Synthesis and characterization of [vinylidene-3H] (-)-α-kainic acid at high specific activity

Article abstract of DOI:10.1002/jlcr.562

[Vinylidene-³H] (-)-α-Kainic acid was prepared by means of a Wittig reaction with a protected ketone precursor and has proved useful as a tool to study neuroexcitatory amino acid receptors. Copyright

Full text of DOI:10.1002/jlcr.562

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 401–405.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.562
Research Article
Synthesis and characterization of
[vinylidene-3H] (À)-a-kainic acid at high
specific activity
David G. Ahern, Richard J. Seguin and Crist N. Filer*
PerkinElmer Life Sciences, Inc., 549 Albany St., Boston, MA 02118, USA
Summary
[Vinylidene-3H] (À)-a-Kainic acid was prepared by means of a Wittig reaction
with a protected ketone precursor and has proved useful as a tool to study
neuroexcitatory amino acid receptors. Copyright # 2002 John Wiley & Sons,
Ltd.
Key Words: tritium; kainic acid; Wittig reaction
Discussion
(À)-a-Kainic acid 1 is a pyrrolidine amino acid with three contiguous
chiral centers, first isolated from the marine algae Digenea simplex1 and
the pronounced neuroexcitatory activity of 1 has prompted extreme
interest in it and related substances.2 Our laboratories had previously
labelled compound 1 with tritium via an exchange procedure with
tritiated water, providing low specific activity material for Kuhar and
co-workers in one of the earliest binding studies in rat brain.3 To
enhance the utility of 1 for the study of the neuroexcitatory amino acid
receptors, it became necessary for us to tritiate it at significantly higher
specific activity, ideally employing a more specific labelling method.
Because of competing functionality, the usual catalytic methods of
*Correspondence to: C. N. Filer, PerkinElmer Life Sciences, Inc., 549 Albany Street, Boston,
Massachusetts 02118, USA. E-mail: crist.filer@perkinelmer.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 5 November 2001
Accepted 21 November 2001

402
D. G. AHERN ET AL.
tritium labelling would be problematic. It seemed to us that 1 might be
labelled with tritium on the vinylidene group via a Wittig reaction
employing a suitable tritiated reagent with an appropriate ketone
derived from 1. Indeed, Wittig reactions on tritiated substrates,
although not an especially common labelling strategy, have been
accomplished in the past and we cite several examples.4–6 Also, the
literature has described the preparation of nitrogen protected ketone 2
from 1 and this appeared to be an attractive entry for a useful Wittig
reaction precursor.7
Therefore, intermediate 2 could be prepared as described by the
literature and methyl esterified to provide ketone precursor 3 or the
latter could be obtained in a three step synthesis from 1 as recently
described by Baldwin.8 Ketone 3 was reacted with freshly prepared
[methyl-3H] methyltriphenylphosphonium iodide to obtain the olefin
intermediate 4 shown below. Penultimate compound 4 could then be
deprotected in straightforward fashion to obtain [vinylidene-3H] (À)-a-
kainic acid 5 in a good yield. Product 5 was radiochemically
homogeneous on both TLC and HPLC and co-chromatographed with
3
authentic 1. A proton decoupled H NMR (D2O) was obtained for 1
and is seen in Figure 1, documenting exclusive and equivalent tritium
labelling at the two vinyl positions (4.76 and 5.05 ppm), in excellent
agreement with chemical shift values reported in the literature.9 This
spectrum, showing equivalent integrals for the two vinyl tritons,
corroborated the retention of both remaining tritons from the originally
fully tritiated Wittig reagent, indicating a theoretical specific activity for
compound 1 as 58 Ci/mmol.
[Vinylidene-3H] (À)-a-Kainic acid 5 has proved to be an immeasur-
ably useful tool to elucidate the neurochemistry of the excitatory amino
acid receptors.10,11
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 401–405

SYNTHESIS AND CHARACTERIZATION OF
403
3
Figure 1. Proton decoupled H NMR (D2O) of 5
Experimental
Evaporations were carried out on a Buchi rotary evaporator in vacuo at
bath temperatures 5408C. TLC was performed on Analtech plates
coated with silica gel (250 mm for analytical and 500 mm for preparative).
Autoradiography was performed at 08C after spraying with PPO and
exposing the plates to X-ray film. TLC plates were also scanned
(ꢀ3 min) for radioactivity (ꢀ10 mCi). Preparative and analytical HPLC
was performed on a Waters instrument and peak detection was done
simultaneously by UV (280 nm-Waters 440 UV detector) and a liquid
scintillation flow monitor.
[Vinylidene-3H] N-BOC-(À)-a-kainic acid, dimethyl ester (4)
To 25 mg (0.062 mmol) of [methyl-3H] methyltriphenylphosphonium
iodide (prepared in situ from the reaction of triphenylphosphine and
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 401–405

