Synthesis of a tritium-labeled, fipronil-based, highly potent, photoaffinity probe for the GABA receptor

Article abstract of DOI:10.1021/jo034520z

3-{4-[1-(2,6-Dichloro-4-trifluoromethylphenyl)pyrazolo]} -3-(trifluoromethyl)diazirine is a fipronil-based (i.e. fiprole), high-affinity probe for the GABA receptor. For synthesis of the tritium-labeled version of this trifluoromethyldiazirinylfiprole ([³H]TDF) the required intermediate, 3-{4-[1-(2,6-dichloro-3-iodo-4-trifluoromethylphenyl)-5-iodopyrazolo]} -3-(trifluoromethyl)diazirine, was prepared in 10 steps from pyrazole and 3,5-dichloro-4-fluorobenzotrifluoride. One of the key transformations was lithiation and subsequent iodination of the 4-(2,2,2-trifluoro-1-hydroxyethyl)pyrazole intermediate. The last step involved reduction of the diiodofiprole with tritium, Pd/C, and triethylamine in ethyl acetate and afforded [³H]TDF with a specific activity of 15 Ci/mmol and 99% radiopurity.

Full text of DOI:10.1021/jo034520z

Syn th esis of a Tr itiu m -La beled , F ip r on il-Ba sed , High ly P oten t,
P h otoa ffin ity P r obe for th e GABA Recep tor
Robert E. Sammelson† and J ohn E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and
Management, University of California, Berkeley, California 94720-3112
ectl@nature.berkeley.edu
Received April 22, 2003
3-{4-[1-(2,6-Dichloro-4-trifluoromethylphenyl)pyrazolo]}-3-(trifluoromethyl)diazirine is a fipronil-
based (i.e. fiprole), high-affinity probe for the GABA receptor. For synthesis of the tritium-labeled
version of this trifluoromethyldiazirinylfiprole ([3H]TDF) the required intermediate, 3-{4-[1-(2,6-
dichloro-3-iodo-4-trifluoromethylphenyl)-5-iodopyrazolo]}-3-(trifluoromethyl)diazirine, was prepared
in 10 steps from pyrazole and 3,5-dichloro-4-fluorobenzotrifluoride. One of the key transformations
was lithiation and subsequent iodination of the 4-(2,2,2-trifluoro-1-hydroxyethyl)pyrazole intermedi-
ate. The last step involved reduction of the diiodofiprole with tritium, Pd/C, and triethylamine in
ethyl acetate and afforded [3H]TDF with a specific activity of 15 Ci/mmol and 99% radiopurity.
Fipronil (1)1 is a major insecticide2 acting as a non-
competitive blocker of the γ-aminobutyric acid (GABA)
receptor/chloride channel.3 It is the most important
example of the phenylpyrazole or fiprole insecticides.
Understanding the noncompetitive blocker site of the
GABA-gated chloride channel would be greatly facilitated
by a fiprole radioligand4 and particularly by a photoaf-
finity probe5 active on both insect and mammalian
systems. We recently introduced a candidate fipronil-
based photoaffinity probe 2, which shows very high
potency at Drosophila and human â3 GABA receptors.6
We envisioned that the radiolabeled portion of our
F IGURE 1. Fipronil and a candidate photoaffinity probe.
photoaffinity probe could be introduced as the final
synthetic step by selective tritium reduction of an io-
doarene.7 Our synthesis began from commercially avail-
able 3,5-dichloro-4-fluorobenzotrifluoride and 4-iodopy-
razole (3), which was prepared in one step from pyrazole
with iodine and ceric ammonium nitrate (CAN) as
depicted in Scheme 1.8 Nucleophilic aromatic substitution
with potassium carbonate in DMF at 100 °C smoothly
produced the desired phenylpyrazole 4. We found that
this route was more reliable than attempting to iodinate,
such as with N-iodosuccinimide or iodine monochloride,
after the phenyl and pyrazole rings had been brought
together. Iodine-magnesium exchange9 of 4 with isopro-
pylmagnesium chloride in THF gave the corresponding
Grignard reagent, which was quenched with freshly
prepared N-(trifluoroacetyl)piperidine. The R-amino alkox-
ide10 generated upon addition of the Grignard reagent
† Current address: Ball State University, Department of Chemistry,
Muncie, Indiana, 47306-0445.
* Corresponding author. Phone: 510-642-5424. Fax: 510-642-6497.
(1) (a) Buntain, I. G.; Hatton, L. R.; Hawkins, D. W.; Pearson, C.
