Based on the 7-hydroxy precursor V, prepared via a 10-step synthesis [32], our first radiosynthetic approach to radioligand [¹⁸F]IV was carried out by applying 2-[¹⁸F]fluoroethyl-4-tosylate [¹⁸F]III(Scheme 1, left) as secondary labeling agent. According to Block et al., who described [¹⁸F]fluoroalkylation of H-acidic compounds [33,34], the tosylate II was substituted by n.c.a. [¹⁸F]fluoride via its K[¹⁸F]F-K2.2.2-carbonate complex. The resulting tosylate [¹⁸F]III was directly used for nucleophilic substitution by the phenolate V, previously activated under basic conditions.

First labeling as well as ¹⁸F-alkylation reactions were carried out in DMF at 110 °C. The reaction was accompanied by many side products and labeling efficiencies resulted in maximum 5% of [¹⁸F]IV. By changing the reaction medium to acetonitrile we achieved labeling efficiencies of 30%–45% for the ¹⁸F-alkylation step. Only a limited number of radioactive by-products occurred, according to radio-TLC and radio-HPLC analyses. However, the purification of [¹⁸F]IV was hindered due to lipophilic non-radioactive by-products. Even after solid phase extraction on alumina oxide and application of isocratic semi-preparative RP-HPLC (Figure 2A), UV-active contents could not be satisfactorily removed from the final product. As result for the two-step radiosyntheses, within 3.5 to 4.5 h for the entire process [¹⁸F]IV was obtained with radiochemical yields of 18%–29% (24% ± 6%, n = 4), but partially insufficient radiochemical purities of 92%–99% (95% ± 4%, n = 4).

According to this, either time consuming purification of the labeling agent [¹⁸F]III, or the more favorable development of a direct radiofluorination of a corresponding precursor was required. Thus, an 11-step synthesis provided the 7-(2-tosyloxyethyl) derivative VI [32]. Based on this, a one-step radiosynthesis of [¹⁸F]IV was established (Scheme 1, right). Most efficient nucleophilic ¹⁸F-for-OTs substitution was performed using the K[¹⁸F]F-K2.2.2-carbonate complex, determined by radio-TLC as well as analytical radio-HPLC. With 2–3 mg of precursor VI in acetonitrile after 15 min at 80 °C, labeling efficiencies of 42% to 72% of [¹⁸F]IV were observed. To reduce the basicity of the reaction mixture and to prevent tosylate decomposition, in an alternative approach K2.2.2 was replaced by tetrabutylammonium hydrogen carbonate as phase transfer catalyst (0.015 mmol, ABX GmbH, Radeberg, Germany; 0.5 mL acetonitrile). After 20 min at 80 °C the conversion of VI with n.c.a. [¹⁸F]fluoride resulted in [¹⁸F]IV with labeling efficiencies of 21%.

Purification of the crude [¹⁸F]IV was successfully carried out by solid phase extraction on a C-18 or silica gel cartridge by means of acetonitrile (methanol could be used as well), followed by isocratic elution on semi-preparative RP-HPLC (Figure 2B). Formulation of the final product for biological testing was done by solvent exchange on a C-18 cartridge, removal of the organic eluent (acetonitrile, ethanol or methanol) and dissolution in physiological saline. Following this process, we obtained the radioligand [¹⁸F]IV in radiochemical yields of 17%–40% (25% ± 9%, n = 10) within 3 to 4 h, with high chemical and radiochemical purities of ≥99%, and high specific activities in the range from 110 to 1,100 GBq/μmol.

To provide an indication of unspecific binding and blood-brain barrier permeability of [^18/19F]IV, we calculated logarithmic distribution coefficients log D. The calculation was done with ACD/LogD (version 4.56, Advanced Chemistry Development Inc., Toronto, Canada) and MarvinSketch (ChemAxon Ltd., Budapest, Hungary). Values, given in Table 1, reached from a log D_7.2 value of 2.27, which is, beside other criteria, pointing to an adequate brain penetration and optimum target to non-target ratio [35], up to log D_7.0–7.4 = 3.52, indicating a high lipophilicity and possibly efficient brain uptake, but high unspecific binding as well.