404
D. G. AHERN ET AL.
[3H] methyl iodide at 87 Ci/mmol in dry THF for 16 h at ambient
temperature) in 0.3 ml of dry THF was added via syringe 40 ml
(0.079 mmol) of 1.98 M n-butyllithium with rapid stirring at ambient
temperature under argon. To the resulting yellow–orange solution was
added a solution of 20.6 mg (0.060 mmol) of ketone 3 in 100 ml of dry THF
via syringe at ambient temperature and the milky white solution was
stirred at ambient temperature for 2 h. After this time labile tritium was
removed by several methanol evaporations and the resulting crude
product (1.9 Ci) was dissolved in 2 ml of THF. The crude product was
purified by preparative TLC on two 500 mm silica gel plates developed
with toluene:dioxane:acetic acid (90:25:4). After development, the main
radioactive band (Rf=0.7) was visualized by UV, scraped and eluted with
ethanol to afford 590 mCi of product 4 which was homogeneous in this
TLC system (where ketone precursor 3 had an Rf of 0.6).
[Vinylidene-3H] (À)-a-kainic acid (5)
A solution of 236 mCi of 4 was stirred in 1 ml of TFA for 16 h at
ambient temperature. After this time excess TFA was removed by
rotary evaporation and the residue was saponified in 2 ml of 1 N sodium
hydroxide for 16 h at ambient temperature. The solution was then
acidified to pH 4–5 with 6N aqueous acetic acid and passed through a
Biorad 50 ion exchange column (hydrogen ion form). The crude product
was eluted from the column with 75 ml of 2 N ammonium hydroxide
affording 198 mCi of reasonably pure product. Final purification was
accomplished by preparative HPLC on a reverse phase column eluted
with 1% aqueous triethylammonium acetate (pH=4). Pure fractions
were combined and dissolved in 2% ethanol in water to afford 79 mCi of
product 1 (a 5% radiochemical yield based on precursor 3) which was
homogeneous on both TLC (silica gel – toluene:dioxane:acetic acid
(90:25:4)) and HPLC (reverse phase – 0.01 M potassium phosphate
(pH=3):methanol (90:10)). Product 5 also co-chromatographed with
3
authentic standard 1 and afforded the proton decoupled H NMR as
seen in Figure 1, demonstrating a specific activity of 58 Ci/mmol.
Acknowledgements
We would like to thank Tom Brewitt of PerkinElmer Life Sciences Inc.
3
for obtaining the H NMR of 5 as seen in Figure 1. Also we want to
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 401–405

SYNTHESIS AND CHARACTERIZATION OF
405
acknowledge Dr James F. Collins of the Department of Chemistry, Sir
John Cass School of Science and Technology (London, England) for an
initial generous gift of precursor ketone 3.
References
1. Ueno Y, Nawa H, Ueyanagi J, Morimoto H, Nakamori R, Matsuoka T.
J Pharm Soc Japan 1955; 75: 807–811.
2. Ben-Ari Y, Cossart R. Trends Neurosci 2000; 23: 580–587.
3. Simon JR, Contrera JF, Kuhar MJ. J Neurochem 1976; 26: 141–147.
4. Kawase A, Ichikawa F, Koike N, Kamachi S, Stumpf WE, Nishi Y,
Kubodera N. Chem Pharm Bull 2000; 48: 215–219.
5. Madden BA, Prestwich GD. J Org Chem 1994; 59: 5488–5491.
6. Chien P-L, Sung M-S, Bailey DB. J Label Compd Radiopharm 1979; 16:
791–797.
7. Goldberg O, Luini A, Teichberg VI. Tetradedron Lett 1980; 21: 2355–2358.
8. Baldwin JE, Fryer AM, Pritchard GJ. J Org Chem 2001; 66: 2588–2596.
9. Hashimoto K, Konno K, Shirahama H. J Org Chem 1996; 61: 4685–4692.
10. Crawford N, Lang TK, Kerr DS, de Vries DJ. J Pharmacol Toxicol 1999;
42: 121–125.
11. Patel S, Meldrum BS, Collins JF. Neurosci Lett 1986; 70: 301–307.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 401–405