Bioconjugate Chem. 2007, 18, 1612−1618
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3-Amino-4-(2-((4-[18F]fluorobenzyl)methylamino)methylphenylsulfanyl)benzonitrile, an F-18 Fluorobenzyl Analogue of DASB: Synthesis, in Vitro Binding, and in ViWo Biodistribution Studies Sudha Garg, Shankar R. Thopate,§ Richard C. Minton, Kimberly W. Black, Andrew J. H. Lynch, and Pradeep K. Garg* PET Center, Department of Radiological Sciences, Wake Forest University Medical Center, Winston Salem, North Carolina 27157. Received April 3, 2007; Revised Manuscript Received July 10, 2007
3-Amino-4-(2[11C]methylaminomethylphenylsulfanyl)benzonitrile (C-11 DASB) exhibits excellent in Vitro and in ViVo properties toward the serotonin transporter. If labeled with a longer physical half-life radioisotope, this ligand could be more attractive to research groups lacking an on-site cyclotron or lacking C-11 synthesis capabilities. We produce p-[18F]fluorobenzyl iodide on a routine basis to synthesize several neuroimaging agents. Therefore, it was straightforward for us to substitute the DASB precursor with this prosthetic group and assess its biological properties. We designed a different synthesis strategy to obtain the DASB precursor (desmethyl DASB). Herein we report an efficient and facile synthetic route that provides higher chemical yields of 3-amino-4-(2aminomethylphenylsulfanyl)benzonitrile and related analogues. In addition, we report our results from incorporating p-[18F]fluorobenzyl iodide in DASB precursor and its affect on the in Vitro and in ViVo biological properties of the DASB.
INTRODUCTION The neurotransmitter serotonin (5-hydroxytryptamine, 5HT) plays an important role in the regulation of a variety of brain functions such as mood, appetite, sleep, behavioral traits, anxiety, depression, sexual disorders, and pain (1, 2). The serotonin transporter (SERT) is located presynaptically on the cell bodies and terminals of the 5-HT neurons. The SERT are distributed throughout the brain in varying densities with the highest density in the raphe and thalamus. The main function of SERT is to regulate serotonin in the synaptic cleft via the reuptake mechanism. SERT transfers 5HT from the synapse into the nerve terminals, thus terminating the 5-HT signal. Diminished synaptic 5HT in turn leads to depression and related disorders. Chemicals that block SERT action have found utility in the treatment of depression, panic disorder, post-traumatic stress disorder, and obsessive compulsive disorder (3-5). Therefore, accurate quantification of SERT density is critical in understanding the role of SERT on serotonin neurotransmission. Noninvasive in ViVo imaging of the SERT with positron emission tomography (PET) would provide accurate quantitative information. PET imaging has been quite effective in assessing drug occupancy on SERT and dopamine transporter (DAT) to evaluate efficacy of novel drugs in humans using specific ligands (6, 7). (+)-[11C]McN5652 was the first PET probe to interrogate SERT in humans (8). A variety of psychiatric disorders including mood disorder, alcoholism, and patients with drug abuse problems were studied using this probe (9, 10). Unfortunately, slow kinetics of this tracer in the brain (11) limited its utility. Therefore, developing another generation of more effective SERT probes became the goal of various laboratories. To achieve that goal, several PET ligands were developed over the * Author to whom correspondence should be addressed. Tel: 336716-5624; fax: 336-716-5639; e-mail:
[email protected]. § Current address: Dr. Shankar R. Thopate, Department of Chemistry, 102 Vasant Vihar; Balikashram Road, Ahmednagar 414 001; Maharashtra, India.