J .; Roberts, D. A. Eur. Pat. Appl. 295117, 1988. (b) Colliot, F.;
Kukorowski, K. A.; Hawkins, D W.; Roberts, D. A. In Brighton Crop
Protection ConferencesPests and Diseases; British Crop Protection
Council: Farnham, UK, 1992; Vol. 1, pp 29-34.
(2) (a) Gant, D. B.; Chalmers, A. E.; Wolff, M. A.; Hoffman, H. B.;
Bushey, D. F. Rev. Toxicol. 1998, 2, 147-156. (b) Bloomquist, J . R. In
Biochemical Sites of Insecticide Action and Resistance; Ishaaya, I., Ed.;
Springer-Verlag: Berlin, Germany, 2001; pp 17-41.
(3) (a) Cole, L. M.; Nicholson, R. A.; Casida, J . E. Pestic. Biochem.
Physiol. 1993, 46, 47-54. (b) Casida, J . E. Arch. Insect Biochem.
Physiol. 1993, 22, 13-23.
(7) For examples of selective reduction of iodoarenes with hydrogen,
deuterium, and tritium in the presence of other reducible groups such
as the diazirine see: (a) Ambroise, Y.; Mioskowski, C.; Dje´ga-Mari-
adassou, G.; Rousseau, B. J . Org. Chem. 2000, 65, 7183-7186. (b)
Ambroise, Y.; Pillon, F.; Mioskowski, C.; Valleix, A.; Rousseau, B. Eur.
J . Org. Chem. 2001, 3961-3964. (c) Faucher, N.; Ambroise, Y.; Cintrat,
J .-C.; Doris, E.; Pillon, F.; Rousseau, B. J . Org. Chem. 2002, 67, 932-
934.
(8) Rodr´ıguez-Franco, M. I.; Dorronsoro, I.; Herna´ndez-Higueras, A.
I.; Antequera, G. Tetrahedron Lett. 2001, 42, 863-865.
(9) Iodine-magnesium exchange is effective at the 4-position of
pyrazoles and avoids competing deprotonations as well as anion
isomerization: Felding, J .; Kristensen, J .; Bjerregaard, T.; Sander, L.;
Vedsø, P.; Begtrup, M. J . Org. Chem. 1999, 64, 4196-4198.
(10) Comins, D. L. Synlett 1992, 615-625.
(4) The 5-amino-4-cyano-3-[3H]methylthiopyrazole analogue of 1 is
reported as a radioligand for the housefly GABA receptor: (a) Dhanoa,
D. S.; Meegalla, S.; Doller, D.; Soll, R. M. U.S. Patent 6,409,988 B1,
2002. (b) Meegalla, S. K.; Doller, D.; Silver, G. M.; Wisnewski, N.; Soll,
R.; Dhanoa, D. 225th American Chemical Society National Meeting,
New Orleans, LA; March 23-27, 2003; MEDI 222.
(5) (a) Hatanaka, Y.; Sadakane, Y. Curr. Top. Med. Chem. 2002, 2,
271-288. (b) Fleming, S. A. Tetrahedron 1995, 51, 12479-12520. (c)
Kotzyba-Hibert, F.; Kapfer, I.; Goeldner, M. Angew. Chem., Int. Ed.
Engl. 1995, 34, 1296-1312.
(6) Sirisoma, N. S.; Ratra, G. S.; Tomizawa, M.; Casida, J . E. Bioorg.
Med. Chem. Lett. 2001, 11, 2979-2981.
10.1021/jo034520z CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/13/2003
J . Org. Chem. 2003, 68, 8075-8079
8075

Sammelson and Casida
SCHEME 1a
SCHEME 3a
a
Reagents and conditions: (a) NaBH4, EtOH, rt, 97%; (b) (i)
LDA, THF, -78 °C, (ii) I2, THF, -78 °C to room temperature, 55%
(10) and 25% (11).
a
Reagents and conditions: (a) I2, CAN, CH3CN, rt, 97%; (b)
3,5-dichloro-4-fluorobenzotrifluoride, K2CO3, DMF, 100 °C, 99%;
(c) (i) i-PrMgCl, THF, 0 °C, (ii) N-(trifluoroacetyl)piperidine, -78
°C to room temperature, 79%.
procedure worked well and predictively on this type of
phenyl ring we moved forward and attempted ortho
lithiations on phenylpyrazole 4. Following the same
procedure as in the synthesis of 6, we obtained bis-TMS
derivative 8 almost exclusively, showing the ease of
metalation of the pyrazole ring in addition to the phenyl
group (Scheme 2). Although not detailed here, we deter-
mined that with limiting base and electrophile, metala-
tion of the pyrazole ring in 4 precedes metalation of the
phenyl ring.
SCHEME 2a
Results with the model systems encouraged us to
continue experimenting with ortho lithiation chemistry.
