Synthesis and Biologic Evaluation of a Novel Serotonin 5-HT_1A Receptor Radioligand, ¹⁸F-Labeled Mefway, in Rodents and Imaging by PET in a Nonhuman Primate

Chemistry

N-{2-[4-(2-Methoxyphenyl)Piperazinyl]Ethyl}-N-(2-Pyridyl)-N-(4-Carboxymethylcyclohexane)Carboxamide (8).

Using reported procedures, 1-(2-methoxyphenyl)-4-[2-(2-pyridylamino)ethyl]piperazine (WAY-100634, 6, Fig. 2 (22)) (96.3 mg, 0.3 mmol) was reacted with 4-carbomethoxycyclohexane-1-carboxylic acid (7, 47.4 mg, 0.3 mmol) in the presence of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP; 132.0 mg, 0.3 mmol), triethylamine (125 μL), and CH₃CN (1.5 mL). The mixture was stirred at room temperature for 24 h. Solvent was removed by rotary evaporation. The residue was then taken up in water (3 mL) and extracted with dichloromethane. The extracts were concentrated and purified on preparative TLC (9:1 CH₂Cl₂:CH₃OH) to provide 8 (58 mg; 40% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.52–8.53 (dd, 1H), 7.76–7.79 (dt, 1H), 7.25–7.31 (m, 2H), 7.02–6.97 (m, 1H), 6.91–6.87 (m, 2H), 6.83–6.85 (m, 1H), 3.97–4.0 (t, 2H), 3.84 (s, 3H, OCH₃), 3.62 (s, 3H, CO₂CH₃), 3.02 (br, 4H), 2.63–2.66 (m, 6H), 2.29–2.32 (m, 1H), 2.04–2.08 (m, 1H), 1.84–1.95 (m, 4H), 1.07–1.64 (m, 4H). MS, m/z, 481 (30%, [M+H]⁺), 503 (10%, [M+Na]⁺).

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FIGURE 2. 

Synthesis scheme of mefway (5), tosylate precursor for ¹⁸F-mefway (10), and ¹⁸F-mefway (11). Reaction conditions include (i) BOP reagent, 2 eq Et₃N, CH₃CN, room temperature for 24 h; (ii) LiAlH₄, THF, 0°C–5°C for 30 min followed by 30 min room temperature; (iii) DAST, CH₂Cl₂ room temperature, 24 h; (iv) p-toluenesulfonyl chloride, CH₂Cl₂, Et₃N, room temperaure, 24 h; (v) ¹⁸F-fluoride, Kryptofix 2.2.2./K₂CO₃, CH₃CN, 30 min. (Left inset) HPLC purification of ¹⁸F-mefway using C₁₈ reverse-phase semipreparative column eluted with 60% acetonitrile/0.1% triethylamine at flow rate of 2.5 mL/min. ¹⁸F-Mefway had retention time of 10.5 min and specific activity of 74–111 GBq/μmol. RA = radioactivity; UV = absorbance at 254 nm.