past decade (12, 13). An F-18 analogue of McN5652 (FMeMcN5652) was also developed showing higher target to nontarget tissue ratio and faster tracer kinetics than the parent (+)-[11C]McN5652 (14, 15). Unfortunately, quantification capabilities using FMe-McN5652 and other biological factors limited the utility of this ligand (14). To obtain a probe with higher affinity and potency, several investigators explored another series of compounds incorporating a diaryl sulfide motif (12, 16, 17). One of the lead compounds from this series, 3-amino-4-(2-[11C]methylaminomethylphenylsulfanyl)benzonitrile (C-11 DASB) showed a higher signal-to-noise ratio and faster brain uptake kinetics than (+)-[11C]McN5652. C-11 DASB was further assessed for its imaging characteristics and found to be a preferred ligand to study SERT (18, 19). Despite excellent specificity and sensitivity of C-11 DASB to SERT, the short half-life of C-11 (20 min) remains an obstacle for many PET groups to utilize this ligand who lack an on-site cyclotron facility. Introducing an F-18 isotope with a longer half-life (t1/2 110 min) while maintaining the DASB template would facilitate its use for longer scanning sessions and more importantly at remote centers without an on-site cyclotron facility. Although previous attempts to incorporate F-18 in a DASB motif were somewhat successful (13), the results were less than convincing to replace C-11 DASB with the newly developed ligands. This kept the search for an effective F-18labeled DASB analogue open. In our laboratories, we routinely synthesize [18F]fluorobenzyl iodide to synthesize various ligands for neuroimaging applications (20-22). It was important for us to assess the feasibility of incorporating the [18F]fluorobenzyl iodide in DASB precursor and study its affect on in Vitro and in ViVo characteristics. Toward that goal, we designed an F-18labeled analogue by replacing one of the N-methyl groups with a p-fluorobenzyl group. During the synthesis of the DASB precursor, 3-amino-4-(2-aminomethylphenylsulfanyl)benzonitrile, we also developed a more efficient and facile synthesis to prepare the intermediate 3-amino-4-(2-aminomethylphenylsulfanyl)benzonitrile and related compounds in higher yields. In addition, we report the synthesis of 3-amino-4-(2-[(4-fluoroben-
10.1021/bc070112g CCC: $37.00 © 2007 American Chemical Society Published on Web 08/18/2007
F-18 Analogue of DASB
zyl)methylamino]methylphenylsulfanyl)benzonitrile (FBASB), its in Vitro binding, and in ViVo biodistribution studies in normal rats and mice.
EXPERIMENTAL PROCEDURES All reactions were carried out under an inert atmosphere with dry solvents, under anhydrous conditions, unless otherwise stated. Reagents and solvents were purchased from commercial suppliers and were used without further purification. Melting points were determined on an Electrothermal “MEL-TEMP” melting point apparatus and are uncorrected. Flash column chromatography was carried out over silica gel (60 Å, Mallinckrodt, Baker). 