Reduction of 4-trifluoromethyl ketone 5 with NaBH4 in
ethanol (Scheme 3) smoothly afforded alcohol 9 and
fortunately this was completely stable to the lithiation
conditions. Reaction of 9 with excess LDA provided
lithiated intermediates as indicated by the formation of
the dark red color. Quenching with iodine followed this
ortho lithiation; however, this reaction was not ideal as
it produced a mixture of substitution products (Scheme
a
Reagents and conditions: (a) (i) LDA, THF, -78 °C, (ii) TMSCl,
-78 °C to room temperature, 89% (6) and 91% (8); (b) (i) LDA,
THF, -78 °C, (ii) I2, THF, -78 °C to room temperature, 94%.
to the amide provided 4-trifluoroacetylpyrazole 5 after
aqueous workup.
Several attempts were made at directed ortho lithia-
tion11 of 5 as well as subsequent synthetic intermediates
up to and including 2. In each case, however, there was
considerable decomposition under the standard condi-
tions for lithiation/iodination12 and the desired products
could not be isolated. We therefore took a closer look at
our system as it pertains to ortho lithiation and meta-
lation chemistry. Reaction of commercially available
3,4,5-trichlorobenzotrifluoride with excess LDA at -78
°C followed by quenching with excess TMSCl gave bis-
TMS derivative 6 in excellent yield (Scheme 2). Following
the same ortho lithiation procedure and then quenching
with iodine provided mono-iodo derivative 7 in nearly
quantitative yield. Presumably, only the monoanion is
formed on reaction of LDA and so the difference in one
versus two substitutions in these metalations is based
on the fact that LDA is stable in the presence of TMSCl13
and the second metalation can occur in situ. Similarly,
with methyl iodide or allyl bromide as the electrophile
two methyl groups or one allyl group was added (not
shown) as would be predicted on the basis of the stability
of LDA with each of these electrophiles. Since this
1
3). H NMR indicated the major product (10) was iodi-
nated on both the phenyl and pyrazole rings. The second
product (11) was iodinated only on the phenyl ring and
there were also traces of an iodinated pyrazole derivative
and starting material that were not isolated from the
crude mixture. We found it most efficient to use diiodo
product 10 and recycle the rest to be iodinated again.
This iodo-containing alcohol was oxidized back to the
corresponding trifluoromethyl ketone (12) most efficiently
with Dess-Martin periodinane (Scheme 4).14 With our
trifluoromethyl ketone containing two iodine atoms in
hand we followed standard protocol for the conversion
to the diazirine.15 The ketone was converted to its oxime
(13) with hydroxylamine hydrochloride in pyridine and
ethanol. This oxime was next reacted with tosyl chloride
and triethylamine with catalytic DMAP16 in dichlo-
romethane to afford the corresponding oxime O-tosylate
14. Conversion of 14 to diaziridine 15 was brought about
with ammonia in ether under pressure in a sealed tube.
(14) (a) Dess, D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155-
4156. (b) Linderman, R. J .; Graves, D. M. J . Org. Chem. 1989, 54, 661-
668.
(15) (a) Li, G.; Samadder, P.; Arthur, G.; Bittman, R. Tetrahedron
2001, 57, 8925-8932. (b) Weber, T.; Brunner, J . J . Am. Chem. Soc.
1995, 117, 3084-3095. (c) Delfino, J . M.; Schreiber, S. L.; Richards,
F. M. J . Am. Chem. Soc. 1993, 115, 3458-3474.
(16) DMAP-catalyzed tosylation was far superior to noncatalyzed
reaction in pyridine.
(11) (a) Snieckus, V. Chem. Rev. 1990, 90, 879-933. (b) Zhang, N.;
Casida, J . E. J . Med. Chem. 2002, 45, 2832-2840.
(12) Leroux, F.; Schlosser, M. Angew. Chem., Int. Ed. 2002, 41,
4272-4274.
(13) (a) Mills, R. J .; Horvath, R. F.; Sibi, M. P.; Snieckus, V.
Tetrahedron Lett. 1985, 26, 1145-1148. (b) Corey, E. J .; Gross, A. W.
Tetrahedron Lett. 1984, 25, 495-498.
8076 J . Org. Chem., Vol. 68, No. 21, 2003

Synthesis of a Photoaffinity Probe for the GABA Receptor
SCHEME 4a
a
Reagents and conditions: (a) Dess-Martin periodinane, dichloromethane, rt, 98%; (b) NH2OH-HCl, pyridine, ethanol, 50 °C, 96%;
(c) TsCl, DMAP, TEA, dichloromethane, rt, 100%; (d) NH3, Et2O, -78 °C to room temperature, 92%; (e) I2, TEA, MeOH, rt, 94%.
