Synthesis and evaluation of [18F] labeled benzamides: High affinity sigma receptor ligands for PET imaging

Nuclear Medicine & Biology, Vol. 24, pp. 333-340, Copyright 0 1997 Elsevier Science Inc.

ISSN 0969-8051/97/$17.00 + 0.00 PII SO969-8051(97)00001-2

1997

ELSEVIER

Synthesis and Evaluation of [18F] Labeled Benzamides: High Affinity Sigma Receptor Ligands for PET Imaging Cm-men S . Dence, ’ Christy S . J~hn,~ Wayne D. Bowen and Michael J. Welch’” ‘MALLINCKRODT KINGSHlCHWAY

INSTITUTE

BLVD.,

20037; ‘NATIONAL

OF RADIOLOGY,

ST. LOUIS,

MO 63110;

INSTITUTE

*THE

OF DIABETES

WASHINGTON GEORGE AND

UNIVERSITY

WASHINGTON

DIGESTIVE

MEDICAL

UNIVERSITY

AND

KIDNEY

SCHOOL, MEDICAL

DISEASES,

NIH,

BOX 8225, 510 SOUTH CENTER, BETHESDA,

WASHINGTON, MD

IX

20892

ABSTRACT. We have synthesized and characterized four new fluorinated halobenzamides as sigma receptor ligands for use with positron emission tomography (PET). All th e compounds were found to have high benzamides were found to be more sigma-1 affinities (& = 0.38-0.98 nM), and the 4-fluoro-substituted potent sigma-2 ligands (Ki = 3.77-4.02 nM) than their corresponding 2-fluoro analogs (Ki = 20.3-22.8 nM). The [18F] radiochemical syntheses of two of the analogs gave overall yields between 3-10% (EOS), radiochemical purities >99%, and specific activities between 800-1200 Ci/mmol (29.6-44.4 TBq/mmol). Rat biodistribution and blocking experiments were performed with 2-[‘sF](N-fluorobenzylpiperidin-4yl)-4iodobenzamide, the analog with the best & value for sigma-l sites (0.38 nM). Results of these experiments demonstrate specific uptake of the compound in tissues believed to contain sigma receptors, such as lungs, kidneys, heart, brain, and spleen and indicate its potential as a candidate for use in PET imaging of tissues NUCL MED BIOL 24;4:333-340, 1997. 0 1997 Elsevier Science Inc. containine these receDtors. KEY WORDS.

Fluorine-18,

Positron

emission

tomography,

INTRODUCTION There is interest in the radiopharmaceutical community in developing receptor-specific molecules labeled with positron- or photonemitting radionuclides for use with positron emission tomography (PET) or single photon-emitting computed tomography (SPECT). We have been interested in the use of sigma (a) receptors as targets for the development of radiopharmaceuticals for imaging tissues containing these receptors. Sigma receptors are non-opiate, nondopaminergic, membrane-bound proteins that possess high affinity for haloperidol and various neuroleptics. Sigma receptors were initially considered to be subtypes of opioid receptors (32). Based upon the pharmacological binding and behavior studies, it has been concluded that u receptors are a unique class of receptors (21). Sigma receptors are present in the central nervous system of guinea pigs and rats, with high concentration of binding sites found in the spinal cord, the pons medulla, throughout the brain stem reticular formation, the cerebellum, the midbrain and hippocampus; moderate density is also found in the hypothalamus and cerebral cortex (5, 17). Sigma receptors have also been found in peripheral tissues such as liver, kidneys, lungs, gonads, and ovaries (7, 8, 34). Sigma receptors have been implicated in posture and movement disorders, as high concentrations of sigma receptors have been found in areas of the brain that control movement, such as the cerebellum and the red nucleus (5, 22, 31). Sigma receptors have recently been shown to be expressed in a variety of human tumors, in particular, those of breast, melanoma, non-small-cell lung carcinoma, brain, prostate and tumors of neural origin (1, ll-15,27-30). It is now known that u receptors exist as two subtypes, o-l and u-2 (7, 26). Sigma-l sites can be selectively labeled by [3H](+)pentazocine (2). Di-o-tolylguanidine (DTG) is a non-subtype selec-