1H NMR data was acquired on a Bruker Advance 300 DPX spectrometer. All chemical shift values are reported in ppm (δ). Elemental analyses (C, H, N) were determined by Atlantic Microlab, Inc., and the analytical results were within (0.4% of the theoretical values for the formula given. Radioactivity levels in tissues were assessed with a Packard Cobra γ-counter. High-pressure liquid chromatography (HPLC) was performed in isocratic mode with a Varian 9010 LC pump, a variable wavelength 9050UV VIS detector (23) (Varian Corp, Palo Alto, CA) set at 254 λ, and a radioisotope detector (Bioscan Inc, Washington, D.C.) attached to Varian’s chromatography software package (Varian Corp, Palo Alto, CA). The HPLC system used for purification of 18F-labeled FBASB was a C-18 reverse-phase ODS, 10 × 250 mm 5µ column (Phenomenex, Torrence, CA) eluted with MeOH: 0.1 M ammonium formate (80:20) at 5 mL/min. The desired peak was eluted at 16 min using this system. Quality control of purified product was performed using a Varian HPLC system and a microsorb reverse phase C-18 column 250 × 4.6 mm eluted with MeOH:0.1 M ammonium formate (80:20) at 1 mL/min and was eluted at 14 min. Preparation of 4-(2-Hydroxymethylphenylsulfanyl)-3-nitrobenzonitrile (1). A mixture of 2-mercaptobenzyl alcohol (2.00 g, 14.28 mmol), 4-chloro-3-nitrobenzonitrile (3.12 g, 17.13 mmol), K2CO3 (3.13 g, 22.64 mmol), and copper powder (0.272 g, 4.28 mmol) was added to DMF (20 mL), and the reaction mixture was stirred under argon for 1 h. Afterward, the reaction mixture was diluted with water (50 mL), and the desired compound was extracted in ethyl acetate (3 × 50 mL). The combined extracts were washed with water (2 × 50 mL) and dried (Na2SO4), and the solvent was removed using a rotary evaporator. A yellow foamy residue thus obtained was purified by flash chromatography using a silica gel column and eluting it with dichloromethane. The yellow solid powder was recrystallized from CH2Cl2/hexane to furnish the desired compound 1 as a yellow solid (3.52 g, 86%): mp 126-130 °C, lit. (23). 120-126 °C, 1H NMR (300 MHz, CDCl3) δ 8.54 (d, J ) 1.8 Hz, 1H, Ar-H), 7.74 (dd, J ) 0.9, 7.8 Hz, 1H, Ar-H), 7.64 (dt, J ) 1.5, 7.5 Hz, 1H, Ar-H), 7.57 (dd, J ) 1.2, 7.8 Hz, 1H, Ar-H), 7.51 (dd, J ) 1.8, 8.4 Hz, 1H, Ar-H), 7.47 (dt, J ) 1.5 and 7.5, Hz, 1H, Ar-H), 6.81 (d, J ) 8.4 Hz, 1H, Ar-H), 4.76 (d, J ) 5.7 Hz, 2H, CH2OH), 1.95 (t, J ) 5.7 Hz, 1H, CH2OH). Preparation of 3-Amino-4-(2-hydroxymethylphenylsulfanyl)benzonitrile (2). Compound 1 (4.10 g, 14.33 mmol) was dissolved in methanol (40 mL), and 12 N HCl (30 mL) was added. The suspension was cooled to 0 °C, and SnCl2 (10.351 g, 45.87 mmol) was added. The reaction mixture was stirred overnight at room temperature. The mixture was diluted with H2O (50 mL), and the compound was extracted in ethyl acetate (3 × 50 mL). The combined organic layers were washed with a saturated aqueous solution of NaHCO3 (2 × 10 mL), followed by water (2 × 20 mL), dried, and concentrated. The yellow solid was purified using flash column chromatography and eluting with ethyl acetate/hexanes (1:2). Further recrystallization of this product in CH2Cl2/hexane yielded the desired compound
Bioconjugate Chem., Vol. 18, No. 5, 2007 1613