SCHEME 5a
Exp er im en ta l Section
1-(2,6-Dich lor o-4-tr iflu or om eth ylp h en yl)-4-iod op yr a -
zole (4). Potassium carbonate (1.39 g, 10 mmol) was added to
a solution of 4-iodopyrazole (1.61 g, 8.32 mmol) in DMF (20
mL). 3,5-Dichloro-4-fluorobenzotrifluoride (1.92 g, 8.24 mmol)
was introduced and the mixture stirred vigorously at 100 °C
for 4 h. As the reaction was cooling, water was added dropwise
until a white precipitate began to form. Additional water was
added to bring the total volume to 80 mL, once the reaction
had cooled to room temperature. The white precipitate was
collected by suction filtration, washed with water, and recrys-
tallized from aqueous methanol to give phenylpyrazole 4 (3.33
g, 99%): mp 118-120 °C; 1H NMR δ (CDCl3) 7.84 (s, 1H), 7.75
(s, 2H), 7.63 (s, 1H); 13C NMR (CDCl3) δ 146.8, 138.6, 135.5,
135.4, 133.3 (q, 35 Hz), 125.9, 122.2 (q, 270 Hz), 58.4. Anal.
Calcd for C10H4Cl2F3IN2: C, 29.51; H, 0.99; N, 6.89. Found:
C, 29.36; H, 0.99; N, 6.78.
a
Reagents and conditions: (a) H2, Pd/C, TEA, EtOAc, rt, 45%;
(b) T2, Pd/C, TEA, EtOAc, rt.
Oxidation of the diaziridine to diazirine 16 was achieved
with iodine and triethylamine in methanol.17
1-(2,6-Dich lor o-4-t r iflu or om et h ylp h en yl)-4-t r iflu or o-
a cetylp yr a zole (5).6 A solution of isopropylmagnesium chlo-
ride in THF (4.9 mL, 9.8 mmol) was added briskly to compound
4 (3.33 g, 8.81 mmol) in THF (30 mL) at 0 °C. The resulting
yellow solution was stirred for 1 h and cooled to -78 °C, and
N-(trifluoroacetyl)piperidine (1.78 g, 1.48 mL, 9.8 mmol) added
quickly.18 The solution was stirred for 2 h as it warmed to room
temperature and quenched with saturated ammonium chlo-
ride, then ethyl acetate (100 mL) was added. The organic layer
was washed with water and brine and then dried with sodium
sulfate. The concentrated oil was purified by column chromo-
tography (5% ethyl acetate in hexane) to give ketone 5 (2.43
g, 79%) as a white solid: mp 59-61 °C; 1H NMR (CDCl3) δ
8.37 (s, 1H), 8.31 (s, 1H), 7.79 (s, 2H); 13C NMR (CDCl3) δ 174.5
(q, J ) 38 Hz), 143.0, 137.9, 136.9, 135.2, 134.2 (q, J ) 35
Hz), 126.2, 122.0 (q, J ) 270 Hz), 117.9, 116.3 (q, J ) 290
Hz).
2,6-Bis(t r im e t h ylsilyl)-3,4,5-t r ich lor ob e n zot r iflu o-
r id e (6). A solution of 3,4,5-trichlorobenzotrifluoride (211 mg,
0.85 mmol) in anhydrous THF (4 mL) was cooled to -78 °C.
LDA prepared from butyllithium and diisopropylamine in THF
or a commercially available solution in heptane/THF/ethyl-
benzene (1.18 mL, 2.0 M, 2.37 mmol) was added dropwise
followed by stirring for 1 h. The reaction was allowed to come
to room temperature after addition of TMSCl (0.236 mL, 1.86
mmol) and stirred for 2 h. The reaction was quenched with
saturated aqueous ammonium chloride (10 mL) and then ethyl
acetate (10 mL) was added. The organic layer was washed with
water and brine, dried, and concentrated to give the crude solid
in almost quantitative yield. Recrystallization from methanol
provided 6 (296 mg, 89%): mp 100-101 °C; 1H NMR δ (CDCl3)
0.48 (q, J ) 1 Hz, 18H); 13C NMR (CDCl3) δ 141.4, 141.2 (q, J
) 32 Hz), 140.7 (q, J ) 5 Hz), 135.5, 123.9 (q, J ) 270 Hz),
Finally reduction of diiodoarene 16 with H2, 10%
Pd/C, and triethylamine in ethyl acetate gave 2 in 45%
isolated yield (Scheme 5). The identical reaction with
tritium gas, however, afforded an intermediate that
contained one tritium atom but also possessed an iodine
atom. Further reduction with tritium gas provided the
desired radiolabeled photoaffinity probe 17 or [3H]TDF.