Benzamides,

tive ligand used for labeling both u-l and u-2 sites (33). No selective radiolabeled ligand exists thus far for labeling u-2 sites. However, u-2 sites can be labeled with [3H]DTG in the presence of dextrallorphan to mask the labeling of u-l sites (26). Radioiodinated u-l ligands, in particular, [1311]N-[2-(piperidino) ethyl]4-iodobenzamide (u-l K, = 2.57 nM, u-2 K, = 205 nM) and N-[2-(diethylaminoethyl)4-iodobenzamide (u-l K, = 11 nM, u-2 K, = 2041 nM) (9), have been used to image human melanoma xenograft, human non-small-cell lung carcinoma, and patients with metastatic melanoma (9-14, 24). Other structurally related iodobenzamides such as (N-benzylpiperidin-4-yl)4-iodobenzamides, 4-IBP, have recently been shown to possess high affinity for both u-l (K, = 1 nM) and u-2 (Ki = 25 nM) receptor subtypes (12, 15). The inhibition constants for the binding affinity of 4-[‘251]IBP to u receptors in membrane preparations from organs such as liver and brain have recently been published (15). We also showed the dose-dependent high-affinity inhibition of binding of radioiodinated 4-[‘251]IBP to human breast cancer cells (12, 15). The structure-activity relationship (SAR) studies of halogenated benzamides revealed that benzylpiperidine was an essential pharmacophore for high affinity at both u-l and u-2 receptor subtypes (C. S. John and W. D. Bowen, unpublished results). We wish to report here the synthesis, characterization, and u receptor affinities for various novel halogen-substituted fluorobenzamides. The fluorine-18 syntheses of these analogs and the in viuo distribution and receptor specificity of 2-[“F](N-fl uorobenzylpiperidin-4-yl)-4-iodobenzamide, the analog with the best K, value for u-l (0.38 + 0.03 nM), are also described. A preliminary report on this work has been published elsewhere (3). MATERIALS

*Author for correspondence. Received 21 November 1996. Accepted 22 January 1997.

Sigma receptors

AND

METHODS

Analytical-grade reagents and solvents were obtained from commercial sources (Sigma-Aldrich, Milwaukee, WI, and Fisher Scientific, Pittsburgh, PA), and unless described, they were used without

334

C. S. Dence et al.

0

0

#aN/\Ph

A)C’C02CH(C’)CH3~

EtOH-HCI,

)

CHC13

X

X

X=Br,l

lb

X=Br

B) Solvent*, Et3N, Heat

2a

X=1. 4-FPF

2c

X=Br, 4-F

2b

X4, 2-F/‘aF

2d

X=Br, 2-F

FIG. 1. Chemical and radiochemical syntheses isolated. (B) Non-radioactive synthesis: ethanol

of compounds used in this study. (A) The intermediate under reflux for 3 h. Radiosynthesis: dimethylformamide

