Synthesis and Biological Evaluation of a New Acyclic Pyrimidine Derivative as a Probe for Imaging Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression

With the idea of finding a more selective radiotracer for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression by means of positron emission tomography (PET), a novel [18F]fluorine radiolabeled pyrimidine with 4-hydroxy-3-(hydroxymethyl)butyl side chain at N-1 (HHB-5-[18F]FEP) was prepared and evaluated as a potential PET probe. Unlabeled reference compound, HHB-5-FEP, was synthesized via a five-step reaction sequence starting from 5-(2-acetoxyethyl)-4-methoxypyrimidin-2-one. The radiosynthesis of HHB-[18F]-FEP was accomplished by nucleophilic radiofluorination of a tosylate precursor using [18F]fluoride-cryptate complex in 45% ± 4 (n = 4) radiochemical yields and high purity (>99%). The biological evaluation indicated the feasibility of using HHB-5-[18F]FEP as a PET radiotracer for monitoring HSV1-tk expression in vivo.


Introduction
Positron emission tomography (PET) is a noninvasive imaging modality for the in vivo visualization of various metabolic processes such as cellular proliferation, HSV1-tk reporter gene expression [1][2][3], determination of receptor concentrations and the assessment of treatment response to therapy [4][5][6]. A paradigm for the non-invasive imaging of transgene expression involves the appropriate combination of a reporter gene and a reporter substrate or probe [7]. In essence, the reporter gene product selectively converts a reporter probe into a negatively charged metabolite that is trapped and accumulates within the transduced cells as it is unable to cross the cell membrane [3]. The accumulation of radioactivity within the transfected cell can be imaged by PET. The most studied reporter gene for the visualization of gene expression in animals and humans is herpes virus type 1 thymidine kinase (HSV1-tk) which is visualized by its enzyme product HSV1-TK. A number of 18 F-labeled pyrimidine ([ 18 F]-FIAU, [ 18 F]-FMAU [8,9], [ 18 F]-FEAU [10]) and purine ([ 18 F]FHBG [11], [ 18 F]FHPG [12]) based nucleosides have shown promise as reporter probes for non-invasive imaging of HSV1-tk gene expression ( Figure 1). In recent years, [ 18 F]FEAU has emerged as a tracer with improved sensitivity and selectivity for imaging HSV1-tk expressing cells [13,14]. Whereas purine [15] and pyrimidine [16] nucleoside analogues have found application as imaging agents for imaging HSV1-tk expression, cellular proliferation is mainly imaged with thymidine analogues [17]. The purine analogue, [ 18 F]FHBG, considered as the gold standard in clinical studies for HSV1-tk reporter gene imaging with PET [18,19] exhibits high abdominal activity due to hepatobiliary elimination [20,21]. When compared with pyrimidines, [ 18 F]FHBG shows less sensitivity towards the native HSV1-TK.
We have previously reported on the synthesis, radiosynthesis and the in vivo evaluation of pyrimidine nucleoside analogues in which acyclic 6-(1,3-dihydroxyisobutyl) and 6-(1,3-dihydroxyisobutenyl) side chains have been attached at the C-6 position rather than at the N-1 position [22][23][24]. More recently, we have also published results on N-Me-[ 18  pyrimidine based nucleosides for the development of novel fraudulent substrates of HSV-1 TK with improved pharmacodynamic and pharmacokinetic profile, we prepared a series of novel C-5 and N-1substituted pyrimidine derivatives [26]. Among these series, a pyrimidine acyclonucleoside bearing a penciclovir (PCV)-like side chain, which was shown to be a substrate of HSV-1 TK, was selected as a lead compound for development as a PET imaging agent for measuring HSV-1 TK expression.
Here we report on the synthesis of unlabeled HHB-5-FEP and the radiosynthesis of its fluorine-18 labeled counterpart, HHB-5-[ 18 F]FEP ( Figure 1). We further present results of the in vitro cellular uptake and the in vivo evaluation of HHB-5-[ 18  The introduction of penciclovir-like chain at N-1 position of pyrimidine scaffold was performed by reaction of 5-(2-acetoxyethyl)-4-methoxypyrimidin-2-one (1) with 4-acetoxy-(3-acetoxymethyl)butyl iodide (2) to give acyclic C-5-substituted pyrimidine derivative 3. Acetyl groups in both N-1 and C-5 side chains were then removed under basic conditions to give the triol 4 [27]. Protection of the 1,3-diols in penciclovir-like side chain was carried out using p-toluenesulfonic acid monohydrate (p-TsOH×H 2 O) and 2,2-dimethoxypropane to afford acetonide 5 in 59% chemical yield. Transformation of compound 5 to the fluorinated derivative 6 was achieved in a one-step reaction in 34% yield using diethylaminosulfur trifluoride (DAST) as fluorinating reagent. Initial attempts to prepare compound 7 from 6 using NaI, TMSCl in MeCN (Method A) afforded 7 in a somewhat lower yield (24%). Besides the formation of several by-products, purification of 7 also proved tedious. Method B which involves the use of concentrated acid provided target compound 7 in an optimal 29% yield.
In order to synthesize tosylate precursor 8 for the radiosynthesis of HHB-5-[ 18 F]FEP, 5-(2-hydroxyethyl)pyrimidine derivative 5 was treated with p-toluenesulfonyl chloride in pyridine to give compound 8 in 68% yield (Scheme 1). The identities of all the synthesized compounds were confirmed by MS and NMR-spectroscopy.