2 as a yellow solid (3.11 g, 85%): mp 122-125 °C, lit. (23) mp 121-124 °C; 1H NMR (300 MHz, CDCl3) δ 7.47 (dd, J ) 1.5, 7.2 Hz, 1H, Ar-H), 7.18-7.31 (m, 3H, Ar-H), 7.03 (dd, J ) 1.2, 7.5 Hz, 1H, Ar-H), 6.92-6.96 (m, 2H, Ar-H), 4.82 (d, J ) 6.0 Hz, 2H, CH2OH), 4.44 (bs, 2H, Ar-NH2), 2.06 (t, J ) 6.0 Hz, 1H, CH2OH). Preparation of 3-Amino-4-(2-chloromethylphenylsulfanyl)benzonitrile (3). To a solution of 2 (2.00 g, 7.81 mmol) in diethyl ether (5 mL + few drops of MeOH) was added a solution of HCl in diethyl ether (5 mL, 2.0 N). After 10 min, the ether was evaporated, and toluene (20 mL) was added followed by a slow addition of thionyl chloride (1.71 mL). After refluxing for 1 h (clear solution) under argon atmosphere, the reaction mixture was cooled to room temperature. The compound was extracted in ethyl acetate (20 mL), the organic layer was washed with a saturated solution of NaHCO3 (5 mL) and dried over Na2SO4. The solvent was removed under vacuum to give solid material 3 in 93% yields. This crude product was used for the next reaction without further purification. Preparation of 3-Amino-4-(2-isoindole-1,3-dionemethylphenylsulfanyl)benzonitrile (4). A mixture of 3 (0.50 g. 1.82 mmol) and potassium phtalimide (0.675 g, 3.64 mmol) in DMF (20 mL) was heated at 100 °C for 1 h. The reaction mixture was cooled to room temperature, and water (20 mL) was added. The product was extracted in ethyl acetate (3 × 25 mL), and the combined organic layer was dried over Na2SO4. After removing the solvent, the yellow solid residue was recrystallized from ethyl acetate/hexanes to yield 4 in 76% yield as a yellowish crystalline solid. Mp 180-183 °C, 1H NMR (300 MHz, CDCl3) δ 7.85-7.89 (m, 2H, Phth-H), 7.73-7.78 (m, 2H, Phth-H), 7.32 (m, 1H, Ar-H), 7.26 (m, 1H, Ar-H), 7.15-7.24 (m, 3H, Ar-H), 7.08 (dd, J ) 1.8, 7.8 Hz, 1H, Ar-H), 6.9 (m, 1H, Ar-H), 5.06 (s, 2H, CH2N), 4.55 (bs, 2H, Ar-NH2). Anal. (C22H15N3O2S) C, H, N. Preparation of 3-Amino-4-(2-aminomethylphenylsulfanyl)benzonitrile (5). A solution of 4 (0.350 g, 0.909 mmol) and hydrazine (0.062 mL, 1.99 mmol) in anhydrous ethanol (15.00 mL) was refluxed for 2 h under argon atmosphere. The reaction mixture was cooled to room temperature, and volatiles were removed using a rotary evaporator. The white residue thus obtained was dissolved in 40% aq KOH (50 mL) and extracted in ether (3 × 25 mL), and the combined organic layer was dried over Na2SO4. After removal of the solvents, the oily residue was purified with flash column chromatography using acetone/ ethyl acetate (1:9) as the solvent to give a thick yellow syrup in 98% yields. Treatment with HCl in ether yielded the desired compound 5 as a white crystalline material. mp 133-137 °C, 1H NMR (300 MHz, DMSO) δ 13.40 (bs, 1H, NH), 8.60 (bs, 1H, NH), 7.50-7.60 (m, 1H, Ar-H), 7.30-7.38 (m, 2H, ArH), 7.20-7.25 (m, 1H, Ar-H), 7.13 (d, J ) 1.5 Hz, 1H, Ar-H), 6.93-7.02 (m, 2H, Ar-H), 4.98 (s, 2H, CH2NH2). Anal. (C14H13N3S‚HCl‚0.5H2O) C, H, N. Preparation of 3-Amino-4-(2-(4-fluorobenzyl)aminomethylphenylsulfanyl)benzonitrile (6). To a solution of 3 (0.800 g, 2.91 mmol) in acetonitrile (10 mL) were added 4-fluorobenzylamine (0.667 mL, 5.83 mmol) and triethylamine (1.22 mL, 8.75 mmol). After refluxing for 1 h, the reaction mixture was cooled to room temperature and the solvent was removed under vacuum. The oily residue was further purified using flash column chromatography on silica gel and eluting with ethyl acetate/hexane (1:4). The compound thus obtained was further recrystallized from CH2Cl2/hexanes to yield 6 as a white fluffy solid in 90% yield. mp 98-99 °C, 1H NMR (300 MHz, CDCl3). δ 7.12-7.36 (m, 6H, Ar-H), 6.98-7.04 (m, 3H, Ar-H), 6.926.95 (m, 2H, Ar-H), 4.59 (bs, 2H, Ar-NH2), 3.92 (s, 2H, benzylic CH2), 3.81 (s, 2H, benzylic CH2), 1.60 (bs, 1H, NH). Anal. (C21H18FN3S) C, H, N.