carbamate was not at 80-90°C for 10

min. further purification. The [3H]DTG (39.1 Ci/mmol) was purchased from DuPont/New England Nuclear (Boston, MA). [sH]( +)-Pentazocine (5 1.7 Ci/mmol) was synthesized as described previously (2). Low-resolution chemical ionization (CI) or electron ionization (EI) mass spectra were obtained on a Finnigan 10 15 mass spectrometer. Proton NMR spectra were recorded on a Bruker 300 AM spectrometer in a mixture of CDCl, + CD,OD. Chemical shifts are reported in ppm downfield from tetramethylsilane. Peak patterns are described by the following abbreviations: br = broad; d = doublet; t = triplet; q = quartet; m = multiplet; arom = aromatic protons. Melting points were determined with a Fisher-Johns apparatus and are not corrected. Elemental analyses were performed by Quantitative Technologies Inc., Whitehouse, NJ. All analyses were within 1% of the calculated values. High-pressure liquid chromatography (HPLC) was performed isocratically with a Spectra-Physics SP 8700 liquid chromatograph, equipped with a UV detector (wavelength at 254 nm) and a Nal(T1) radioactive detector. HPLC purification of the [‘“Flfluorobenzamides was performed on a preparative silica column, Whatman Partisil M-9, 500 mm X 9 mm; the eluting solvent was a mixture of 5% 2-propanol in dichloromethane and hexanes (92.8 v/v) at 5 mL/min. Radiochemical purity of the intermediates and final products was assayed on a reverse-phase, analytical-size Alltech C-18 Versapack column, 10-p particle size, 300 mm X 4.1 mm; the eluting solvent was a mixture of acetonitrile in O.OlM KHzPO, (65:35 v/v) at 1 mL/min. Thin-layer chromatography (TLC) was performed on Analtech uniplate silica gel plates, 250 I*, 10 cm X 20 cm, with fluorescent indicator; the plates were developed with chloroform/ methanol (90: 10 v/v). [ ‘sF]Chromatograms were read on a Bioscan System 200 imaging scanner provided with automatic plate changer and integration software. Specific activity (SA) was determined by

HPLC on the analytical-size Cl8 column previously described. A decayed sample of known initial radioactivity was used to determine the amount of mass associated with the radioactive peak when compared to a known amount of the nonradioactive compound used as standard.

Synthesis The overall

of Precursors chemical

and Authentic

synthesis is illustrated

Standards in Fig. 1.

N-(PIPERIDIN-4-YL)4-IODOBENZAMIDE (1A). To a solution of N-(Nbenzylpiperidin-4-yl)4-iodobenzamide (1.0 g, 2.38 mmol) in chloroform (50 mL) was added a solution of l-chloroethyl chloroformate (0.37 g, 2.62 mmol) in chloroform (10 mL). The mixture was heated at reflux for 5 h. The volatiles were removed and the mixture was heated at reflux with ethanolic HCl for another hour. The volatiles were removed and the residue was dissolved in chloroform/ methanol (80:20 v/v) and purified by column chromatography by elution with 50 mL of chloroform/methanol (80:20 v/v) followed by 100 mL of methanol. The fractions containing the desired compound (TLC silica gel; R, = 0.1 in chloroform/methanol, 9O:lO v/v) were combined, and the volatiles removed to give a white solid (0.66 g, 77%). ‘H NMR: 1.48-1.61 (q, J = 12 Hz; CH,); 1.83-1.88 (d, ZH, CH,); 2.77-2.86 (t, 2H, CH,); 2.97-3.12 (m, 2H, NCH,); 3.16 (s, lH, NH); 3.23-3.24 (d, 2H, NCH,); 3.77-3.85 (m, lH, CH); 7.25-7.28 (d, 2H, arom); 7.50-7.52 (d, 2H, arom). Anal. C,H,N, C,,H,,N,OI, m/e = 331 (M + l)+, m.p. > 280°C. N-(PIPERIDIN-4-YL)4-BROMOBENZAMIDE (1B). A procedure similar to the one above was used. To a solution of N-(N-benzylpiperidin-4yl)4-bromobenzamide (1.0 g, 2.68 mmol) in 50 mL of chloroform was added a solution of 1-chloroethyl chloroformate (0.39 g, 2.68 mmol) in 10 mL of chloroform. The mixture was heated at

Evaluation

of [‘sF] Labeled

335

Benzamides

CHO

CHO

a

b

NO2

‘8F

‘8F

70--80%

S---90%

FIG. 2. Radiochemical syntheses of the intermediate [‘*F]fluorobenzyl iodides ([‘aF]FBI). (a)“F/K 2.2.2./K+, 5-7 set in microwave cavity; C-18 SepPak@ purification and extraction with dichloromethane. (b) Diiodosilane reagent (18) (prepared from 790 pmol iodine, 891 pmol phenylsilane, and 61.4 nmol ethylacetate immediately before use) was added to the “Fbenzaldehyde and allowed to react at room temperature for 3 to 4 min. The crude mixture was purified by the use of a solid-phase medium consisting of sodium bicarbonate/sodium thiosulfate supported on silica gel.