Comparison of radiolabeled probe 17 with the cold
standard 2 showed them to be identical by reverse-phase
HPLC and normal-phase TLC. The specific activity of the
radiolabeled photoaffinity probe was determined to be 15
Ci/mmol. Radioflow chromatogram analysis provided a
radiopurity of 99.3% and this was corroborated by
normal-phase TLC followed by scintillation counting of
the labeled region of silica from the chromatoplate.
Radiolabeled photoaffinity probe 17 was stable for several
months when stored as a dilute solution in the dark at
-20 °C.
In conclusion, a more efficient synthetic route to
intermediate 5 was described and the directed ortho-
metalation chemistry for this system has been developed.
This methodology allowed for the synthesis of the novel,
tritium-labeled, fipronil-based, highly potent, photoaf-
finity probe 17 via several diiodo intermediates. Target
compound 17 ([3H]TDF) contains both the radiolabel
(tritium) and the photoreactive substituent (diazirine)
necessary for a useful photoaffinity probe. The synthesis
of [3H]TDF should now allow for the photoaffinity label-
ing of the GABA receptor and its insecticide binding site.
(18) The yield was lower with slow addition of isopropylmagnesium
chloride and N-(trifluoroacetyl)piperidine compared with the optimized
conditions.
(17) Silver(I) oxide gave comparable yield and purity but it was
essential that it be prepared fresh each time.
J . Org. Chem, Vol. 68, No. 21, 2003 8077

Sammelson and Casida
2.4. Anal. Calcd for C13H18Cl3F3Si2: C, 39.65; H, 4.61. Found:
C, 39.26; H, 4.75.
Martin periodinane (1.15 g, 2.7 mmol) and this mixture was
stirred overnight at room temperature. The reaction mixture
was diluted with ether (20 mL) and washed with sodium
thiosulfate in saturated aqueous sodium bicarbonate, then
sodium bicarbonate, and water. The combined aqueous phases
were extrated with ether (20 mL) and the organic phases were
dried with sodium sulfate, filtered, and evaporated to give
ketone 12 (560 mg, 98%) as an oil, which was pure according
to NMR and used as such for further synthesis. 1H NMR
(CDCl3) δ 8.33 (q, J ) 2 Hz, 1H), 7.86 (s, 1H); 13C NMR (CDCl3)
δ 174.0 (q, J ) 38 Hz), 144.7, 143.4, 139.0 (q, J ) 34 Hz), 137.2,
135.5, 127.3 (q, J ) 7 Hz), 126.0, 121.1 (q, J ) 280 Hz), 116.1
(q, J ) 290 Hz), 96.1, 93.9. An analytical sample was
crystallized from methanol as its methyl hemiacetal. Anal.
2-Iod o-3,4,5-tr ich lor oben zotr iflu or id e (7). The proce-
dure for the synthesis of 6 was followed to scale with the noted
differences: 3,4,5-trichlorobenzotrifluoride (847 mg, 3.4 mmol),
10 mmol of LDA, iodine (2.58 g, 10 mmol), and washing with
5% aqueous sodium bisulfite to give 7 on recrystallization (1.19
1
g, 94%): mp 30-31 °C; H NMR δ (CDCl3) 7.69 (s, 1H); 13C
NMR (CDCl3) δ 141.9, 135.0, 134.7 (q, J ) 32 Hz), 134.5, 126.9
(q, J ) 7 Hz), 121.5 (q, J ) 270 Hz), 95.5. Anal. Calcd for
C7HCl3F3I: C, 22.40; H, 0.27. Found: C, 22.53; H, 0.25.
1-(2,6-Dich lor o-4-tr iflu or om eth yl-3-tr im eth ylsilylp h e-
n yl)-4-iod o-5-tr im eth ylsilylp yr a zole (8). The procedure for
the synthesis of 6 was followed to scale with the noted
differences: 4 (200 mg, 0.49 mmol), 1.62 mmol of LDA, and
TMSCl (187 mg, 1.72 mmol) to give 8 (246 mg, 91%) after
preparative TLC (3% ethyl acetate in hexane): mp 107-109
Calcd for
C13H6Cl2F6I2N2O2: C, 23.63; H, 0.92; N, 4.24.
Found: C, 23.83; H, 0.89; N, 4.14.
