| Dopamine tranporter (DAT) in the central nervous sysytem is related to many neurological and psychiatric disorders. Great attention has been paid to theraputic and diagnostic drug design targeted to DAT. Molecular imaging with DAT tracer provides valuable information for Parkinson's diease. In this study, a DAT imaging agent, [18F]-2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane (18F-FECNT), was developed and evaluated for molecular imaging for PD. The synthesis and characterization of a new precursor, a automated high-radiochemical yield radiosyntheisis of 18F-FECNT, a series of preclinical studies including microPET imaging were reported to meet the requirement of the clinical application.A new mesylate precursor of 18F-FECNT, 2β-Carbomethoxy-3β-(4- chlorophenyl)-8-(2- mesyloxyethyl)nortropane (MsOECNT), was designed and synthesized. The synthesis began with hydrolysis of cocaine in hydrochloride solution and gave ecognine. Intramolecular dehydration of ecognine in POCl3 followed by treatment with methanol produced anhydraecognine methyl ester. The key intermediate, 2β-carbomethoxy-3β-(4- chlorophenyl)tropane (CClT), was obtained by selective 1,4-addition reaction of anhydraecognine methyl ester with p-chlorophenyl magnesium bromide followed by purification. The stereo structure of CClT was confirmed by single crystal x-diffraction. Demethylation of CClT afforded 2β-carbomethoxy-3β-(4-chlorophenyl)nortropane (nor-CClT). The new intermediate, 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2- hydroxyethyl)-nortropane (HOECNT), was prepared by hydroxyethylation of nor-CClT followed by purification by chromatography. Finally, MsOECNT was synthesized by mesylation of HOECNT with methanesulfonic anhydride. In addition, 2β-carbomethoxy-3β- (4-chlorophenyl)-8-(2-fluoroethyl)nortropane (FECNT) was synthesized by fluoroethylation of nor-CClT as the reference substance of 18F-FECNT. Structures of MsOECNT, FECNT and all intermediates were confirmed by IR, MS, 1HNMR and elemental analysis.A one-step automated high-radiochemical-yield synthesis of 18F-FECNT from the mesylate precursor was established. [18F]fluorination was performed by heating 4 mg mesylate precursor and K18F in 1.0 ml anhydrous acetonitrile at 90℃for 20 min. The crude 18F-FECNT was obtained with a radio labeling yield of 48%. After purification by preparative high performance liquid chromatography (HPLC), the final 18F-FECNT product was obtained with a radiochemical purity of 98.4% and a decay corrected radiochemical yield of 33±9% (and the uncorrected radiochemical yield was 19±5%, n=5). The procedure of the [18F]fluorination was automated and the duration of the total radiosynthesis was 80-100 min.A series of preclinical studies of 18F-FECNT was finished to testify its clinical potential. The octanol-water partition coefficients of 18F-FECNT were 34.1 and 56.4 at pH 7.0 and pH7.4 respectively. Biodistribution in mice showed that 18F-FECNT penetrated the blood-brain barrier (BBB) rapidly and had favorable retention in the brain. (2.22, 1.20, 1.02, 0.78, 0.71, 0.67 %ID at 5, 15, 30, 60, 120, 180min respectively post i.v. injection). In the brain, 18F-FECNT concentrated in the target area, the striatum (ST). The ratios of striatum to cerebellum (CB), hippocampus, parietal cortex and temporal cortex reached as high as 3.47, 3.55, 4.40 and 3.47, respectively, at 15 min post injection, while the ratios of striatum to frontal cortex, occipital cortex were 3.17, 3.35, respectively, at 30 min post injection. The selective striatum uptake of 18F-FECNT decreased dramatically to the background when the DAT was blocked withβ-CFT. The autoradiography showed that 18F-FECNT was concentrated in the striatum highly and symmetrically in normal rat brains (STleft/CB = 5.42,STright/CB = 5.52), while in left-sided lesioned PD rat brains, the striatal uptake of 18F-FECNT bilaterally decreased and no significant uptake was visible in the 6-OHDA lesioned-sided striatal areas (STunlesioned/CB = 2.57, STlesioned/CB = 1.05). These results indicated that 18F-FECNT had high affinity and selectivity to DAT. The major radioligand was metabolized by the hepatic system. Toxicity trial displayed that the acceptable dose per kilogram to mice was 625 times greater than that to human, which implied safty of 18F-FECNT for human.The microPET imaging of the normal and PD rats was carried out to verify the clinical potential of 18F-FECNT in living animals. The normal rat's striatum exhibited the highest uptake of the radio ligand and prolonged retention of the radioligand in the brain (ST/CB=2.18±0.16,STleft/STright=1.00±0.05). Radiouptake in the striatum remained highest in 5-125 min post injection and the ratio of ST to CB peaked to 3.44 at 25 min. As for the 6-OHDA lesioned PD rats, the highest uptake was observed in the unlesioned side striatum ( STunlesioned/CB = 2.01±0.23, 5-125 min), whereas no significant uptake was visible in lesioned side (STlesioned /CB = 1.04±0.05). Time-activity curves demonstrate that uptake of the unlesioned side striatum remained higher than CB but lower than the normal rat's striatum in 5-125 min post injection, whereas the lesioned side keep similar to CB. |
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