Radiochemistry

The radiosynthesis of ¹⁸F-mefway was performed using an automated CPCU. ¹⁸F in H₂¹⁸O from an MC-17 cyclotron was passed through a QMA-light Sep-Pak (Waters Corp.), preconditioned with 3 mL of K₂CO₃, 140 mg/mL, followed by 3 mL of anhydrous acetonitrile. The ¹⁸F trapped in the QMA-light Sep-Pak was then eluted with 1 mL Kryptofix 2.2.2./K₂CO₃ (360 mg/75 mg in 1 mL of water and 24 mL of acetonitrile) and transferred to the CPCU reaction vessel. The SYNTH1 program in the CPCU was used for the synthesis. This involved initial drying of the ¹⁸F-fluoride, Kryptofix 2.2.2., and K₂CO₃ mixture at 120°C for 10 min. Subsequently, acetonitrile (2 mL) from CPCU reagent vial 2 was added and evaporated at 120°C for 7 min to ensure dryness of the ¹⁸F-fluoride mixture. After this, the precursor, N-{2-[4-(2-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4-tosyloxymethylcyclohexane)carboxamide, 10 (1–2 mg in 0.5 mL of anhydrous acetonitrile in CPCU reagent vial 3), was added and the reaction was continued for 15–30 min at 96°C. Subsequent to the reaction, CH₃OH (7 mL contained in CPCU reagent vial 4) was added to the mixture and the CH₃OH contents were passed through a neutral alumina Sep-Pak (prewashed with methanol) to remove any unreacted ¹⁸F-fluoride. The collected CH₃OH solution coming out of the CPCU now contained N-{2-[4-(2-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4-¹⁸F-fluoromethylcyclohexane)carboxamide, 11 (¹⁸F-mefway). The CH₃OH was removed in vacuo, and the residue was taken for HPLC purification. The product was purified in a reverse-phase HPLC C₁₈ Econosil column (250 × 10 mm; Alltech Assoc. Inc.) with 60% acetonitrile:40% water containing 0.1% triethylamine with a flow rate of 2.5 mL/min. The retention time of ¹⁸F-mefway was found to be 10.5 min (Fig. 2, HPLC chromatogram). The ¹⁸F-mefway fraction was collected into a flask and the solvent was removed in vacuo using a rotary evaporator to dryness. The radiosynthesis was accomplished in 1.5–2 h, with an overall radiochemical yield of 20%–30% decay corrected. Specific activity was measured to be 74–111 GBq/μmol.

Lipophilicity

Lipophilicity (log P) was measured to evaluate the lipid solubility of ¹⁸F-mefway by partitioning between n-octanol and 50 mmol/L Tris-HCl (pH 7.4) buffer. Log P was taken as the concentration of ¹⁸F-mefway in n-octanol over the concentration in buffer.

In Vitro Studies

In vitro binding affinities of WAY-100635 and mefway (5) to 5-HT_1A receptor sites were determined from a competitive binding assay using ¹⁸F-mefway (11) as the radioligand. The brains from Sprague–Dawley rats were removed from the skull and frozen in isopentane at −20°C. Several 10-μm-thick horizontal rat brain slices were preincubated at room temperature for 15 min in 50 mmol/L Tris-HCl buffer (pH 7.4). The brain slices were then treated with increasing concentrations of WAY-100635 and mefway in the presence of 167 kBq/mL of ¹⁸F-mefway at 37°C for 1 h. After incubation, slices were washed twice with cold buffer for 1 min each, dipped in cold water, air dried, and exposed to phosphor storage screens for 24 h. To calculate the binding affinity (inhibitory concentration of 50% [IC₅₀]) of WAY-100635 and mefway to 5-HT_1A receptor sites, nonspecific binding in the cerebellum was subtracted from all samples and changes in specific binding under different concentrations of WAY-100635 and mefway were calculated. Binding affinity was computed using the KELL program (BioSoft Inc.).

For serotonin competition studies, brain slices (10 μm) were preincubated in 50 mmol/L Tris-HCl buffer (pH 7.4) for 10 min and then incubated with 130–148 kBq/mL of ¹⁸F-mefway at 37°C for 1 h. Nonspecific binding was measured in the presence of 10 μmol/L of WAY-100635. Increasing amounts of serotonin (1 nmol/L to 10 μmol/L) competed with 130–148 kBq/mL of ¹⁸F-mefway at 37°C for 1 h. After incubation, slides were washed twice (1-min washes) with ice-cold buffer. Slides were then quickly dipped in cold deionized water, air dried, and exposed to a phosphor screen for 24 h. Binding affinity (IC₅₀) was computed as described earlier.

Monkey PET Study

A male rhesus monkey was anesthetized using ketamine (10 mg/kg) and xylazine (0.5 mg/kg) and maintained on 1%–1.5% isoflurane. Two intravenous catheters were placed, one on each arm, for administration of the radiopharmaceutical and for obtaining blood samples during the study. Vital signs of the monkey were monitored and did not show any unusual deviations from baseline values. The head of the animal was placed in the gantry of the ECAT EXACT HR+ PET scanner and positioned in place with adhesive tape. A transmission scan using a ⁶⁸Ge/⁶⁸Ga rod source was acquired before administration of the radiopharmaceutical to correct for tissue attenuation of the coincident radiation. A dynamic sequence of scans was acquired for 180 min after intravenous administration of 130 MBq of ¹⁸F-mefway (specific activity, 74–111 GBq/μmol). Data in the final form were expressed in units of percentage injected dose per milliliter (%ID/mL) or kBq/mL. Areas showing maximal radioligand binding in the frontal cortex, temporal cortex, dorsal raphe, and other brain regions were delineated on the images. The PET images were coregistered with an MR image template of the rhesus brain as reported previously (23). A PET image summed for a duration of 120 min was used for the PET/MRI coregistration, which was performed using the VINCI program (CPS Innovations, Inc.). Time–activity curves were obtained for all of these brain regions.