reflux for 5 h. The volatiles were removed and the mixture was heated at reflux with ethanolic HCI for another hour. The volatiles were removed and the residue dissolved in chloroform/methanol (8020 v/v) and purified by column chromatography by eluting with 50 mL of chloroform/methanol (80:20 v/v) followed with 100 mL of methanol. The later fractions were combined with the volatiles removed to give the desired debenzylated benzamide (0.55 g, 72%). ‘H NMR: 1.55-1.68 (m, 2H, CH,); 1.85-1.94 (d, 2H, NCH,); 3.1-3.15 (d, 2H, NCH,); 3.80-3.90 (m, 1H. NCH); 7.31-7.38 (d, 2H, arom); 7.46-7.50 (d, 2H, arom). Anal. C,H,N, C,zH,,BrN,O, m.p. = 255-258°C. N-W-4-FLUOROBENZYLPIPERlDIN-4~YL~4JODOBENZAMlDE (ZA). A mixture of N-(piperidin-4-yl)4-iodobenzamide (1.0 g, 2.84 mmol) and 4-fluorobenzylbromide (0.54 g, 2.84 mmol) and triethylamine (2 mL) in ethanol (40 mL) was heated at reflux for 3 h. The volatiles were removed in wacuo and the residue was dissolved in chloroform and washed with water (30 mL). The organic layer was separated, dried, and volatiles removed to give a white solid (0.8 g, 60%). ‘H NMR: 1.53-1.61 (m, ZH, CH,); 1.97-2.18 (m, 4H, CH,); 2.812.85 (d, 2H, CH,); 3.47 (s, 2H, benzyl CH,); 3.95-3.99 (m, lH, methyne); 5.96-5.99 (d, lH, NH); 6.95-7.01 (t, 2H. arom); 7.26-7.29 (t, 2H, arom); 7.43-7.46 (d, 2H, arom); 7.73-7.76 (d, 2H, arom). Anal. C,H,N, C,,H,,FN,OI, m/e = 439 (M + l)‘, m.p. = 201-203°C. N-(N-2-FLUOROBENZYLPIPERlDlN-4-YL)4-IODOBENZAMIDE (ZB). Aprocedure similar to the one described above was used with 2-fluorobenzylhromtde. The desired compound was obtained in 66% yield. ‘H NMR: 1.55-1.62 (m, 2H, CH,); 1.98-2.12 (d, 2H, CH,); 2.21-2.30 (m, 2H, CH,); 2.81-2.84 (d, 2H, CH,); 3.55 (s, 2H, benzyl CH,); 3.95-4.01 (m, lH, methyne); 5.96-5.99 (d, lH, NH); 7.01-7.16 (m, 2H. arom); 7.21-7.28 (m, lH, arom); 7.31-7.39 (m, lH, arom); 7.44-7.46 (d, 2H, arom); 7.73-7.76 (d, ZH, arom). Anal. C,H,N, C,,H,,FN,OI, m/e = 439 (M + I)‘, m.p. =

225-227°C. N-~N-4-FLUOROBENZYLPIPERIDIN-4-YL)4-BROMZAMIDE (ZC). A mixture of N-(piperidin-4-yl)4-bromobenzamide (hydrochloride salt) (1.0 g, 3.13 mmol) and 4-fluorobenzylbromide (0.60 g, 3.13 mmol) and triethylamine (2 mL) in ethanol (40 mL) was heated at reflux for 3 h. The volatiles were removed in vacua and the residue was dissolved in chloroform and washed with water (30 mL). The organic layer was separated, dried, and volatiles removed to give a white solid (0.92 g, 77%). ‘H NMR: 1.50-1.59 (m, 2H, CH,); 1.96-2.09 (d, 2H, CH,); 2.12-2.17 (m, 2H, CH,); 2.79-2.83 (d, 2H, CH,); 3.45 (s, 2H, benzyl CH,); 3.93-3.98 (m, lH, methyne);