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Scheme 1. Synthesis of Substituted Benzyl Chloride 3, a Key Intermediate in the Preparation of Various Substituted Benzyl Amines
Preparation of 3-Amino-4-(2-methylaminomethylphenylsulfanyl)benzonitrile (8). To a solution of 3 (0.400 g, 1.45 mmol) in acetonitrile (5 mL) was added 40% aqueous methylamine (1.132 mL, 14.59 mmol), followed by triethylamine (0.610 mL, 4.37 mmol). After the reaction mixture was refluxed for 1 h, it was cooled to room temperature and the solvent was removed. The residue was dissolved in ethyl acetate (20 mL), and the organic layer was washed with water (3 × 5 mL) and dried over Na2SO4. The solvent was removed in Vacuo, and the oily residue was further purified using flash column chromatography and eluting with acetone as a thick viscous oil. Recrystallation of this oily residue using CH2Cl2/hexanes yielded 8 as a white crystalline compound in 72% yields. mp 93-96 °C, 1H NMR (300 MHz, CDCl3) δ 7.32-7.37 (m, 2H, Ar-H), 7.21-7.27 (m, 1H, Ar-H), 7.12-7.19 (m, 1H, Ar-H), 7.00 (dd, J ) 1.5, 7.8 Hz, 1H, Ar-H), 6.91-6.94 (m, 2H, Ar-H), 4.73 (bs, 2H, Ar-NH2), 3.89 (s, 2H, CH2NHMe), 2.47 (s, 3H, CH2NHMe), 1.53 (bs, 1H, CH2NHMe). Anal. (C15H15N3S) C, H, N. Preparation of 3-Amino-4-(2-(4-fluorobenzyl)methylaminomethylphenylsulfanyl)benzonitrile FBASB (9). To a solution of 8 (0.025 g, 0.092 mmol) and DBU (0.042 mL, 0.278 mmol) in dry acetonitrile (2 mL) was added 4-fluorobenzyl bromide (0.014 mL, 0.111 mmol). The reaction mixture was stirred at room temperature for 12 h under argon. The solvent was removed, and the oily residue was purified using column chromatography and eluting with ethyl acetate/hexane (1:6) to yield colorless oil in 72% yields. Compound 9 was obtained as an HCl salt by treating the oily residue with HCl in ether. Mp 166-170 °C, 1H NMR (300 MHz, CDCl3) δ 7.44 (d, J ) 7.8 Hz, 1H, Ar-H), 7.30-7.35 (m, 3H, Ar-H), 7.12-7.18 (m, 2H, Ar-H), 6.92-7.02 (m, 5H, Ar-H), 4.61 (bs, 2H, Ar-NH2), 3.67 (s, 2H, benzylic CH2), 3.56 (s, 2H, benzylic CH2), 2.10 (s, 3H, NCH3). Anal. for HCl salt. (C22H20FN3S‚HCl‚H2O) C, H, N. Radiochemical Synthesis of [18F]FBASB. The F-18 FBASB was obtained via N-alkylation of 3-amino-4-(2-methylaminomethylphenylsulfanyl)benzonitrile (8) using [18F]fluorobenzyl iodide ([18F]FBI) as the intermediate. The alkylating group [18F]FBI was prepared as previously described (24). Briefly, [18F]FBI was reacted with 3-amino-4-(2-methylaminomethylphenylsulfanyl)benzonitrile in DMF and triethylamine at 106 °C for 10 min followed by HPLC purification using a C-18 column eluted with MeOH: 0.1 M ammonium formate (80:20) to yield the desired product in 36 ( 8% radiochemical yields (EOS) and >99% radiochemical purity. The specific activity of FBASB was 4900 ( 1100 Ci/mmol (n ) 7). In Vitro Binding. Affinity of FBASB to 5-HT transporter sites was determined using cortical membranes from rat brains as described (25). The binding affinity was determined from binding of 0.4 nM [3H]paroxetine with 50 mg of the rat cortical membrane. The nonspecific binding was determined from binding of 10 µM fluoxetine to the membrane. Potencies were calculated from displacement curves using 7-10 concentrations of unlabeled compound. The Ki values were calculated using the Cheng-Prusoff (26). Biodistribution Studies. In ViVo biodistribution of [18F]FBASB was performed in normal rats and mice. A group of animals was injected with 20 µCi of [18F]FBASB each via a tail vein injection. At 30, 60, and 120 min postinjection, groups of five animals were killed and the tissue of interest excised, weighed, and counted for radioactivity on a gamma counter.
Radioactivity uptake in various tissues was calculated as a percent-injected dose per gram of tissue (%ID/g tissue) by counting the dose standard of appropriate count rate along with the tissues.
