5-Amino-1-(2,6-dichloro-4-trifluoromethyl-phenyl)-3-[3H 3]-methylsulfanyl-1H-pyrazole-4-carbonitrile (CTOM): Synthesis and characterization of a novel and selective insect GABA receptor radioligand

Article abstract of DOI:10.1016/j.bmcl.2003.08.042

Pyrazole 2a is a novel, potent ligand for insect GABA receptors obtained from housefly head membrane preparations (K_i=8 nM). It is 500-fold selective against the mammalian receptor (mouse brain preparations). Its specifically tritiated version (2b) was synthesized by reduction of disulfide 10 with NaBH₄ followed by alkylation with [³H ₃]-CH₃I.

Full text of DOI:10.1016/j.bmcl.2003.08.042

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4035–4037
5-Amino-1-(2,6-dichloro-4-trifluoromethyl-phenyl)-3-[3H3]-
methylsulfanyl-1H-pyrazole-4-carbonitrile (CTOM):
Synthesis and Characterization of a Novel and Selective Insect
GABA Receptor Radioligand
Sanath K. Meegalla,a,* Dario Doller,a Gary M. Silver,b Nancy Wisnewski,b
Richard M. Solla and Dale Dhanoaa
a3-Dimensional Pharmaceuticals, Inc., 665 Stockton Drive, Suite 104, Exton, PA 19341, USA
bHeska Corporation, 1613 Prospect Parkway, Fort Collins, CO 80525, USA
Received 6 January 2003; revised 1 May 2003; accepted 22 August 2003
Abstract—Pyrazole 2a is a novel, potent ligand for insect GABA receptors obtained from housefly head membrane preparations
(Ki=8 nM). It is 500-fold selective against the mammalian receptor (mouse brain preparations). Its specifically tritiated version (2b)
was synthesized by reduction of disulfide 10 with NaBH4 followed by alkylation with [3H3]-CH3I.
# 2003 Elsevier Ltd. All rights reserved.
g-Aminobutyric acid (GABA) receptors are membrane-
bound glycoproteins that belong to the ligand-gated ion
channel receptor family. These receptors play a major
role in the inhibition of central nervous system (CNS)
neuronal activity in vertebrates and invertebrates.
GABA receptors are also the target for a relatively new
class of highly effective insecticides, the arylpyrazoles,
which are used for the chemical control of economically
important pests and insects such as fleas, ticks, house-
flies, grasshoppers and cockroaches.1 Arguably the most
important member of this family, fipronil (1), has
attained the position of world leader in sales for this
market. An important characteristic of the arylpyr-
azoles as a class is their high selectivity, which provides
them with a favorable toxicity profile for insects relative
to their safety against vertebrates. At the molecular
level, the selectivity of the arylpyrazoles is a reflection of
the target-site specificity between the GABA receptors
of insects and vertebrates. Within the family of different
insect GABA receptors three distinct binding sites have
been identified: a GABA agonist binding site, a benzo-
diazepine binding site and a convulsant binding site.
The convulsant binding site of GABA receptors in
insects is the major target site for many of the drugs and
insecticides currently on the market. Mechanism of
action studies demonstrated that 1 is a non-competitive
GABA antagonist in insects, acting either with an
allosteric binding site or by irreversible binding.2
The development of suitable radioligands constitutes a
major step in furthering the understanding of GABA
receptor pharmacology. An effective insect GABA
radioligand should satisfy several important require-
ments. It should have high affinity for the insect GABA
receptors, with high species selectivity, preferably high
insecticidal activity, high specific activity upon chemical
labeling and chemical stability upon standard storage
conditions [e.g., (À18 ꢀC) dimethyl sulfoxide solutions
in freezer]. Radiolabeled ligands 3–6, among others,
*Corresponding author. Tel.:+1-610-458-8959; fax: +1-610-458-
8249; e-mail: meegalla@3dp.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.042

