Original Articles
Radiosynthesis and autoradiographic evaluation of [11C]NAD-299, a radioligand for visualization of the 5-HT1A receptor

https://doi.org/10.1016/S0969-8051(98)00091-2Get rights and content

Abstract

The selective 5-HT1A receptor antagonist NAD-299 ([R]-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide) was labeled with the positron-emitting radionuclide carbon-11. The radioligand was synthesized from NAD-195 ([R]-3-N,N-dicyclobutylamino-8-fluoro-5-trifluoromethylsulfonyloxy-3,4-dihydro-2H-1-benzopyran) in two radiochemical steps. A palladium-catalyzed reaction of NAD-195 and [11C]cyanide was followed by hydrolysis of the carbon-11-labeled nitrile intermediate with basic hydrogen peroxide. The total radiochemical yield, based on [11C]CO2 and corrected for decay, was 20–40%. The specific radioactivity was 24 GBq/μmol (900 Ci/mmol) at end of synthesis, with a radiochemical purity better than 99% and a total synthesis time of 40–45 min. Autoradiographic examination of [11C]NAD-299 binding in human brain postmortem demonstrated high binding in hippocampus, raphe nuclei, and neocortex. The binding in the hippocampus was higher than in the neocortex. Within the hippocampus, the densest binding was observed in the CA1 region. [11C]NAD-299 binding was inhibited by addition of the 5-HT1A receptor ligands WAY-100635, pindolol, (±)-8-OH-DPAT, 5-HT, and buspirone, leaving a low background of nonspecific binding. The results indicate that [11C]NAD-299 binds specifically to 5-HT1A receptors in the human brain in vitro and is a potential radioligand for positron emission tomography (PET) examination of 5-HT1A receptors in vivo.

Introduction

The 5-HT1A receptor is a serotonin receptor subtype that has been studied extensively due to its implicated role in several major neuropsychiatric disorders such as anxiety, depression, and schizophrenia (28). [11C]WAY-100635 is the only carbon-11-labeled radioligand that has, so far, been proven useful for in vivo imaging of the 5-HT1A receptor with positron emission tomography (PET) 9, 19, 25, 26, 27. Despite being a selective and potent 5-HT1A receptor antagonist giving a high signal-to-noise ratio, this radioligand has a drawback due to the formation of small amounts of labeled metabolites that pass the blood–brain barrier 10, 21, 22. Moreover, the very low background of unchanged radioligand in reference tissue obstructs reliable use of simplified quantitative methods such as the transient equilibrium analysis for clinical studies 8, 10.

NAD-299 ([R]-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide, Fig. 1) is a 5-HT1A receptor antagonist developed at Astra Arcus AB, Sweden. NAD-299 represents a structurally new chemical class of compounds with 5-HT1A receptor affinity and has high affinity (Ki = 0.6 nM) and selectivity for the 5-HT1A receptor in vitro 7, 17. The lipophilicity of NAD-299 (log P = 2.5 at pH 7.4) should allow for rapid diffusion across the blood–brain barrier and low nonspecific binding (6).

In the present study, we developed a synthesis for the labeling of [11C]NAD-299 with a high radiochemical yield and high specific radioactivity starting with [11C]cyanide as the labeled precursor. The anatomical distribution and specificity of [11C]NAD-299 binding to 5-HT1A receptors was examined by autoradiography on human brain whole-hemisphere cryosections.

Section snippets

General

NAD-299 ([R]-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate, Fig. 1) and NAD-195 ([R]-3-N,N-dicyclobutylamino-8-fluoro-5-trifluoromethylsulfonyloxy-3,4-dihydro-2H-1-benzopyran) were supplied by Astra Arcus AB (Södertälje, Sweden). The synthesis of NAD-299 and NAD-195 is described elsewhere (7). Tris(dibenzylideneacetone)-dipalladium(0)-chloroform adduct ([Pd2(dba)3] · CHCl3), 1,1′-bis-(diphenylphosphino)-ferrocene (DPPF) and

Chemistry

After 3 min of heating, the incorporation of [11C]cyanide to the nitrile intermediate (II) was higher than 98% using 4 mg of the precursor NAD-195. The subsequent hydrolysis of the nitrile intermediate (II) to the corresponding amide [11C]NAD-299 resulted in 30–50% conversion (Fig. 2). The total radiochemical yield of [11C]NAD-299 based on [11C]CO2 calculated from end of bombardment (EOB) and corrected for decay was 20–40% with a total synthesis time of 40–45 min. Purification of [11C]NAD-299

Chemistry

The key step of the preparation of [11C]NAD-299 is a palladium(0)-catalyzed nucleophilic displacement reaction of the aryl triflate precursor NAD-195 (I) with [11C]cyanide. The incorporation of [11C]cyanide yielded an aryl cyanide intermediate (II). The reaction was catalyzed by [Pd2(dba)3] · CHCl3-DPPF and NMP was used as solvent.

It has been shown that an exchange of the catalyst ligand from conventional triphenylphosphine (PPh3) to DPPF improves the catalytic efficiency of Pd(0) to a great

Conclusion

The 5-HT1A receptor antagonist NAD-299 was labeled with carbon-11 with high yield and high specific radioactivity. Autoradiography on postmortem human brain whole hemisphere cryosections demonstrated specific binding of [11C]NAD-299 to regions known to have a high density of 5-HT1A receptors. NAD-299 represents a structurally new chemical class for binding studies of the 5-HT1A receptor. The high affinity and selectivity combined with its favorable lipophilicity makes [11C]NAD-299 a potential

Acknowledgements

The authors would like to thank Mr. Göran Printz and Mr. Peter Söderholm for assistance with the radionuclide production and Ms. Carita Rask and Mrs. Siv Eriksson for technical assistance. This work was supported by grants from the Swedish Medical Research Council (03560, 09114, and 11640) and the Swedish Natural Science Research Council (K-KU 9973-308), the National Institute of Mental Health USA (NIMH, 41205 and 44814), and Karolinska Institutet.

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