A blood analysis of the monkey PET study subsequent to administration of ¹⁸F-mefway was performed to observe levels of breakdown in the blood similar to methods described for ¹⁸F-fallypride (24). Venous whole blood (∼1 mL) was obtained at various time points (5, 10, 15, 30, 45, 60, 90, 120, 150, and 180 min) during the course of the PET study. The samples were spun in a Microfuge centrifuge (Eppendorf centrifuge 5415C) at 12,000 rpm for 5 min. Plasma (0.6 mL) was separated from each sample, and 0.1 mL was counted. The remaining 0.5 mL of plasma at each time point was combined with 0.1 mL NaHCO₃, mixed well, and subsequently extracted with 0.4 mL of ethyl acetate. The ethyl acetate was separated, and 0.1 mL of ethyl acetate layer and 0.1 mL of aqueous layer were counted for analysis of ¹⁸F-mefway in blood. The aqueous layer contains hydrophilic metabolites of ¹⁸F-mefway, whereas the ethyl acetate fraction contains ¹⁸F-mefway and other lipophilic metabolites. Each of the ethyl acetate extracts was concentrated to a small volume (20 μL) and spotted (along with standard, ¹⁸F-mefway) on a large TLC plate. The plate was eluted with 9:1 CH₂Cl₂:CH₃OH to analyze for parent ¹⁸F-mefway and metabolites. The developed TLC plate was dried, apposed overnight to a phosphor screen, and read using the Phosphor Cyclone Imager. All blood samples were counted for radioactivity in a Packard 5000 series Gamma Counter.

Synthesis

Production of mefway, shown in Figure 2, took place in a 3-step simplified procedure. The reaction of WAY-100634 (6) with the commercially available cyclohexane-1,4-dicarboxylic acid monomethyl ester was performed using BOP to provide N-{2-[4-(2-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4-carboxymethylcyclohexane)carboxamide (8). The yields of this coupling step were moderate (30%–40%); the acid chloride procedure as described for the synthesis of WAY-100635 may provide higher yields (22). This ester (8) was reduced with LiAlH₄ more efficiently than with NaBH_4, although in both cases there was significant breakdown of the amide bond. Conversion of the alcohol to the corresponding fluoro- compound (mefway) proceeded in modest yields (∼40%). The alcohol (9) was converted to the tosylate (10), with approximately >60% yield.

Radiosynthesis of ¹⁸F-Mefway

The reaction of the tosylate (10) in acetonitrile with ¹⁸F-fluoride from a MC-17 cyclotron using Kryptofix 2.2.2. and K₂CO₃ at 96°C for 30 min in the CPCU proceeded efficiently, to provide ¹⁸F-mefway in a single step with a radiochemical yield of 20%–30%. Semipreparative HPLC chromatographic separation of the ¹⁸F-mefway (11) product mixture gave the product radioactive peak ¹⁸F-mefway at ∼10.5 min, as seen in the HPLC chromatogram in Figure 2, with >95% radiochemical purity. This product peak was clearly separated from other mass peaks. Radiochemical yields were lower than those typically observed with tosylate-to-¹⁸F exchange reactions as previously reported (25). A large radioactive peak (seen in HPLC profile in Fig. 2) close to the dead volume remains to be identified. Stability of the product to the basic radiolabeling conditions (such as cleavage of the amide bond) needs further investigation. The specific activity of ¹⁸F-mefway was ∼74–111 GBq/μmol, which is high enough to occupy only a small fraction of the receptors and, therefore, is not expected to cause any biologic effects.