1-(2,6-Dich lor o-3-iodo-4-tr iflu or om eth ylph en yl)-5-iodo-
4-(tr iflu or oa cetyl)p yr a zole Oxim e (13). Trifluoromethyl
ketone derivative 12 (550 mg, 0.87 mmol) was dissolved in
pyridine (4 mL) and ethanol (1.5 mL). Hydroxylamine hydro-
chloride (67 mg, 0.98 mmol) was added and the reaction
temperature increased to 50 °C, then the mixture was stirred
for 14 h. Ethyl acetate (30 mL) and water (10 mL) were added
to the cooled mixture and the organic layer was washed twice
with 0.1 N hydrochloric acid and once with brine. Drying,
filtration, and evaporation provided oxime 13 (539 mg, 96%)
as a solid. 1H NMR (acetone-d6) δ 12.18 (br s, 1H), 8.16 (s,
1H), 7.98 (s, 1H); 13C NMR (acetone-d6) δ 144.4, 143.8, 139.9
(q, J ) 32 Hz), 139.3, 138.9 (q, J ) 32 Hz), 136.7, 128.6 (q, J
) 5 Hz), 122.7 (q, J ) 280 Hz), 120.4, 119.2 (q, J ) 280 Hz),
97.5, 89.9. Anal. Calcd for C12H3Cl2F6I2N3O: C, 22.38; H, 0.47;
N, 6.53. Found: C, 22.48; H, 0.32; N, 6.29.
1-(2,6-Dich lor o-3-iodo-4-tr iflu or om eth ylph en yl)-5-iodo-
4-(tr iflu or oacetyl)pyr azole O-(p-Tolylsu lfon yl)oxim e (14).
To oxime 13 (339 mg, 0.53 mmol) in dichloromethane (4 mL)
was added triethylamine (0.22 mL, 1.6 mmol) at room tem-
perature. Tosyl chloride (121 mg, 0.63 mmol) addition was
followed by a catalytic amount of DMAP (6.5 mg, 53 µmol).
After 2 h ethyl acetate and water were added, the layers
separated, and the organic phase washed with sodium bicar-
bonate, water, and brine. After drying with sodium sulfate,
filtration and evaporation provided tosylate 14 (421 mg, 100%)
as a solid. 1H NMR (CDCl3) δ 7.93 (d, J ) 8.2 Hz, 2H), 7.84 (s,
2H), 7.40 (d, 8.2 Hz, 2H), 2.49 (s, 3H); 13C NMR (CDCl3) δ 146.6
(q, J ) 36 Hz), 146.3, 143.7, 142.6, 138.7 (q, J ) 32 Hz), 137.5,
135.8, 131.3, 129.8, 129.4, 127.2 (q, J ) 5 Hz), 121.3 (q, J )
280 Hz), 119.3 (q, J ) 280 Hz), 115.1, 96.0, 89.4, 21.7. Anal.
Calcd for C19H9Cl2F6I2N2O3S: C, 28.59; H, 1.14; N, 5.27.
Found: C, 28.89; H, 0.91; N, 5.03.
1
°C; H NMR (CDCl3) δ 7.85 (s, 1H), 7.82 (s, 1H), 0.49 (q, J )
1.5 Hz, 9H), 0.15 (s, 9H); 13C NMR (CDCl3) δ 147.6, 144.8,
144.1, 140.9, 139.3, 137.9 (q, J ) 32 Hz), 136.0, 126.2 (q, J )
7 Hz), 122.7 (q, J ) 270 Hz), 68.7, 2.1, 1.1. Anal. Calcd for
C
16H20Cl2F3IN2Si2: C, 34.86; H 3.66; N, 5.08. Found: C, 34.80;
H, 3.82; N, 5.01.
1-(2,6-Dich lor o-4-tr iflu or om eth ylph en yl)-4-[1-h ydr oxy-
(2,2,2-tr iflu or oeth yl)]p yr a zole (9). Purified ketone 5 (1.92
g, 5.1 mmol)19 in ethanol (30 mL) at 5 °C was treated
portionwise with sodium borohydride (0.22 g, 5.8 mmol). After
being stirred for 3 h at room temperature the reaction was
quenched with ice and water (100 mL). An ethyl acetate (100
mL) extract was washed with water and brine, concentrated,
and triturated with dichloromethane to give the compound as
a white solid upon filtration. The mother liquor was concen-
trated and triturated with hexane to give a second crop. These
white solids were dried under vacuum to give 9 (1.86 g, 97%):
mp 145-146 °C; 1H NMR (CDCl3) δ 7.92 (s, 1H), 7.75 (s, 2H),
7.71 (s, 1H), 5.18 (p, J ) 6 Hz, 1H), 2.86 (d, J ) 6 Hz, 1H); 13
C
NMR (acetone-d6) δ 141.2, 140.6, 136.3, 133.6 (q, J ) 34 Hz),
132.5, 127.0, 126.0 (q, J ) 280 Hz), 123.5 (q, J ) 270 Hz),
119.5, 66.3 (q, J ) 33 Hz). Anal. Calcd for C12H6Cl2F6N2O: C,
38.02; H, 1.60; N, 7.39. Found: C, 37.93; H, 1.62; N, 7.44.
