Synthesis, resolution, and in vitro evaluation of three vesicular acetylcholine transporter ligands and evaluation of the lead fluorine-18 radioligand in a nonhuman primate

Abstract

Vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing cholinergic dysfunction associated with dementia. We recently reported three new potent and selective carbon-11 labeled VAChT radiotracers. Herein, we report the resolution with a Chiralcel OD column of three additional fluorine containing VAChT ligands in which a fluoroethoxy or fluoroethylamino moiety was substituted for the methoxy group. An in vitro competitive binding assay showed (−)-7 had high potency for VAChT (Ki-VAChT = 0.31 ± 0.03 nM) and excellent selectivity for VAChT versus σ receptors (Ki-σ1= 1869 ± 248 nM, Ki-σ2 = 5482 ± 144 nM). Three different radiolabeling approaches were explored; the radiosynthesis. (−)-[18F]7 was successfully accomplished via a stepwise two-pot, three-step method with moderate yield (11 ± 2%) and high radiochemical purity (> 98%). PET imaging studies in a nonhuman primate indicated that (−)-[18F]7 rapidly entered the brain and accumulated in the VAChT-enriched striatum. Uptake of (−)-[18F]7 in the target striatal area peaked at 10 min and displayed improved clearance kinetics compared to our VAChT tracer [18F]VAT, which has been approved by the Food and Drug Administration (FDA) for first-in-man studies. These studies justify further investigation of (−)-[18F]7 and exploration of the structure-activity relationships of these fluoroethoxy and fluoroethylamino analogs.

In Vivo and In Vitro Characteristics of Radiolabeled Vesamicol Analogs as the Vesicular Acetylcholine Transporter Imaging Agents

“…The influence of enantioselectivity of the vesamicol analogs FEOBV ( 2 ) [ 45 ], MIBT ( 8 ) [ 55 ], FBT ( 9 ) [ 60 ], oIV ( 10 ) [ 62 ], OMV ( 11 ) [ 63 ], OIDV ( 13 ) [ 67 ], FBBV ( 18 ) [ 78 ], 19 [ 81 ], TZ659 ( 20 ) [ 82 ], VAT ( 21 ) [ 83 ], and 22 [ 84 ] on the binding affinity for VAChT, σ 1 , and σ 2 receptors was reported. The investigated vesmicol analogs except for MIBT ( 8 ) and FBT ( 9 ) showed that (−)-enantiomers had about 3- to 29-fold higher binding affinity for VAChT than (+)-enantiomers.…”

“…22 is a vesamicol analog that incorporated the fluoroethyl amino group instead of the methylamino group into (−)-TZ659 ( 20 ) [ 84 ]. The affinity (Ki) of ( 22 ) to VAChT, σ 1, and σ 2 was 0.31 nM (PC12 A123.7 cells which express human VAChT), 1,870 nM (Guinea pig brain), and 5,480 nM (rat liver), respectively.…”

“…However, because the affinity of vesamicol, as a reference, for VAChT, σ 1 , and σ 2 and the affinity of sigma ligands such as pentazocine ( σ 1 ) and DTG ( σ 2 ), as a reference, for σ 1 and σ 2 were not investigated by the same in vitro binding assay method, it is difficult to compare between other vesamicol analogs ( 1 – 17 ) and these compounds ( 18 – 22 ) regarding the affinity for VAChT and sigma receptors ( σ 1 and σ 2 ). These compounds ( 18 – 22 ) displayed low accumulation in the brain against the physicochemical characteristics such as molecular weight (MW = 354–405) and lipophilicity (ALog D or Log P = 2–3) [ 71 , 81 , 83 , 84 ]. Tu et al reported that the accumulation of (−)-[ 11 C]TZ659 ( 20 ) in the brain increased 2.2-fold by pretreatment with cyclosporine A (CycA), which inhibits a P-glycoprotein related to the drug excretion mechanism, 30 min prior to injection of a radiotracer [ 83 ].…”

Exploration of Sulfur‐Containing Analogues for Imaging Vesicular Acetylcholine Transporter in the Brain

“…Reactions were monitored by thin-layer chromatography (TLC) and visualized under ultraviolet light (l = 254). Flash column chromatography was performed using Silica Flash P60 silica gel (40-63 mm) from Silicycle Inc. 1 Ha nd 13 CNMR spectra were recorded on Varian 400 and 300 MHz instruments. Chemical shifts were reported in parts per million (ppm) and calibrated using the residual undeuterated solvent as an internal reference (CDCl 3 : d = 7.26 ppm;C D 3 OD: d = 3.31 ppm;[ D 6 ]DMSO: d = 2.50 ppm).…”

“…[10] To test the hypothesis that incorporating sulfur into VAChT radiotracer would alter the in vivo pharmacologic properties, we interposed sulfur-containing moieties such as thioether, sulfoxide, sulfone, and sulfamide into the carbonyl-containing pharmacophore structures represented by lead VAChT radiotracers, (-)-[ 18 F] 4 and (-)-[ 11 C] 5 (Figure 1). [11, 12, 13] In current manuscript, we reported our efforts on the syntheses and in vitro biological evaluation of these new sulfur-containing analogues, the radiosyntheses and in vivo evaluation in rodent of representative (-)-[ 11 C] 8 and (-)-[ 18 F] 14a , as well as microPET study of (-)-[ 11 C] 8 in the brain of non-human primate (NHP).…”