RESULTS AND DISCUSSION In order to synthesize the desired compound 9, we prepared several intermediate compounds. Another compound of interest for us was compound 3 since this would provide a convenient route to place a variety of radiolabeled prosthetic groups. Compound 4 would provide a convenient single step route to synthesize a wide array of substituted amines. In addition, compound 8 (3-amino-4-(2-methylaminomethylphenylsulfanyl)benzonitrile) was also of special interest since this compound is widely used as a precursor to serotonin transporter ligand DASB and is commonly known as MASB. Our first goal was to obtain compound 3. This would be a common intermediate to obtain several compounds of our interest. The synthetic strategy is shown in Scheme 1. To accomplish this, we started our synthesis with commercially available 2-thiobenzyl alcohol. Copper-catalyzed coupling of thiobenzyl alcohol with 4-chloro-3-nitrobenzonitrile using K2CO3 as a base and DMF as a reaction solvent provided product 1 in 86% yields (Scheme 1). The next step was to reduce the nitro group on the benzonitrile ring (Figure 1, ring B) to the corresponding amine. Wilson et al. reported such reduction by heating a corresponding nitro compound with hydrazine hydrate and ferric chloride in ethanol for 3 days to obtain the desired compound in 49% yield (23). In an effort to improve chemical yields and to reduce the overall reaction time, we explored various other reduction methods. We found that a reduction of nitro compound 1 by using tin(II) chloride in HCl and stirring the reaction mixture at room temperature overnight was a simple, fast, and high yielding
Figure 1. Structure of FBASB. The designation of rings A and B represent the assignments referred to in the discussion section.
F-18 Analogue of DASB
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Scheme 2. Synthesis of FBASB, DASB, and Precursor of DASBa
a This scheme depicts an improved synthesis of precursor to DASB, and FBASB. this scheme highlights the significance of compound 3 as a key intermediate in the synthesis of various dialkylamines. It also shows the utility of compounds 6 and 8 as important intermediates to synthesize a variety of substituted amines.
procedure. Using this procedure, followed by a routine workup of the reaction mixture, we obtained the desired compound 2 in 85% yields. Others have also successfully used tin chloride for facile reduction of the nitro groups while use of hydrazine hydrate resulted in incomplete reduction of nitro compound (27). Benzyl alcohol 2 was refluxed with thionyl chloride in toluene to provide corresponding benzyl chloride 3 in 93% yields. Our next goal was to synthesize compound 5 with a methylamine group at 2-position on the phenyl ring A. A previously reported method (28) used coupling of thiosalicylic acid with chlorobenzenes followed by reaction with thionyl chloride to produce acid chloride. Subsequent reaction with ammonia yielded the corresponding amide. Reduction of this amide with BH3/THF complex delivered the desired benzylamine (28). To simplify and improve the reaction conditions and overall scheme, we adopted a different synthetic route. We explored conversion of benzyl chloride to a thalimide intermediate. As shown in Scheme 2, heating benzyl chloride 3 with potassium thalimide at 100 °C in DMF for 1 h gave 76% of thalimide 4 which on refluxing with hydrazine in ethanol for 2 h produced the target compound 5 in 98% yield. For our synthetic scheme, we found the use of thalimide as a free amine synthon to be a rapid, efficient, and high yielding intermediate. Reacting 3 with substituted amines yielded a wide selection of analogues of 5 viz. compounds 6, 8, 9, 10. Reaction of compound 5 in acetonitrile with excess pfluorobenzyl bromide and DBU as a base did not yield the expected N,N-bis(4-fluorobenzyl)amino compound 7. Instead, a small amount of compound 6 (a monobenzylated derivative)