To clarify these findings, we determined the ligand distribution coefficients for [^18/19F]IV experimentally, under physiological pH conditions in solid-liquid phase systems via retention on RP-HPLC, and in liquid-liquid phase systems by conventional shake-flask method. In three n-octanol/aqueous buffer solution systems at a pH range of 7.2 to 7.4, log D values of up to 2.73 were obtained for the radioligand (Table 1). With a retention time based capacity factor k in relation to those of standard substances [36], lipophilicity of IV was determined on two reversed phase/neutral buffered eluent systems with log D = 2.63. All in one, an experimental log D_7.0–7.4 value of ~2.7 promised a good potential for brain uptake of [¹⁸F]IV in vivo, attended by low unspecific binding.

Furthermore, the chemical stability of [¹⁸F]IV in vitro under physiological conditions was investigated. After incubation at 40 °C for up to 1.5 h, the radioligand remained intact with ≥99% of native [¹⁸F]IV in 0.9% sodium chloride solution (pH 7.2) and in 0.01 M TRIS-HCl buffer (pH 7.4 at 21 °C), respectively, and showed a sufficient stability with 97% in physiological phosphate-saline (pH 7.2), verified by radio-TLC on silica gel and alumina oxide plates. For formulation and storage, radioligand stability in acetonitrile (98%, 1 h, 80 °C) and in ethanol (98%, 1 h, 80 °C; ≥99%, 1.5 h, 40 °C) was proven.

The dissociation constant K_D of [¹⁸F]IV was determined in homologous competitive binding experiments by nonlinear regression analysis [37]. Cell membrane homogenates from PDE10A-transfected SF21 cells (100 µg protein/mL incubation volume) were incubated with solutions of six concentrations of IV (working concentration 10 pM–10 µM), each spiked with a fixed concentration of [¹⁸F]IV (working concentration 0.05–0.1 nM). Based on an IC₅₀ value of 39.3 (n = 2; 38.3 nM, 40.4 nM), a K_D of 13.8 nM (n = 2; 15.9 nM, 11.6 nM) was calculated.

Besides, inhibition of recombinant PDEs expressed in a baculovirus-SF21 cell system by measuring degradation of [³H]-cAMP revealed inhibitory potency of IV not only towards PDE10A (IC₅₀ = 105 nM) but also towards PDE3A (IC₅₀ = 88.7 nM) and PDE4A (IC₅₀ = 562 nM), while the enzymatic activity of the PDEs 1B, 2A, 5A, 6, 7B, 8A, 9A, and 11A was not affected (IC₅₀ > 1000 nM) [32]. Therefore, the resultant functional selectivity is moderate. Nevertheless, the distinctive expression of PDE10A with levels of mRNA much higher than that of PDE3A or PDE4A in striatum [8] was assumed to allow the detection of PDE10A expression by [¹⁸F]IV.

Contrary to the numerous investigations regarding the inhibitory potency of PDE10A ligands [18,19,20,21,22], so far no data have been published concerning the affinity of PDE10A inhibitors. Although the available data on PDE10A transcript and protein expression did not allow a pre-estimation of the B_max value of the target, with respect to the sub-nanomolar to nanomolar affinity values of successful PET-radioligands in neuroimaging [38], we assumed the nanomolar affinity of [¹⁸F]IV to be sufficient for specific visualization of PDE10A expression in the brain. However, an only recent report including data which allow the calculation of B_max values of PDE10A very likely attenuates the imaging potential of [¹⁸F]IV due to an estimated B_max value of PDE10A binding sites of approximately 2 nM in the striatum [25].

In vitro co-incubation of rat brain slices with [¹⁸F]IV and IV revealed saturable binding of [¹⁸F]IV with an accumulation in PDE10A specific brain regions like caudate putamen, nucleus accumbens, substantia nigra, olfactory tubercle and cerebellum (Figure 3A,B). This pattern corresponds with the immunohistochemical localization of PDE10A in rat brain described in Seeger et al. [9].