4036
S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4035–4037
have proven to be only partially satisfactory tools in
insect receptor studies, probably due in part to their low
to moderate insecticidal activity. Radioligand 3 ([3H]-
EBOB), which is currently widely used, also has the
drawback of being fairly chemically unstable, pre-
sumably due to the hydrolytic nature of its orthoester
functionality, and therefore requiring periodical puri-
fications prior to being used.
obtain disulfide 10, arising from air-oxidation of the
intermediate sulfide. Reduction of disulfide 10 with
NaBH4 followed by in situalkylation with CH I furn-
3
ished unlabeled 2a in good yields (27% overall yield).5
When the final step was carried out with radiolabeled
[3H3]-CH3I we were able to obtain labeled compound
2b with 82.5 Ci/mmol specific activity. HPLC analysis
showed greater than 99.5% radiochemical purity.
Continuous use of 2b has demonstrated its chemical
stability.
Unlabeled compound 2a exhibited 8 nM binding affinity
for GABA receptors in house fly head preparations and
4 mM binding affinity in mouse brain preparations,
resulting in a 500-fold selectivity for insect GABA
receptors.4 Despite its high in vitro binding affinity and
selectivity, this compound did not show significant in
vivo insecticidal activity when tested in either flea or tick
contact assays or when fed to fleas in a blood meal using
an artificial feeding apparatus. We currently have a
limited amount of experimental information to explain
this fact. One possibility may be rapid metabolic inac-
tivation upon absorption, as the corresponding sulfox-
ide 2c, which was isolated from flea feces after blood
feeding and showed no activity in flea contact assays.
Other possible causes that could contribute to the lack
of in vivo activity include binding to a non-insecticidal
site of GABA receptors, high non-specific protein
binding and poor bioavailability of this compound in
fleas and ticks. One further possibility is that com-
pound 2a is acting as an antagonist of compound 1,
binding at the same allosteric site without having
insecticidal properties. To address this potential sce-
nario, compound 1 and 2a were co-administered in a
flea contact assay to assess the blocking potential of 2a.
Compound 2a, which alone showed no insecticidal
activity, administered at 10-, 20-, and 100- fold excess
of three concentration titration of compound 1, which
alone showed 50–100% flea mortality, exhibited no
effect on the insecticidal properties of compound 1.
Thus, it is unlikely that compound 2a is acting as a
conventional antagonist of compound 1.
Our structure-insecticidal activity relationship studies
among arylpyrazoles yielded compound 2a [5-amino-1-
(2,6-dichloro-4-trifluoromethylphenyl)-3-methylsulfanyl-
1H-pyrazole-4-carbonitrile, CTOM in its radiolabeled
version 2b] as a potent (housefly Ki=8 nM) and selec-
tive (500-fold selectivity vs mouse receptor) GABA
receptor ligand. Since this compound satisfied the
requirements of high affinity and insect selectivity, we
envisioned a synthetic route to introduce a tritiated thio-
methyl group at the 3-position of the pyrazole ring, which
would enable us to investigate the additional requirements
for a new, stable, high specific activity radioligand.
The synthetic methodology employed in our investiga-
tions is shown in Scheme 1. In order to introduce a tri-
tiated thiomethyl group at C-3 we contemplated the use
of a reduction/alkylation sequence with [3H3]-methyl
iodide on disulfide 10. Precursor disulfide 10 was pre-
pared starting with commercially available mal-
ononitrile 7. The anion of malononitrile was reacted
with carbon disulfide, and the resulting ketenedithio-
enolate intermediate was alkylated in situby quenching
with p-methoxybenzylchloride (PMBCl) to obtain
bisalkylated ketenedithioacetal 8.3 Cyclocondensation
of 8 with 2,6-dichloro-4-trifluoromethylphenylhydrazine
afforded arylpyrazole 9. Removal of the protecting
PMB group from 9 was carried out using Hg(OAc)2 to
In summary, we have developed a new, highly-selective
and stable GABA receptor radioligand, CTOM. This
was prepared in high specific activity and radiochemical
purity from readily available commercial materials in
four steps. We believe compound 2b will be a useful tool
in studying in vitro insect GABA receptor biology.
Acknowledgements
We wish to thank Mike Kolpak, Stephen Eisnnagel,
Heidi Ott, Joely Maddux, Victor Ozols, and Scott
Walmsley for analytical and technical support.
References and Notes
1. Hainzl, D.; Cole, L. M.; Casida, J. E. Chem. Res. Toxicol.
1998, 11, 1529.
Scheme 1.