5.97-6.00 (d, lH, NH); 6.94-7.00 (m, 2H, arom); 7.24-7.30 (m, 2H, arom); 7.51-7.54 (m, 2H, arom); 7.57-7.60 (m, 2H, arom). Anal. C,H,N, C,,H,,BrFN,O, m/e = 391 (M + l)‘, m.p. =

205-207°C. N-(N-2-FLUOROBENZYLPIPERIDIN-4-YL~4-BROMOBENZAMIDE (LD). A procedure similar to the one for 4fluorobenzamide described above was used with 2-fluorobenzylbromide. The desired compound was obtained in 73% yield. ‘H NMR: 1.53-1.58 (m, 2H, CH,); 1.97-2.19 (m, 2H, CH,); 2.23-2.27 (t, 2H, CH,); 2.85-2.88 (d, 2H, CH,); 3.58 (s, 2H, benzyl CH,); 5.95-5.98 (d, lH, NH); 7.01-7.11 (m, 2H. arom); 7.21-7.24 (m, 2H, arom); 7.32-7.37 (m, lH, arom); 7.51-7.54 (m, 2H, arom); 7.56-7.60 (m, ZH, arom). Anal. C,H,N, C,,H,,BrFN,O; m/e = 391 (M + l)‘, m.p. =

203-206°C. Radiochemical

Syntheses

ilsFIFLUORIDE. [ isF]Fluoride was produced from 95% enriched [‘sOlwater by the ‘80(p,n)‘8F nuclear reaction as previously described (16), using the Washington University CS-15 or the Japan Steel Works (JSW) 16/8 cyclotron. An anion-exchange column in the carbonate form (26) was used to separate the enriched [ ‘sO]water from the [‘“F] radioactivity. The latter was obtained as a 0.02 M solution of potassium carbonate (K&O,) and was then resolubilized in the presence of Kryptofix@ 2.2.2 (1.1 mg per 100 FL of the 0.02 M carbonate solution). Azeotropic drying in a Vacutainera under nitrogen with acetoitrile (3 X 0.5 mL) furnished the dry potassium fluoride-Kryptofix@ complex, which was then redissolved in 200-300 p,L of anhydrous dimethylsulfoxide.

2- or 4-[18F]Eluoroben~l

Iodide

The radiochemical synthesis is indicated in Fig. 2. The syntheses of the 2- and 4-[“Flfluorobenzyl iodides ([“F]FBI) from the 2- and 4-NOz-benzaldehydes were performed by the one-step reductioniodination method with diiodosilane (DIS) (18). The starting intermediates, 2- and 4-[‘8F]fluorobenzaldehydes, were first synthesized as follows. The resolubilized [“Flfluoride in dimethysulfoxide was added to a Pyrex test tube 10 cm X 15 mm o.d. containing 2-3 mg of the desired substrate; the test tube was capped and heated for 5 to 7 set in a microwave cavity (4). After cooling, the mixture was purified on a C-18 Sep-Pak@ cartridge and extracted with 3 mL of dichloromethane. The 2- and 4-[‘*F]fluorobenzaldehydes were analyzed by HPLC (analytical C-18 column, retention time about 5.2 min), had a radiochemical purity higher than 99.5%, and were

336

C. S. Dence et nl.

obtained in radiochemical yields (EOS) between 50-80% (n = 16). Conversion of the intermediate [‘8F]fluorobenzaldehyde to the [‘sF]fluorobenzyl iodide was performed by the one-step reductioniodination reaction with DIS. The DIS reagent was prepared just prior to use in a l- X 15-cm test tube under nitrogen atmosphere according to a published procedure (18). The freshly prepared reagent was added to the [‘8F]fluorobenzaldehyde previously eluted with 3 mL of dichloromethane in a 15mL test tube and allowed to react for 3 to 4 min. The crude reaction mixture was then purified by the use of a new solid-phase medium as described below. The [‘8F]fluorobenzyl iodides were analyzed by HPLC (analytical C-18 column, retention time about 10.7 min), had a radiochemical purity of 85-95%, and were obtained in radiochemical yields (EOS) between 63-80% (n = 14).