However, [¹⁸F]IV binding was not completely inhibited by the highly PDE10A specific MP-10 (Figure 3C). In particular, non-displaceable binding of [¹⁸F]IV was observed in olfactory tubercle and cerebral regions. This result might indicate additional binding of [¹⁸F]IV to PDE3A, because of a non-negligible inhibitory potency of IV towards PDE3A (IC_50,PDE3A = 88.7 nM [32]) in association with a high expression of PDE3A mRNA in the olfactory bulb, cerebral cortex, cerebellum and the pontine nuclei of the rat brain, described by Reinhardt et al. [39]. Notably, Lakicz et al. [8] did not detect expression of PDE3A/B mRNA in human brain. Therefore, species-specific patterns of PDE expression have to be considered in the development of PDE10A targeting PET ligands based on the herein reported quinazoline structures. Furthermore, computer-assisted prediction of bioactivity of IV using Molinformation Cheminformatics software (Molinspiration Cheminformatics, Slovensky Grob, Slovak Republic) indicate high activity of compound IV as kinase inhibitor (score 0.92) and moderate activity as general enzyme inhibitor (score 0.57). Therefore, future work on structurally related PDE10 targeting ligands will have to focus on target selectivity in terms of kinase inhibitory potency.

The metabolic fate of [¹⁸F]IV was investigated in brain, plasma and urine samples of CD1-mice at 30 and 60 min p.i. Proteins were precipitated with acetonitrile and extraction efficiencies of 96.4% for brain homogenate, 90.2% for plasma and 89.4% for urine were obtained, indicating sufficient recovery rates. The supernatants of each material were analyzed by radio-TLC and analytical radio-HPLC, whereof two representative chromatograms are given in Figure 4.

In the brain, [¹⁸F]IV accounted for 94.3% and 93.1% of total radioactivity at 30 and 60 min p.i., respectively. A single lipophilic metabolite was detected representing only 4.2% of total radioactivity at 60 min p.i. Therefore a major impairment of the imaging properties of [¹⁸F]IV in vivo should not be expected. The same radiometabolite was detected in plasma with 23.5% and 22.0% of the total radioactivity at 30 and 60 min p.i., while the parent radiotracer [¹⁸F]IV accounted for 69.5% and 70.1%, respectively.

In urine the metabolic profile is distinctly different. While the non-metabolized [¹⁸F]IV and the single plasma- and brain-related radiometabolite accounted for only ~1% of the total radioactivity in urine, further radiometabolites were detected. The main three of them represent 11%, 12% and 44% of the total radioactivity. In contrast to brain and plasma samples, in urine free [¹⁸F]fluoride could be detected, although at a low level of only ~5% of total radioactivity.

The organ distribution of [¹⁸F]IV after i.v. injection was investigated in female CD-1 mice. Time-activity data for ¹⁸F corresponding uptake in peripheral organs, in the brain and in particular in the striatum at 5, 30, 60 and 120 min p.i. are presented in Table 2.

An initial brain uptake of ~2% ID/g was observed (brain: 1.99% ID/g; striatum: 2.32% ID/g) providing evidence for a moderate blood-brain barrier permeability of [¹⁸F]IV, as suggested by a lipophilicity value of log D_7.0–7.4~2.7. However, striatum-to-blood ratios of 1.0 at 30 min p.i. and 0.8 at 60 and 120 min p.i. indicate insufficient uptake of [¹⁸F]IV in the mouse brain. This might be due to efflux mediated by multidrug resistance proteins expressed at the blood-brain barrier or due to insufficient target binding. A low uptake of radioactivity in femur of ~2% ID/g, which was reduced to about 1% ID/g after removal of highly lipophilic bone narrow, indicates negligible defluorination of the radioligand. The high uptake of radioactivity in urine with values of 56.4% and 30.9% ID/g at 5 and 120 min p.i., respectively, points to renal elimination as one major excretory pathway of [¹⁸F]IV and its metabolites, as already suggested by the metabolic studies discussed above. Furthermore, a major part of the radiotracer is excreted hepatobiliary, as indicated by the radioactivity uptake in liver (20.5% ID/g at 5 min p.i. to 7.05% ID/g at 120 min p.i.) and intestine (13.5% ID/g and 42.8% ID/g, 5 and 60 min p.i., respectively). The high accumulation of radioactivity in adrenals of up to 48.5% ID/g at 30 min p.i. points to the involvement of cytochrome P450 oxygenases in radiotracer metabolism, as these enzymes are highly expressed in adrenals [40].