was detected with the majority of the recovered compound as the starting material. Thereafter, we decided to synthesize compound 6 and 7 in a step by step manner using an alternate strategy as depicted in Scheme 2. Reaction of the benzyl chloride 3 with p-fluorobenzyl amine in refluxing acetonitrile using Et3N as the base provided compound 6 in 90% yields (Scheme 2). Our subsequent efforts to react compound 6 with p-fluorobenzyl bromide and DBU or Et3N as a base in acetonitrile failed to yield compound 7 (Scheme 2). One possible explanation for this failed reaction could be the presence of bulky functionality (i.e. p-fluorobenzyl group on benzylic nitrogen) causing steric hindrance that in turn hampered the second p-fluorobenzyl group substitution. The other factor responsible for these failed attempts could be due to a highly electronegative fluorine atom (Pauling scale of electronegativity: F ) 4.0, H ) 2.1) rendering benzylic nitrogen less basic through negative inductive effect, this in turn preventing benzylation. These factors may have been also responsible for our previous failed attempts in synthesizing compound 7 from compound 5 (Scheme 2), where only a small quantity of compound 6 was obtained instead. Refluxing compound 3 with 40% aqueous methylamine in acetonitrile for 1 h produced compound 8 in 74% yields. Compound 8 is an important compound since this is used as a precursor to synthesize C-11-labeled DASB. Wilson et al. (23) reported synthesizing compound 8 by directly reacting the corresponding benzyl alcohol (compound 2, Scheme 2) first with thionyl chloride to generate 3 in situ and then reacting it with 40% aqueous methylamine for 24 h at room temperature. The
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Scheme 3. Radiochemical Synthesis of [18F]FBASB Using [18F]FBI as a Synthon
yields for the desired product remained modest (37%). We preferred isolating compound 3 and purifying this product before its subsequent reaction with methyl amine. Using this approach, we obtained 8 in higher purity and yields. Despite isolating and purifying benzyl chloride 3 followed by reaction with methylamine, our method remains a convenient and high yielding procedure to synthesize compound 8 (precursor for DASB). Following this procedure, it is simple to perform a large-scale synthesis of DASB precursor. Compound 9 was obtained in 72% yield by reacting compound 8 with p-fluorobenzyl bromide using DBU as a base. Reacting 3 or 8 with aqueous dimethylamine and refluxing in acetonitrile provided compound 10. Using this strategy, we were able to synthesize compounds 6, 8, 9, and 10 in high overall yields. In addition, we synthesized compound 4 that serves as a useful intermediate to synthesize various substituted amines. In Vitro binding of FBASB, i.e., 9, to SERT as assessed by rat membrane homogenate was 0.51 ( 0.11 µM. Although the in Vitro binding of FBASB was significantly lower than that for the DASB (19), the in Vitro binding for the desmethyl analogue of FBASB, i.e., compound 6 was 4.04 ( 0.95 nM, a value several fold higher than the N-methylated analogue 9. Similarly, binding for the unsubstituted benzylamine 5 was 0.070 ( 0.015 µM, a 7-fold higher binding affinity toward SERT than the substituted analogue 9. These in Vitro results further echo earlier findings on compounds with a tropane template that the N-substitution reduces 5HT potency and a reduced 5HT affinity for methylated analogues of compounds with similar motif as ours (25). Although demethylation of compound 9 lead to reduced affinity toward SERT, we did not screen this compound against other receptors and transporters. Radiochemical synthesis of [18F]FBASB was accomplished as shown in Scheme 3. Reaction of [18F]FBI with compound 8 provided [18F]FBASB in 36 ( 8% radiochemical yield (EOS). These radiochemical yields are comparable to other radiotracers routinely synthesized in our laboratory using the [18F]FBI synthon. After HPLC purification, the radiochemical purity of this product was >99%. The total synthesis time to produce [18F]FBASB was ∼140 ( 20 min including the reformulation and quality control assays on the final product. The entire synthesis was a remote operated process and required minimal intervention from the production staff. In our earlier attempts to modify chemical structure of a routinely used dopamine transporter ligand FCT by replacing the chloro group on the phenyl ring with alkyl, despite a high in Vitro binding affinity of the newer analogue, we observed a complete blockade of brain penetration (29). Therefore, for the current study, our interest was to first ascertain if this compound penetrates into the rat brain. The results from the biodistribution studies show 0.34 ( 0.02% of the injected dose in rat brain at 30 min. In comparison, [11C]DASB uptake was 0.89 ( 0.39% and 0.33 ( 0.07% injected dose per gram of brain at 30 and 60 min, respectively (30). These results swayed us to investigate if there is any preferential distribution of this ligand between target and nontarget sites within the brain. In addition, we