Radiosynthesis of HHB-5-[ 18 F]FEP ([ 18 F]7)
The radiosynthesis of [ 18 F]7 was carried out using a two-step procedure and consisted of [ 18  HHB-5-[ 18 F]FEP was purified by a semipreparative radio-HPLC within 25 min (t R = 21.08 min) and formulated for in vitro and in vivo studies. The radiochemical yield was 45% ± 4, (n = 4) decay corrected. The total amount of radioactivity at the end of the synthesis (EOS) was up to 7.36 GBq in a radiochemical purity of > 99%. The specific activity ranged between 50 and 135 GBq/µmol after a total synthesis time of approx. 90 min. The chemical identity of HHB-5-[ 18 F]FEP was confirmed by coinjection with the non-radiolabeled reference compound HHB-5-FEP (7).

Cell Uptake Studies
The in vitro uptake of HHB-5-[ 18 F]FEP was performed on HEK293TK+ cells and compared to wild type HEK293 cells. The uptake of HHB-5-[ 18 F]FEP was at all time points (60, 120, 240 min) higher in HEK293TK+ cells than in control cells. For the investigated time points, the ratio of radioactivity uptake in HEK293TK+ and HEK293 cells was 35-41-fold higher than in wild type cells ( Figure 2). This favorable in vitro properties encouraged the further in vivo testing of HHB-5-[ 18 F]FEP.

Biodistribution
Similar to the in vitro cell uptake studies, a higher uptake of HHB-5-[ 18 F]FEP in TK-positive xenograft was observed compared to HEK293 control xenograft. The SUV PET for the TK-positive xenograft was 0.32. Uptake in the control xenograft was slightly higher (SUV PET = 0.17) compared to background activity (SUV PET = 0.08). Compared to [ 18 F]FHBG, HHB-5-[ 18 F]FEP revealed a higher background activity but a lower abdominal radioactivity. [ 18 F]FHBG showed similar imaging characteristics as previously shown [28]. The SUV PET for HHB-5-[ 18 F]FEP was higher than the SUV PET for [ 18 F]FHBG, however, due to the higher background activity of HHB-5-[ 18 F]FEP, a lower TK+/control ratio was obtained (Table 1). The region with the highest uptake radioactivity was the abdomen followed by the gallbladder and the TK-positive xenograft. F]FEP in TK-positive xenografts was significantly higher than in the control xenografts (p = 0.004 and 0.033, respectively, Student's t-Test). The uptake ratio of TK+ to control xenograft was 5.7 ± 1.9 for [ 18 F]FHBG (n = 3) and 3.4 ± 1.1 for HHB-5-[ 18 F]FEP (n = 3). The distribution patterns of the two tracers were similar, however, HHB-5-[ 18 F]FEP exhibited lower radioactivity levels in the intestine, liver and spleen and higher activity in the muscles, blood, kidneys, thyroid gland and brain area. High accumulation of radioactivity in kidneys, gallbladder and urine suggested renal excretion pathway and resulted in lower intestine activity when compared to [ 18 F]FHBG. Both radiotracers showed very low bone uptake indicating negligible in vivo defluorination.