The results of the blocking experiments, performed by pre-injection of the potent and selective PDE10A inhibitor MP-10 or of compound I indicated low specificity of [¹⁸F]IV binding in the brain and other tissues because no significant changes in the amount of accumulated radioactivity could be determined in all organs known to express PDE10A (p > 0.05). The observed slightly increase in the uptake of radioactivity in most organs might be caused by an increase in the free fraction of [¹⁸F]IV in plasma or changes in physiological parameters such as blood flow due to high concentrations of the blocking compounds.

To determine the regional distribution of [¹⁸F]IV in the brain in vivo, we also performed ex vivo autoradiography. In CD-1 mice brain, heterogeneous distribution of radioactivity was noticed at 30 min after [¹⁸F]IV injection (Figure 5), but the localization of radioligand did neither correlate with the spatial distribution of PDE10A in the brain known from immunohistochemical [9] and in vivo PET studies [25] nor with the pattern of [¹⁸F]IV binding in the brain in vitro (Figure 3A). While increased radioligand binding was detected in cortex, periaqueductal gray, thalamus, substantia nigra and medulla, PDE10A-expressing brain regions such as caudate putamen, olfactory tubercle or cerebellum were free of significant accumulation of [¹⁸F]IV in vivo. The discrepancy in the pattern of [¹⁸F]IV distribution between in vitro and ex vivo autoradiography most probably indicates off-target binding of the radiotracer in combination with to low target affinity. While the dissociation rate underlying low affinity towards PDE10A allows a detection in the closed system of an in vitro study, the dynamic processes in vivo make binding of [¹⁸F]IV towards PDE10A non-detectable. Instead, the off-target binding of [¹⁸F]IV as already indicated by bioactivity score analyses has to be taken into account and will be considered in detail in future studies of potential PET ligands for PDE10A imaging. Altogether, the autoradiographic results verify the non-target binding of [¹⁸F]IV already indicated by the biodistribution studies and do not advocate the use of [¹⁸F]IV for in vivo imaging of PDE10A.

No-carrier-added [¹⁸F]fluoride (half-life: 109.8 min) was produced via the [¹⁸O(p, n)¹⁸F] nuclear reaction by irradiation of a [¹⁸O]water target (>97% enriched, 2 mL) on a PETtrace cyclotron (16.5 MeV proton beam), GE Healthcare. The final radiolabeled product was purified by semi-preparative HPLC on a TRACERlab^TM FX F-N synthesizer (GE Healthcare, Waukesha, WI, USA) containing a S1021 pump (SYKAM Chromatographie Vertriebs GmbH, Fürstenfeldbruck, Germany); WellChrom K-2001 UV detector (KNAUER GmbH, Berlin, Germany); NaI(Tl)-counter; automated data acquisition, NINA, Nuclear Interface) and by solid phase extraction on SepPak^®Plus cartridges (Waters Corporation, Milford, MA, USA). Analyses by TLC were performed on POLYGRAM® SIL G/UV254 and POLYGRAM^® ALOX N/UV254 plates, 40 × 80 mm (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Analytical HPLC was carried out on a computer assisted LC-2000Plus system from JASCO International Co., Ltd. (including LC-NetII/ADC Netbox, DG-2080-54 4-Line Degasser, LG-2080-04S quaternary gradient unit, PU-2080Plus HPLC Pump, AS-2055Plus Sampler, UV-2070Plus UV/Vis Detector), coupled with a radioactivity-HPLC-flow-monitor (Gabi Star, Raytest GmbH, Straubenhardt, Germany).

Processed TLC plates and organ sections were exposed to ¹⁸F-sensitive storage phosphor screens (BAS-TR2025, FujiFilm Co., Tokyo, Japan), and image plates were analyzed using a bioimaging analyzer system (BAS-1800 II, BASReader 2.26 Beta and AIDA 2.31 Image Analyzer software; Raytest GmbH, Straubenhardt, Germany, and FujiFilm Co., Tokyo, Japan).

In vivo studies were carried out in 3-months-old female CD-1 mice (20–25 g). Animals were obtained from the Medizinisch-Experimentelles Zentrum, Universität Leipzig, maintained on a 12 h light-dark cycle, and habituated for at least two days before experiments. All procedures involving animals were approved by the respective State Animal Care and Use Committee and conducted in accordance with the German Law for the Protection of Animals.