pore at the sec-
ond transmembrane segment and a b subunit mayplay
an important role. The Drosophila GABA receptor has
two a and one b subunits (one of which has been cloned
and expressed).25ꢀ27 A single point mutation in Droso-
phila greatlyreduces the binding affinityfor the major
GABAergic insecticides, suggesting that this A302S
We report here that 1-[2,6-dichloro-4-(trifluoromethyl)-
phenyl]-4-[(trifluoromethyl)diazirinyl]-1H-pyrazole (3)
(Fig. 1) has interesting properties as a candidate photo-
affinityprobe. This compound was based on 1 and
selected for studyfor two reasons. First, moderate to
high receptor potencyis retained on deleting the cyano
and amino substituents of 1, as in compound 2 (Fig. 1),8
and on replacing –S(O)CF3 at the 4-position with –
S(O2)CF3, –SCF3 or –CF3.9ꢀ11 Second, the (tri-
fluoromethyl)diazirinyl moiety, with many structural
features in common with this series of substituents, is a
favorable carbene generating group for photolabeling
reagents.12
Figure 1. Structures of fipronil (1), a potent analogue lacking the amino
and cyano substituents (2) and the candidate photoaffinityprobe ( 3).
*Corresponding author. Tel.: +1-510-642-5424; fax: +1-510-642-
6497; e-mail: ectl@nature.berkeley.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00604-7

2980
N. S. Sirisoma et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2979–2981
ꢂ
.
Scheme 1. (a) nBuLi, EtOC(O)CF3, THF, ꢀ78 C, 40%; (b) (1) NH2OH HCl, pyridine; (2) TsCl, pyridine, 64%; (c) NH3, Et2O, 72%; (d) Ag2O,
Et2O 97%.
Figure 2. Potencies of fipronil (1) and the candidate photoaffinityprobe ( 3) as inhibitors of [3H]EBOB binding in Drosophila and human b3 GABA
receptors. IC50 values (as an index of potencyfor the insecticide binding site of GABA receptors) are given as nM ꢁSD (n=3). Data points include
SD. Hill coefficients (nH) for 1 with the Drosophila and human b3 receptors and for 3 with the b3 receptor are 1.01ꢁ0.07, 1.02ꢁ0.02 and 1.06ꢁ0.04,
respectively, suggesting a similar type of action. Interestingly, a nH of 0.61ꢁ0.02 is obtained for 3 with Drosophila receptor, implying a multiple class
of action or negative cooperativity.
modification is at or near the insecticide binding
site.25,26,28 This region of the Drosophila GABA recep-
tor has a close sequence homologyto that of the human
b3 subunit.22,24,25 The mammalian b3 homooligomeric
receptor displays binding activity of radiolabeled
blocker(s) appropriate for the insecticide binding site
when expressed in either insect Sf9 cells23,24 or human
embryonic kidney cells.29,30 The human recombinant b3
receptor from Sf9 cells is more sensitive to insecticide
action than a receptor with anyother single subunit or
combination of subunits examined.23 Not onlythe
insecticide sensitivitybut also the specificityare con-
served, so it is proposed that the insecticide and/or
picrotoxinin binding site of the human b3 homo-
oligomer can be used as a model for the Drosophila
receptor and vice versa.24 A single photoaffinityprobe
for both the Drosophila and human GABA receptors
would allow a more direct test of this hypothesis. It
would also help define the precise location of the insec-
ticide binding site in insect and mammalian GABA
receptors and potentiallythe basis for selective toxicity.
inhibition (IC50 values) by 1 and 3 with both receptors
fall in the 1–28 nM range. The structural changes on
modifying 1 to give 3 apparentlyhave little effect on fit
at the receptor, with a potencyloss of only1.5- to 16-
fold, that is candidate photoaffinityprobe 3 is a suitable
mimic of 1 at the binding site in the GABA receptors of
both insects and mammals. The final step in converting
the candidate photoaffinityprobe into a practical photo-
affinityradioligand will require the incorporation of one
or two tritium atoms, either at the phenyl substituent or
the position of newlyintroduced protons on converting
1 to 3.
Acknowledgements
This studywas supported in part bygrant R01 ES08419
from the National Institute of Environmental Health
Sciences (NIEHS), National Institutes of Health (NIH),
and its contents are solelythe responsibilityof the
authors and do not necessarilyrepresent the official
views of the NIEHS, NIH. We thank GaryQuistad
and Nanjing Zhang of this laboratoryfor helpful
suggestions.