General
Melting points (uncorrected) were determined with Kofler micro hot-stage (Reichert, Wien, Austria). Precoated Merck (Darmstadt, Germany) silica gel 60F-254 plates were used for thin layer chromatography and the spots were detected under UV light (254 nm). Column chromatography was performed using silica gel (0.063-0.2 mm) Fluka (Buchs, Switzerland); glass column was slurry-packed under gravity. 1 H-and 13 C-NMR spectra were acquired on a Bruker 300 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany). All data were recorded in DMSO-d 6  Purification of the radiolabeled products was performed using a semi-preparative HPLC system, equipped with a Smartline Pump 1000, Smartline Manager 5000, Smartline UV detector 2500 (Knauer, city, state abbrev if US, country), 3 mL-loop and a GabiStar radiodetector (Raytest, Straubenhardt, Germany). A semi-preparative HPLC column with precolumn (Phenomenex, Gemini, C18, 110 Å, 5 µm, 250 × 10 mm) was used and UV absorption was monitored at 254 nm wavelength. For the purification of HHB-5-[ 18  Specific activity was calculated based on the integrated UV peak by using a calibration curve, derived from different concentrations of the reference compound HHB-5-FEP and FHBG, respectively.

Cell Culture
Cell uptake and internalization experiments were performed as previously described [25]. In brief, HEK293 human embryonic kidney cells and HEK293 stable transfected with nonmutant HSV1-tk (HEK293TK+ cells) were cultured in high glucose DMEM media supplemented with 10% FBS and 1% Penicillin/Streptomycin. Cells were grown in humidified atmosphere with 5% CO 2 at 37 °C. Thymidine kinase expression in the HEK293TK+ cells was maintained with 0.3 mg/mL G418 in the culture medium and was routinely verified by visualization of the cotransfected RFP with a fluorescence microscope.

Animals
All animal experiments were approved by the local veterinarian department and complied with Swiss and local laws on animal protection. Six week-old female NMRI nude mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Under 2-3% isoflurane anesthesia, animals were injected subcutaneously with 5 × 10 6 cells in Matrigel (100 µL). Transfected HEK293TK+ cells were injected on the right side of the shoulder region, control HEK293 cells on the left side. Xenograft growth and body weight were monitored regularly. Experiments were conducted when the xenografts reached a volume of 1-2 cm 3 , which was approx. four weeks after inoculation.

In Vivo PET Scan
For PET imaging, eXplore VISTA PET/CT tomograph (Sedecal/GE Healthcare, Madrid, Spain) was used. Nude mice, bearing HEK293TK+ xenografts on the right shoulder and HEK293 xenografts (control) on the left shoulder were injected with HHB-5-[ 18 F]FEP or [ 18 F]FHBG formulations (100 μL per injection) via lateral tail vein injection (t = 0). After injection of the radiotracer, mice were anesthetized by inhalation of isoflurane in an air/oxygen mixture approximately 5 min prior to PET data acquisition and scanned as described previously [30]. In dynamic PET mode, one animal was scanned from 0-90 min. For the second animal, data were acquired from 60-150 min p.i. For static PET scans, mice were scanned from 60-90 min. as it revealed to be the optimal time frame. After acquisition, PET data were reconstructed and fused datasets of PET and CT were analyzed with PMOD software (version 3.4).  (4) were prepared according to a previously published procedure [27]. (5). To a stirred solution of compound 4 (726 mg, 2.67 mmol) in dry DMF (9 mL), 2,2,-dimethoxypropane (0.61 mL, 3.89 mmol) and p-toluenesulfonic acid monohydrate (15.17 mg, 0.08 mmol) were added. The stirring was continued for 2 h at room temperature. The reaction mixture was then neutralized by triethylamine. The solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (CH 2 Cl 2 -CH 3 OH = 10:1) to give compound 5 as white crystals (494.7 mg, 59%, mp: 120-121 °C). 1 (6). A solution of compound 5 (296.7 mg, 0.95 mmol) in anhydrous CH 2 Cl 2 (7 mL) was cooled to −68 °C and diethylaminosulfur trifluoride (DAST) (0.27 mL, 2.04 mmol) was added under argon atmosphere. The reaction mixture was stirred at −40 °C for 3 h and then allowed to warm up to room temperature. The solvent was evaporated and the residue chromatographed (CH 2 Cl 2 -CH 3 OH = 15:1). Compound 6 was isolated as a colourless oil (100.2 mg, 34%). 1 (HHB-5-FEP, 7).
Method B. Compound 6 (37.3 mg, 0.12 mmol) was dissolved in concentrated 37% HCl (1.6 mL) and the mixture was stirred at reflux for 10 min. Then, it was neutralized by the addition of 6 M NaOH. The salt was filtered of, the solvent was evaporated under reduced pressure and the residue chromatographed (CH 2 Cl 2 -CH 3 OH = 10:1). Compound 7 was obtained as colourless oil (9.0 mg, 29%). 1 (8). p-Toluenesulfonyl chloride (TsCl, 357 mg, 1.87 mmol) dissolved in pyridine (3 mL) was added to a cooled solution (0 °C) of compound 5 (100 mg, 0.32 mmol) in anhydrous pyridine (5 mL). The reaction mixture was stirred at 0 °C for 1 h then allowed to warm to room temperature and additionally stirred for 1 h. Ethyl acetate (50 mL) was added to the reaction mixture and the organic layer was extracted three times with water (2 × 30 mL). The aqueous washings were extracted again with ethyl acetate (2 × 30 mL) and the combined organic layer was dried over anhydrous MgSO 4 . The drying agent was filtered off, the solvent was removed by rotary evaporation and the residue chromatographed on silica gel (CH 2 Cl 2 -CH 3 OH = 15:1). Compound 8 was isolated as a colourless oil (102.2 mg, 68%). 1