wanted to investigate if this compound exhibits resistance to in ViVo dehalogenation. Therefore, we performed biodistribution studies in mice to ascertain F-18 distribution in the whole body as well as differential distribution in various brain tissues. The results from these in ViVo biodistribution studies are shown in Figure 2. Although the radioactivity accumulation in all organs remained low, the major sites of retention of F-18 at 60 and 120 min were the kidneys, liver, and muscles. Each of these organs showed less than 0.9% of the injected dose per gram of tissue. The F-18 uptake in the next highest uptake organ was the lung with 0.37 ( 0.09% and 0.23 ( 0.08% injected dose per gram of tissue at 60 and 120 min, respectively. F-18 uptake in the muscles was 0.32 ( 0.09% ID/g at 120 min, and muscle remained the tissue with the third highest uptake of radioactivity, perhaps from increased lipophilicity of FBASB due to the fluorobenzyl group in the molecule. Similar to that seen from our rat studies, [18F]FBASB showed resistance to in ViVo dehalogenation in mice. At 120 min, the F-18 uptake in the bones was 0.14 ( 0.07 %ID/g. Further studies were performed to assess a differential distribution pattern of F-18 within brain tissues. In these studies, cerebellum was chosen as tissue with nonspecific binding (30). As seen from F-18 uptake data, the radioactivity distribution remained quite homogeneous throughout the brain with slightly higher uptake in the thalamus when compared to that for the cerebellum. For example, at 60 min the F-18 uptake in the rat cerebellum was 0.16 ( 0.03 %ID/g as compared to 0.21 ( 0.08, %ID/g in the hypothalamus. Specific to nonspecific ratio in the mouse brain at 60 min time points is shown in Figure 3. Although, at 60 min, the specific to nonspecific uptake ratio remained higher than 1 for most SERT rich tissue, the amplitude of this ratio was significantly lower than DASB and other related SERT specific compounds. While all tissues show specific to nonspecific ratios better than 1 at 60 min, cerebellum to tissue ratio for the cortex, thalamus, and hypothalamus improved slightly with time.
Figure 2. Tissue distribution of F-18 in mice at 60 and 120 min after injecting F-18 FBASB via the tail vein injection. An overall low radioactivity in all normal tissue and further reduction in accumulation at a 2 h time point supports rapid clearance of radiotracer from the body. Low bone uptake at 1 and 2 h further supports its resistance to dehalogenation.
F-18 Analogue of DASB
Figure 3. Tissue to cerebellum ratio for the F-18 FBASB at 60 min as derived from F-18 uptake in the mouse brain.
Despite slightly better ratios in SERT specific tissues, the properties of this ligand remained marginal for the imaging application. Nonetheless, in ViVo results are in agreement with in Vitro binding affinity results toward the SERT. Owing to its higher in Vitro affinity, the desmethyl analogue 8 may exhibit better biodistribution properties and selectivity toward SERT than that seen with the [18F]FBASB. It is also likely that the substitution of less bulky prosthetic groups, such as fluoromethyl or fluoroethyl would yield a more promising SERT ligand. In summary, we synthesized an F-18 analogue of DASB using a simple and facile radiochemical synthesis procedure. Low binding affinity of this compound resulted in low brain uptake along with low selectivity toward SERT rich tissues, resulting in a poor specific to nonspecific uptake ratio. In conclusion, we herein describe a rapid and high yielding synthesis to prepare a key intermediate precursor necessary to synthesize [11C]DASB. In addition, this paper describes a facile and high yielding synthesis of another key intermediate that could aid in the synthesis of a wide array of compounds with application in PET and SPECT.
ACKNOWLEDGMENT We thank Jessica Stukes for her assistance with tissue biodistribution studies and the cyclotron staff for providing help with obtaining F-18 radioisotope. We are thankful to Drs. Steve Childers and Li Wu for their help with performing the in Vitro binding assay. This research was supported in part through a grant from National Institutes of Health (CA105382 to P.K.G.).
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