1-(2,6-Dich lor o-3-iod o-4-t r iflu or om et h ylp h en yl)-4-[1-
h yd r oxy-(2,2,2-tr iflu or oeth yl)]-5-iod op yr a zole (10). The
procedure for the synthesis of 6 was followed to scale with the
noted differences: 9 (600 mg, 1.58 mmol), 9.5 mmol of LDA,
iodine (2.58 g, 10 mmol), and washing with 5% sodium bisulfite
to afford a crude brown tar. Flash chromatography with ethyl
acetate/hexane (1/9 slowly increased to 1/4) provided 10 (552
mg, 55%) as a solid: 1H NMR (acetone-d6) δ 8.13 (s, 1H), 7.98
(s, 1H), 6.06 (d, J ) 6.2 Hz 1H), 5.10 (p, J ) 6.7 Hz 1H); 13C
NMR (acetone-d6) δ 144.6, 142.9, 139.4, 138.9 (q, J ) 32 Hz),
136.9, 128.6 (q, J ) 7 Hz), 126.1 (q, J ) 280 Hz), 124.8, 122.7
(q, J ) 275 Hz), 97.4, 89.3, 67.9 (q, J ) 32 Hz). Anal. Calcd
for C12H4Cl2F6I2N2O: C, 22.84; H, 0.64; N, 4.44. Found: C,
22.71; H, 0.82; N, 4.07.
3-{4-[1-(2,6-Dich lor o-3-iod o-4-tr iflu or om eth ylp h en yl)-
5-iodopyr azolo]}-3-(tr iflu or om eth yl)diazir idin e (15). Ether
(2 mL) was added to tosylate 14 (421 mg, 0.53 mmol) in a thick-
walled glass tube and the mixture was cooled to -78 °C. A
stream of ammonia gas was bubbled in until about 1 mL had
condensed at which time the tube was sealed. The reaction
was allowed to warm to room temperature and stirred over-
night and then cooled again before opening the tube. Removal
of the ammonium tosylate via filtration and evaporation of the
solvent under reduced pressure provided diaziridine 15 (311
mg, 92%) after flash chromatography (10% ethyl acetate in
hexane) as a white solid. 1H NMR (CDCl3) δ 8.14 (s, 1H), 8.04
1-(2,6-Dich lor o-3-iod o-4-t r iflu or om et h ylp h en yl)-4-[1-
h yd r oxy-(2,2,2-tr iflu or oeth yl)]p yr a zole (11). Continued
flash chromatography of the reaction that afforded 10 with
ethyl acetate/hexane (1/3) gave 11 (199 mg, 25%) as a solid:
1H NMR (CDCl3) δ 7.88 (s, 1H), 7.80 (s, 1H), 7.67 (s, 1H), 5.13
(q, J ) 6.3 Hz, 1H), 3.53 (br s, 1H); 13C NMR (CDCl3) δ 142.9,
140.5, 138.3, 137.9 (q, J ) 32 Hz), 134.9, 130.7, 127.3 (q, J )
7 Hz), 124.2 (q, J ) 280 Hz), 121.4 (q, J ) 280 Hz), 117.6,
96.2, 66.2 (q, J ) 34 Hz). Anal. Calcd for C12H5Cl2F6IN2O: C,
28.54; H, 1.00; N, 5.55. Found: C, 28.75; H, 1.07; N, 5.28.
1-(2,6-Dich lor o-3-iodo-4-tr iflu or om eth ylph en yl)-5-iodo-
4-(tr iflu or oa cetyl)p yr a zole (12). To a solution of 10 (560
mg, 0.89 mmol) in dichloromethane (8 mL) was added Dess-
(s, 1H), 3.73 (d, J ) 8.7 Hz, 1H), 3.51 (d, J ) 8.7 Hz, 1H); 13
C
NMR (CDCl3) δ 145.0, 139.8 (q, J ) 32 Hz), 139.4, 137.3 (q, J
) 7 Hz), 128.7 (q, J ) 5 Hz), 127.4, 125.2 (q, J ) 280 Hz),
123.1(q, J ) 270 Hz), 122.1, 97.9, 91.1, 53.5 (q, J ) 36 Hz).
Anal. Calcd for C12H4Cl2F6I2N4: C, 22.42; H, 0.63; N, 8.71.
Found: C, 22.67; H, 0.77; N, 8.53.