Solid-Phase Medium for the Purification of Iodination Products A 10% solution of sodium bicarbonate (NaHCO,) and a 10% solution of sodium thiosulfate (Na,S,O, * 5HzO) were prepared. Each solution (100 mL) was added to an individual 25 g of silica gel (Si-60, 230-400 mesh), and after mixing, the excess of water was rotary-evaporated and the solids dried overnight at 90°C under vacuum. The resulting solid-phase medium was carefully homogenized to the free-flowing appearance of the original silica gel and stored in tightly closed plastic bottles until ready to use. The purification column (Biorad disposable columns 1 cm X 25 cm) was provided with a 0.45-p nylon filter. Silica gel (0.5 g) was placed at the bottom of the column, followed by 2 g of silica-bound Na,S,O, and 2 g of silica-bound NaHCO, media. The crude radioactive mixture obtained after the iodination reaction in about 3 mL dichloromethane was then added to the purification column and the products eluted with additional dichloromethane for a total volume of 3-4 mL. The clear eluate was used directly for the alkylation step.

[“F]Alkylation

Reaction

The purified [‘*F]fluorobenzyl iodide in dichloromethane was added to a lo-mL conical flask containing 2 mg of the desired norbenzylated substrate, followed by 200-300 PL of dimethylformamide and 20-30 p,L of triethylamine (Fig. 1). The dichloromethane was then evaporated under a stream of nitrogen in a 80-90°C oil bath, and the reaction flask was heated for an additional 10 min. After cooling, the mixture was purified on a C-18 Sep-Pak@ cartridge and extracted with 3 mL of dichloromethane. The organic phase was dried with magnesium sulfate before injection onto the semipreparative HPLC silica column (retention time about 16 min). The non-optimized total radiochemical synthesis including HPLC purification time took about 2.5 h. The overall radiochemical yield (EOS) was 3-10% (n = lo), the radiochemical purity >99% (analytical C-18 column, retention time about 12 min), and the specific activity between 800-1200 Ci/mmol (29.6-44.4 TBq/ mmol). After HPLC collection of the desired [18F]benzamide, the mobile phase was removed in ~acuo and the product redissolved in 15% ethanol/saline and filtered through a 0.22-p. sterile filter. In a typical experiment, starting with about 50 mCi of [‘8F]fluoride, one will have between 1.5 and 5 mCi ready for injection.

Pharmacology IN VITRO u-1 BINDING ASSAY. The a-l receptors were assayed using the highly selective o-l receptor probe [3H](+),pentazocine in guinea pig brain membranes (2). Guinea pig brain membranes (300-500 kg protein) were incubated with 3 nM [‘HI(+)-pentazocine (51.7 Ci/mmol) in 0.5 mL of 50 mM Tris-HCl, pH 8.0, for 120 min at 25°C. Assays were terminated by the addition of 5 mL ice-cold 10 mM Tris-HCl, pH 8.0, and filtered through a glass fiber filter using a Brandel cell harvester (Gaithersburg, MD). The filters were then washed twice with 5 mL of ice-cold 10 mM Tris-HCl pH 8.0. The non-specific binding was determined in the presence of 10 FM of (+)-pentazocine or 10 p,M of haloperidol. The filters were soaked in 0.5% polyethylenimine prior to use. Scintillation counting after overnight extraction of radioactivity was carried out in Ecoscint (National Diagnostics, Manville, NJ). Guinea pig brain membranes were prepared as previously described (2). Protein was determined by the method of Lowry et al. (19). IN VITRO U-Z BINDING ASSAY. The u-2 receptors were assayed using [3H]DTG and membranes from rat liver, a tissue previously shown to be rich in u-2 sites (8). Rat liver membranes (150-200 pg of protein) were incubated with 3 nM [3H]DTG (33) (39.4 Ci/mmol) in the presence of 1 PM dextrallorphan to mask a-l sites. The procedure was the same as above except that the non-specific binding was determined in the presence of 10 PM haloperidol. Rat liver membranes were prepared as previously described (8).