Production of Dried [ 18 F]fluoride
No-carrier-added [ 18 6. The reactor was additionally rinsed with MeCN (1 mL) and the organic phase was also passed through the Sep-Pak light silica cartridge. The combined MeCN phase was evaporated to dryness under reduced pressure and a gentle stream of nitrogen at 90 °C. For deprotection, concentrated hydrochloric acid (0.5 mL) was added to the reaction vessel and the solution was heated for 10 min at 100 °C to give [ 18 F]7. After cooling for 5 min, 5 M NaOH (1 mL) was added to neutralize the acidic solution and 0.6 M PBS (1.0 mL) and water (0.5 mL) were added for dilution to a total volume of 3 mL. The final radiolabeled product was purified by using a semi-preparative radio-HPLC. The product fraction was collected and passed through a sterile filter into a sterile pyrogen-free vial ready to use for further experiments. The specific activity ranged between 50 and 135 GB q/µmol. A chromatogram of the HPLC analysis of the formulated radiotracer is available in the Supplementary Materials.

Radiosynthesis of [ 18 F]FHBG ([ 18 F]11).
For preparation of [ 18 F]FHBG, the tosyl precursor (3 mg, 3.15 µmol) was dissolved in anhydrous MeCN (400 µL) and added to the azeotropically dried [ 18 F]fluoride-cryptate complex (30)(31)(32)(33)(34)(35)(36)(37)(38). The solution was heated at 115 °C for 20 min and was allowed to cool down for 10 min. A mixture of 15% MeOH in CH 2 Cl 2 (1 mL) was added and the solution was passed through a Sep-Pak light silica (preconditioned with 5 mL Et 2 O). The reactor was additionally rinsed with 15% MeOH in CH 2 Cl 2 (3 mL) and the solution was also passed through the Sep-Pak light silica cartridge. The organic phase was evaporated to dryness under reduced pressure and a gentle stream of nitrogen at 90 °C. For deprotection, 1 M hydrochloric acid (0.6 mL) was added to the reaction vessel and the solution was heated for 10 min at 115 °C to give [ 18 F]FHBG. After cooling for 5 min, 1 M NaOH (0.6 mL) was added to neutralize the acidic solution. Then, 0.6 M PBS (1 mL) and water (0.8 mL) were added for dilution to a total volume of 3 mL. The radioproduct was purified by using a semi-preparative radio-HPLC. The product fraction was collected and passed through a sterile filter into a sterile pyrogen-free vial.
Cell uptake studies of HHB-5-[ 18 F]FEP showed 35-41-fold higher accumulation of radioactivity in TK+ cells than in control cells. HHB-5-[ 18 F]FEP in tumor bearing mice clearly visualized HSV1-tk expressing tumors but the contrast between transduced and non-transduced xenografts was higher for [ 18