S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4035–4037
4037
2. Bloomquist, J. R. Annu. Rev. Entomol. 1996, 41, 163.
3. Villemin, D.; Ben Alloum, A. Synthesis 1991, 301.
(2H, d, J=8.7 Hz), 4.41 (2H, broad), 4.23 (2H, s). MS:
M+1=473 (calcd 473).
4. Dhanoa, D. S.; Meegalla, S.; Doller, D.; Soli, R. M. US
Patent 6,409,988.
5-Amino-3-({5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]
-4-cyano-pyrazol-3-yl}disulfanyl)-1-[2,6-dichloro-4-(trifluoro-
methyl)phenyl]pyrazole-4-carbo-nitrile (10). Compound 9 (473
mg, 1.0 mmol) was dissolved in trifluoroacetic acid (6.5 mL)
containing anisole (0.2 mL) at 0 ꢀC, and Hg(OAc)2 (383 mg,
1.2 mmol) was added. The resulting mixture was stirred at 0 ꢀC
for 30 min, and the volatiles evaporated under reduced pres-
sure. The residue was dried under high-vacuum and dissolved
in ethanol. H2S (g) was bubbled through the solution for 30
min. The black precipitate was filtered through a Celite pad.
The filtrate was concentrated, dissolved in methylene chloride,
and thoroughly washed with NaHCO3 (satd soln, 30 mL). The
residue was dissolved in methanol (20 mL) and stirred open to
the air for 24 h. Methanol was removed under reduced pres-
sure, and the residue purified by flash chromatography (25%
ethyl acetate–hexanes) to yield the desired disulfide 10 (229
5. 1H NMR spectra were recorded on a Bruker AC 300 NMR
spectrometer, in CDCl3 as solvent. Chemical shifts are repor-
ted in ppm relative to tetramethylsilane as an internal stan-
dard. Mass spectra were recorded on a LCQ Finnigan mass
spectrometer. Commercially available solvents and reagents
were used as received. Flash chromatography was carried out
using Merck Kieselgel 60 silica gel.
{Bis[(4 - methoxyphenyl)methylthio]methylene}methane - 1,1 -
dicarbonitrile (8). Anhydrous dimethyl formamide (40 mL)
was slowly added to a rapidly stirred mixture of malononitrile
(13.2 g, 0.2 mol), NaH (9.6 g, 0.4 mol) and carbon disulfide
(22.8 g, 0.3 mol) in anhydrous benzene (200 mL) at room
temperature. The reaction mixture was stirred for 30 min and
4-methoxybenzyl chloride (93.6 g, 0.6 mol) was added. The
resulting mixture was stirred for 12 h, and benzene (50 mL)
and ice-water (200 mL) were added. The organic layer was
separated, dried, and concentrated under vacuum. The pro-
duct was purified by flash chromato-graphy on silica gel, elut-
ing with ethyl acetate/hexanes (15:85). Yield: 45.8 g (0.12 mol,
60%). 1H NMR (CDCl3, 300 MHz): d 7.22 (4H, d, J=8.6 Hz),
6.85 (4H, d, J=8.6 Hz), 4.33 (4H, s); 3.79 (6H, s).
5-Amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-3-[(4-meth-
oxyphenyl)-methylthio]pyrazole-4-carbonitrile (9). A solution of
2,6-dichloro-4-trifluoromethyl phenyl hydrazine (245 mg, 1.0
mmol) and {bis[(4-methoxyphenyl)methylthio] methylene}-
methane-1,1-dicarbonitrile (382 mg, 1.0 mmol) in isopropyl
alcohol (15 mL) was heated at reflux temperature for 16 h. The
solvent was removed under reduced pressure and the desired
product obtained after chromatographic separation (silica gel)
using ethyl acetate–hexanes (331 mg, yield: 70%). 1H NMR
(CDCl3, 300 MHz): d 7.7 (2H, s), 7.2 (2H, d, J=8.7 Hz), 6.8
1
mg, 65% yield). H NMR (CDCl3, 300 MHz): d 7.75 (4H, s),
4.7, (4H, broad). MS: M+1=703 (calcd 703).
5-Amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-3-[3H3]-
methylthio-pyrazole-4-carbonitrile (2b). A solution of disulfide
9 (20 mg, 29 mmol) in ethanol (5 mL) was treated with excess
NaBH4 (15 mg) at room temperature. After 10 min excess
[3H3]-methyl iodide (50 mL) was added and the mixture
stirred at room temperature for 2 h. The reaction mixture
was cooled down to 0 ꢀC (ice-water bath) and unreacted
NaBH4 was destroyed by dropwise addition of aqueous
HCl (10%, 1 mL). The mixture was neutralized and extracted
with ethyl ether (3Â10 mL). The organic fractions were com-
bined and washed with brine, dried, and the solvent removed
under reduced pressure. Column chromatography yielded pure
compound 2b (0.77 mCi, 82.5 Ci/mmol). MS: 367.2 and 369.2
(calcd 367). 1H NMR (for (2a), CDCl3 300 MHz): d 7.77 (2H, s),
4.55, (2H, broad), 2.54 (3H, s).