In Vitro Autoradiographic Study of ¹⁸F-Mefway

As seen in Figure 3A, in vitro autoradiography in horizontal rat brain slices displayed selective binding of ¹⁸F-mefway to the cortex, hippocampus, and colliculus, all regions rich in serotonin 5-HT_1A receptors. The cerebellum showed little or no selective binding and, therefore, was used as a measure of nonspecific binding. The hippocampus gave the highest ratio of specific to nonspecific binding, 82:1, consistent with other reported WAY-100635 analogs. The colliculus and cortex, brain regions known to be less densely packed with 5-HT_1A receptors, gave ratios of 46:1 and 40:1, respectively. Binding of ¹⁸F-mefway was displaced (>95% in hippocampus and cortex) in the presence of 10 μmol/L of WAY-100635 (Fig. 3A), suggesting binding of ¹⁸F-mefway to areas rich in the 5-HT_1A receptor.

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FIGURE 3. 

(A) In vitro horizontal brain slices of rat brain show binding of ¹⁸F-mefway (red = highest binding and white = lowest binding). (Left) Rat brain slice, 10 μm thick, shows brain regions. (Center) Same rat brain slice after treatment with 130–148 kBq/mL of ¹⁸F-mefway. (Right) Rat brain slice with nonspecific binding in presence of 10 μmol/L WAY-100635. (B) Horizontal rat brain slices (with dorsal hippocampus) show in vitro binding in competitive study of ¹⁸F-mefway with increasing concentrations of serotonin (5-HT): (a) 1 nmol/L; (b) 10 nmol/L; (c) 100 nmol/L; (d) 1 μmol/L; (e) 10 μmol/L). (C) (Left) Competition curves of WAY-100635 and mefway against ¹⁸F-mefway measured autoradiographically in hippocampus of rat brain slices. Inhibition constant (IC₅₀) of WAY-100635 = 23.2 ± 2.8 nmol/L and of mefway = 25.7 ± 2.4 nmol/L. (Right) Inhibition curves of ¹⁸F-mefway binding by serotonin measured autoradiographically in different brain regions of rat brain slices shown in B. Ctx = cortex; St = striata; Hp = hippocampus; Cb = cerebellum; Col = colliculus.

Competition Study of Serotonin with ¹⁸F-Mefway

Serotonin competition was studied in increasing concentrations of 1–10 μmol/L (Fig. 3B). It was found that 10 μmol/L serotonin displaced >90% of ¹⁸F-mefway binding. Competition curves for the change in specific binding of these brain regions with increasing concentrations of serotonin are shown in Figure 3C. Inhibition constants (IC₅₀ values) of 243.5 ± 2.0 nmol/L (colliculus), 169.4 ± 5.0 nmol/L (hippocampus), and 218.3 ± 15 nmol/L (cortex) were measured.

Binding Affinity and Lipophilicity

Binding of ¹⁸F-mefway to the 5-HT_1A receptor site was inhibited by both WAY-100635 and mefway (Fig. 3C). As WAY-100635 and mefway reached micromolar concentrations, the amount of ¹⁸F-mefway bound to the 5-HT_1A receptors was <5% in all brain regions. Mefway exhibited an IC₅₀ of 25.7 ± 2.4 nmol/L, which was similar to the affinity of WAY-100535 (IC₅₀ of 23.2 ± 2.8 nmol/L). Lipophilicity (log P) of ¹⁸F-mefway was found to be 2.62 ± 0.06. This compares well with the reported log P of ¹¹C-WAY-100635 and ¹¹C-desmethyl-WAY-100635 (26), suggesting that ¹⁸F-mefway would demonstrate good brain uptake.

Monkey PET Studies

Uptake of ¹⁸F-mefway in various regions was rapid and in <2 min reached levels of >0.03 %ID/mL. This is comparable to levels attained by ¹⁸F-MPPF (16) and other related WAY-100635 derivatives in monkey PET studies (12,17). After 3 h, about 6% of the initial activity in the cerebellum was still present. A number of brain regions exhibited retention of ¹⁸F-mefway and were consistent with the presence of 5-HT_1A receptors. Coregistered PET/MR images showed a high degree of binding in the hippocampus as well as other regions of the temporal cortex (Fig. 4). Several other regions in the cortex exhibited a high degree of binding. The striatum and thalamus exhibited some binding, greater than that found in the cerebellum. Discrete binding was observed in the raphe, as seen in Figure 4.