The insecticide binding site can be readilyexamined
with 40-ethynyl-4-[2,3-3H2]propylbicycloorthobenzoate
([3H]EBOB), which has similar affinityat Drosophila
head and human b3 homooligomeric receptors.23,24,28,31
The potencies of 1 and 3 in displacing specific
[3H]EBOB binding were therefore compared with Dro-
sophila and human b3 receptors32 with the fortuitous
finding that high potencyis retained for 3 on both
receptors (Fig. 2). The molar concentrations for 50%
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ꢃ0.5 mL NH3 was condensed into it. The screw cap was
closed tightlyand the mixture was stirred overnight at room
temperature. The residue after removing the white precipitate
was chromatographed with 15% ethyl acetate in hexane to
obtain 7 (oil, 202 mg, 0.516 mmol, 72%) which is stable for
several months at ꢀ20 ꢂC.
19. To 7 (202 mg, 0.516 mmol) in dryether (4 mL) was added
14
freshlyprepared
Ag2O (500 mg, 2.16 mmol). After 45 min,
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(3% ethyl acetate in hexane) to obtain 3 (oil, 194 mg, 0.50
mmol, 97%). Compound 3 is much less stable than immediate
precursor 7, so the final conversion step should be carried out
shortlybefore use.
20. 1H NMR (CDCl3, 300 MHz, d) for pyrazole H3 and H5,
respectively: 3 7.68 and 7.55; 4 7.80 and 7.61; 5 8.38 and 8.30;
6 8.44 and 8.26; 7 7.96 and 7.82. 13C NMR (CDCl3, 75 MHz,
d) for CF3 and pyrazole C3, C4 and C5, respectively: 3, 121.8,
139.8, 113.5, and 126.0; 4, no CF3, 142.4, 95.4, and 125.9; 5
116.3, 143.0, 117.9, and 126.2; 6 119.9, 146.3, 108.2 and 126.0;
7 123.3, 140.4, 115.8, and 126.6.
21. 3: HRMS (EI) m/z C12H5Cl2F6N4, [MH]+ calcd 388.9796,
found 388.9798; UV (ethanol) mmax 358 nm.
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16. Bromopyrazole 4 (1.264 g, 3.51 mmol) in dryTHF (12
mL) at ꢀ78 ꢂC was added slowlyto n-butyllithium (1.6 M, 2.6
mL, 4.16 mmol) in dryTHF (20 mL) at ꢀ78 ꢂC. After stirring
for 15 min, EtOC(O)CF3 (1.0 g, 7.0 mmol) was added in THF
(5 mL). The solution was allowed to warm to 20 ꢂC and stirred
for 1 h and 1 mL of saturated NaCl was added. The reaction
mixture was diluted with ethyl acetate (20 mL) and washed
with half-saturated NaCl (50 mL) and saturated NaCl (50 mL)
then the organic layer was dried over MgSO4, filtered, and
concentrated. The residue was chromatographed on silica gel
with 5% ethyl acetate in hexane to obtain 5 (529 mg, 1.40
mmol, 40%).
17. Trifluoromethylketone derivative 5 (529 mg, 1.40 mmol)
was dissolved in pyridine (4.4 mL, dried over CaH2) and eth-
anol (1.8 mL). NH2OH HCl (100 mg, 1.44 mmol) was added
32. The receptor preparations were Drosophila head mem-
branes (300 mg protein/assay) and Sf9 cell membranes expres-
sing recombinant human b3 receptor (250 mg protein/assay).
Assaymixtures contained membrane fraction, various con-
centrations of 1 or 3 dissolved in dimethyl sulfoxide (final
concentration 1%), and 0.8 nM (Drosophila) or 1.0 nM (b3)
[3H]EBOB (30 Ci/mmol) in a total volume of 0.5 mL of 300
mM (for Drosophila) or 200 mM (for b3) sodium chloride/10
mM sodium phosphate buffer (pH 7.5). After 70 min incuba-
tion at 25 ꢂC, the binding reaction was terminated byfiltration
on a GF/B filter (presoaked in 0.1% polyethyleneimine) and
the filter was rinsed three times with ice cold water containing
0.9% sodium chloride/2% ethanol. Specific binding was
defined as the difference in radioactivityon the filter in the
absence and presence of 5 mM a-endosulfan.
.
and the mixture was stirred overnight at 60 ꢂC. The residue
from evaporation was dissolved in ethyl acetate with work up
involving washing with water, 0.1 N HCl and saturated NaCl,
then drying, filtration and concentration. The crude oxime
intermediate (401 mg, 1.02 mmol) was dissolved in anhydrous
pyridine (3.5 mL) at room temperature, tosyl chloride (278
mg, 1.46 mmol) was added and the mixture was refluxed for 4
h. The solvent was evaporated and the residue chromato-
graphed with 4–6% ethyl acetate in hexane to give 6 (490 mg,
0.90 mmol, overall 64%) as a white solid.
18. A solution of 6 (390 mg, 0.714 mmol) in dryether (1.5
mL) in a thick-walled glass tube was cooled to ꢀ78 ꢂC and