Original ArticlesSynthesis and Evaluation of 11C-Labeled Nonpeptide Antagonists for Cholecystokinin Receptors: [11C]L-365,260 and [11C]L-365,346
Introduction
Cholecystokinin (CCK) is a polypeptide that is found in the gut and the central nervous system (CNS) [12]. CCK plays important roles in hormonal control of pancreatic enzyme secretion and gall bladder contraction. CCK has only recently come under investigation and has been shown to be implicated in satiety, anxiety, analgesia and other brain functions as a neurotransmitter or a neuromodulator [12]. Two receptor subtypes for CCK were initially discovered in rat pancreas and brain, and these were termed peripheral (CCK-A) receptors and central (CCK-B) receptors, respectively. CCK-A receptors are primarily distributed in pancreatic acinar cells, gall bladder and ileal muscle, but are also present within a few brain regions. CCK-B receptors are widely distributed in the brain. It has recently been demonstrated that CCK coexists with dopamine in mammalian midbrain dopamine neurons and modulates dopamine function 10, 11, 19, 27. Alterations of CCK and/or CCK receptors in human diseases linked to dopamine such as Parkinson’s disease, schizophrenia and Huntington’s disease have also been reported [12]. Although the CCK–dopamine interactions are suggestive of a role for CCK in certain CNS functions in which CCK exerts its actions via dopamine neurons, the precise molecular mechanisms and the respective roles of the two CCK receptors in mammalian physiological events are still obscure.
Recent development of receptor imaging technique using a positron emission tomography (PET) has made it possible to investigate the molecular mechanisms of biomolecules such as neurotransmitters and hormones in living humans 15, 28. The application of this technique, using positron emitter–labeled CCK receptor radioligands, to CCK receptor studies should help to elucidate the human physiological functions of CCK and its receptors. PET radioligands for studying binding sites in the brain should have high permeability of the blood–brain barrier (BBB), as well as a high affinity constant and high specificity to the binding sites. Several potent and subtype selective nonpeptide CCK antagonists, which have the ability to cross the BBB, are now available 3, 13. Among them, two benzodiazepine derivatives, MK-329 (or L-364,718) and L-365,260, are the most widely used antagonists for CCK pharmacological studies. MK-329 shows picomolar CCK-A receptor affinity (IC50 = 0.07 nM) and >103 selectivity over CCK-B receptors. L-365,260 shows nanomolar CCK-B receptor affinity (IC50 = 2 nM) and >102 selectivity over CCK-A receptors. The stereochemistry at the C-3 position of L-365,260 is critical for determining the subtype selectivity for the CCK receptors [5]. The optical antipode (S-enantiomer) of L-365,260, namely L-365,346, binds to CCK-A receptor (IC50 = 3 nM) with 50-fold selectivity over CCK-B receptors. These nonpeptide antagonists display suitable in vitro binding characteristics as target molecules for developing PET radioligands and are capable of labeling on the N1-methyl position with positron emitter 11C. 11C-LABELED MK-329 ([11C]MK-329) has recently been synthesized as a potential imaging agent for peripheral CCK-A receptors [8]. Here we have synthesized both 11C-labeled L-365,260 and L-365,346 with high specific activities to develop an enantiomeric pair of radioligands useful for CCK receptor studies with PET. Brain permeabilities of benzodiazepine analogs, including MK-329 and L-365,260, have previously been measured using the technique of Oldendorf [23], and significantly lower brain incorporation of L-365,260 compared with MK-329 has been reported [21]. Although this previous study could predict the low BBB permeability of 11C-labeled L-365,260 ([11C]L-365,260), we are interested in the in vivo behaviors of these isomeric compounds, such as regional brain distributions and peripheral organ distributions. Thus, we have examined the different brain and organ uptake characteristics of the two enantiomers in rodents.
Section snippets
General
All melting points (mp) are uncorrected. Nuclear magnetic resonance (1H-NMR) spectra were recorded on a JNM-GX-270 spectrometer with tetramethylsilane as an internal standard. All chemical shifts (δ) are reported in parts per million (ppm) downfield from the standard. Fast atom bombardment mass spectra (FAB-MS) were obtained on a JEOL NMS-SX102 spectrometer. Infrared (IR) spectra were recorded on a JASCO IR-700 spectrometer. Column chromatography was done on Merck Kieselgel gel 60 F254 (70–230
Results
Reaction of the precursor (2) with CH3I by the method described in the literature 1, 14, and subsequent optical resolution with chiral HPLC gave L-365,260 and L-365,346 in high yields as shown in Fig. 1. The nonradioactive analog of 3, which was obtained in 89% isolated yield after the methylation, had analytical and spectral data identical with those of the authentic sample prepared by an alternative procedure 4, 6that included N1-methylation of a 3-amino group protected analog of 1 with CH3I.
Discussion
A variety of biologically active neuropeptides are known to exist in the mammalian brain and to have neurotransmitter-like properties [7]. It is also well established that most neuropeptides coexist with classical low molecular weight neurotransmitters, like dopamine, within neurons in the brain [10]. These peptides are released together with the classical transmitters and act as their own receptors. Functional studies of these neuropeptides, however, are less advanced than those of classical
Acknowledgements
We thank Mr. T. Nakano and N. Takai for their assistance during the animal experiments and the cyclotron crew of the National Institute of Radiological Sciences for their help in radioisotope production. We also thank Mr. N. Ibii of Shionogi and Co. for supplying an authentic sample. This work was supported by the International Joint Research Program of Japan Science and Technology Corporation.
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