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FIGURE 4. 

Distribution of ¹⁸F-mefway in rhesus monkey brain. Coregistered MR images with summed PET images show localization of ¹⁸F-mefway. (First and second rows) Coregistered MR images and PET images, respectively, show binding in various cortical regions, including distinct hot spot (Hs) seen in red near insular cortex. (Third and fourth rows) Coregistered MR images and PET images, respectively, show hippocampus, seen in red in 3 slices, and raphe. St = striata; Hs = hot spot; Cg = cingulate; Oc = occipital cortex; Cb = cerebellum; Th = thalamus; Tc = temporal cortex; Hp = hippocampus; Rp = raphe.

Time–activity curves for the various brain regions are shown in Figure 5A. There were 4 sets of regions with different activity levels. The highest binding regions were the hippocampus and an area in the cortex (hot spot), most probably associated with the insular cortex. Clearance from this cortical region was faster than that observed in the hippocampus. The second group was composed of the temporal cortex, cingulate gyrus, frontal cortex, and occipital cortex. The third group was the striatum, thalamus, and raphe. Fourth, the lowest binding region was the cerebellum. The cerebellum, containing little or no 5-HT_1A receptors, was taken as the reference region similar to the other 5-HT_1A radiotracers. Ratios of the various brain regions against the cerebellum are shown in Figure 5B. The highest ratio, between 8 and 10, was found for the hippocampus and the hot spot in the cortex. These ratios decreased after plateaus around 80 min, suggesting that a 90- to 120-min PET study should be sufficient to obtain quantitative information on receptor concentration. The second group of regions, with ratios between 5 and 8, was found for the temporal cortex, frontal cortex, occipital cortex, and anterior cingulate. All ratios decreased after peaking at ∼80 min. The third group of regions—namely, the thalamus, striatum, and raphe—had ratios of 2–3.5. At the end of the 3-h scan, ∼60% of the initial activity in the hippocampus was still specifically bound and the hippocampus-to-cerebellum ratio had decreased from a peak of 9.7 to 8.4.

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FIGURE 5. 

(A) Time–activity curve of ¹⁸F-mefway in various monkey brain regions obtained after intravenous administration of ¹⁸F-mefway. (B). Ratio plot of various brain regions (shown in A) against cerebellum. Hp = hippocampus; Hs = hot spot; Cg = cingulate; Fc = frontal cortex; Tc = temporal cortex; Oc = occipital cortex; St = striata; Rp = raphe; Th = thalamus; Cb = cerebellum.

Hydrophilic and lipophilic components were observed in the plasma during the PET study with ¹⁸F-mefway (Fig. 6A). The aqueous fraction most likely consists of the amide-hydrolysis product, 4-¹⁸F-fluoromethylcyclohexane carboxylic acid, analogous to other reported radiotracers, such as ¹⁸F-FCWAY (14). Formation of this acid seems to plateau at around 50%–60% after 50 min following injection. Lipophilic components that were extracted into ethyl acetate indicated primarily the parent, ¹⁸F-mefway (Fig. 6B), suggesting that radiolabeled ¹⁸F-mefway is likely the principal component in the brain. The standard ¹⁸F-mefway spotted on the plate was ∼80% pure. At 3 h after injection, ∼30% of ¹⁸F-mefway still remained unmetabolized in plasma. These levels are greater than those reported for some previous 5-HT_1A radioligands.

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FIGURE 6. 

(A) Blood analysis of ¹⁸F-mefway in rhesus monkey shows ¹⁸F activity in ethyl acetate and aqueous fractions. In ethyl acetate fractions, at 3 h after injection, ∼30% of ¹⁸F-mefway remained unmetabolized in plasma. (B) Thin-layer chromatographic analysis of ethyl acetate fraction of plasma obtained during PET study (0–180 min) shows presence of ¹⁸F-mefway (standard [Std] ¹⁸F-mefway shown for comparison).