3-{4-[1-(2,6-Dich lor o-3-iod o-4-tr iflu or om eth ylp h en yl)-
5-iod op yr a zolo]}-3-(tr iflu or om eth yl)d ia zir in e (16). To di-
aziridine 15 (67.3 mg, 0.10 mmol) in methanol (1 mL) was
added triethylamine (42 µL, 0.30 mmol) followed by iodine (31
mg, 0.12 mmol). This dark solution was stirred at room
(19) An equivalent amount of crude ketone can also be used with
excess NaBH4 to reduce the remaining N-(trifluoroacetyl)piperidine
to piperidine and 2,2,2-trifluoroethanol. For an example of trifluoro-
acetamide reduction see: Bukownik, R. R.; Wilcox, C. S. J . Org. Chem.
1988, 53, 463-471.
8078 J . Org. Chem., Vol. 68, No. 21, 2003

Synthesis of a Photoaffinity Probe for the GABA Receptor
temperature for 2 h and then ethyl acetate was added followed
by washing with aqueous sodium bisulfate, sodium carbonate,
and brine. The organic layer was dried with sodium sulfate,
filtered, and concentrated to give 16 (63 mg, 94%) as a solid
after flash chromatography with 3% ethyl acetate in hexane.
1H NMR (CDCl3) δ 8.04 (s, 1H), 7.82 (s, 1H); 13C NMR (CDCl3)
δ 144.8, 144.5, 138.6 (q, J ) 32 Hz), 135.9, 127.2, (q, J ) 4.5
Hz), 125.9, 121.3 (q, J ) 280 Hz), 118.2 (q, J ) 260 Hz), 96.0,
86.3, 22.9 (q, J ) 40 Hz). FAB-MS 641 (MH)+; HRMS calcd
for (C12H2Cl2F6I2N4 + H)+ 640.7735, found 640.7729.
3-{4-[1-(2,6-Dich lor o-4-tr iflu or om eth ylp h en yl)p yr a zo-
lo]}-3-(tr iflu or om eth yl)d ia zir in e (2). The compound was
synthesized from 5 as previously described.6 In addition, 16
(4.0 mg, 6 µmol) was dissolved in ethyl acetate (200 µL) and
to this was added triethylamine (20 µL) and 10% Pd/C (4.0
mg). The septum-sealed flask was briefly purged with hydro-
gen gas and then a balloon filled with hydrogen gas (1.03 atm)
was attached. After 4 h the system was opened and flash
chromatography (3% ethyl acetate in hexane) of the reaction
mixture gave 3 (1.1 mg, 45%) as an oil. 1H NMR (CDCl3) δ
7.76 (s, 2H), 7.68 (s, 1H), 7.55 (s, 1H); 13C NMR (CDCl3) δ
139.8, 138.5, 135.4, 133.7 (q, J ) 34 Hz), 131.0, 126.0, 122.1
(q, J ) 270 Hz), 121.8 (q, J ) 270 Hz), 113.5, 24.2 (q, J ) 45
Hz).
(20 × 100 mm) with 30% water (with 0.05% TFA) and 70%
acetonitrile. The purified probe (7.8 mCi) had a specific activity
of 15 Ci/mmol and radiopurity of >99% determined by TLC
cochromatography and radioautography. 17 was stored as a
solution (680 µCi/mL) in ethyl acetate in the dark at -20 °C.
After several months of storage under these conditions the
radiopurity had remained >95%.
Ack n ow led gm en t. We thank current or former
laboratory colleagues Nanjing Zhang, Pierluigi Caboni,
and Nilantha Sirisoma for helpful suggestions. Robert
Fazio and Robert Andresini at ViTrax (Placentia, CA)
carried out the radiosynthesis and radioligand purifica-
tion. The project described was supported by Grant R01
ES008419 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 official views of the NIEHS,
NIH.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal procedures, synthesis of 3 and N-(trifluoroacetyl)piperidine,
reverse-phase HPLC chromatogram of 2, 1H NMR and reverse-
phase HPLC chromatogram of 16, and radioflow chromato-
gram of 17. This material is available free of charge via the
Internet at http://pubs.acs.org.
3-{4-[1-(2,6-Dich lor o-3-tr itio-4-tr iflu or om eth ylp h en yl)-
5-tr itiop yr a zolo]}-3-(tr iflu or om eth yl)d ia zir in e (17). Fol-
lowing the above procedure for 2 replacing hydrogen with
tritium gas and properly modifying the reactor to handle the
radioactive atmosphere provided [3H]TDF (17). [3H]TDF was
purified by reverse-phase HPLC on a YMC ODSA C18 column
J O034520Z
J . Org. Chem, Vol. 68, No. 21, 2003 8079