Animal

Distribution

Study

Animal experiments were carried out in accordance with U. S. federal regulations concerned with the conduct of animal experiments. For the in oivo tissue distribution study we used the HPLC-purified 2-[18F](N-fluorobenzylpiperidin-4-yl)-4-iodobenzamide (Fig. 1, 2b) Anesthetized adult female Sprague-Dawley rats weighing between 150-170 g (7 to 8 weeks old) were injected into the tail vein with 14-20 $Zi (- 100 pL) of the labeled compound. The animals (n = 4/time-point) were sacrificed at 0.5, 1, 2, and 4 h postinjection, a blood sample was obtained, and the tissues were rapidly removed. Standards were prepared by diluting an identical dose of radioactivity to 100 mL with water and withdrawing a 1-mL aliquot of this solution into a vial. Samples and standards were counted in a Beckman Gamma 8000 counter and the percent of injected dose per gram of tissue (%ID/g) was calculated by comparison with standards. A blocking experiment was performed by co-injecting haloperidol (-5 pg/animal) with the no-carrier-added radioactive tracer. The animals (n = 4) were sacrificed at 1 h and the tissues removed and counted as before.

Statistical

Analysis

The statistical tests normally distributed ric Mann-Whitney normally distributed for significance was

used were Student’s t- test when the data was and had equal variance, and the non-parametrank-sum test when the data was either not or the variances were not equal. The criterion p < 0.05.

RESULTS AND DISCUSSION Phuwnacological

Evaluation

Previously, we reported the inhibition constants (K, = nM) for the binding affinities of the (N-benzylpiperidin-4-yl)-2-, 3- and 4-iodobenzamides for various receptors (12). All three isomers showed a

Evaluation

of [18F] Labeled

337

Benzamides

Table 1. Sigma-l and Sigma-2 Receptor Fluorinated and Nonfluorinated Benzamides

Substitution Compound no.

x

2a 2b la 2c 2d lb

I 1 I Br Br Br

F

Inhibition

Constants

high affinity for the u-l receptor (ortho = 1.64 nM, meta = 3.02 nM, and para = 1.70 nM) and moderate affinity for u-2 (29.6 nM, 84.6 nM, and 25.2 nM, respectively). Assays for dopamine-D2, PCP, and muscarinic receptors were carried out using the radioligand and tissues as previously described (12). 4-Iodobenzylpiperidine (4sIBP, Table 1, la) showed the lowest dopamine D-2 receptor affinity, 382 nM, compared to 63.4 nM for the ortho and 24.8 nM for the meta isomer. None of the isomers had any affinity for the PCP and muscarinic receptor sites (K, > lo4 nM). Based on the results of this previous study, we synthesized the 2and 4-[F](N-fluorobenzyl-piperidin-4-yl)4-iodobenzamides, [F](4IBP) and the 2- and 4-[F](hJ-fluorobenzylpiperidin-4-yl)4-bromobenzamides [F](4-BrBP) with th e intent to evaluate their in vitro a-l and u-2 activities and to label them with [“Flfluorine for use as possible probes for PET studies involving areceptors. All the compounds were prepared according to the scheme shown (Fig. 1) in yields between 60-77%, and were characterized by proton NMR, MS, and elemental analyses. Inhibition constants (K, = nM) for the binding affinity to u-l and u-2 receptors of the fluorinated vs. the non-fluorinated analogs synthesized for this work are presented in Table 1. The benzamides were evaluated in vitro for their u-l receptor affinity in guinea pig brain membranes using [‘HI-( +)-pentazocine. Likewise, u-2 receptor affinity was determined in rat liver membranes using [3H]DTG in the presence of dextrallorphan to mask u-l sites. As seen in Table 1 all the compounds were found to have high u-l affinities (K, = 0.38-1.70 nM) and the 4-fl uoro-substituted benzamides (2a and 2c) were found to be more potent at a-2 sites (K, = 3.77 and 4.02 nM, respectively) than their corresponding 2-fluoro (2b and 2d) analogs (K, = 20.3 and 22.8 nM, respectively). The u-2 affinity for compound lb was not determined. Our results compare favorable with published data for the u-l binding affinities of (+)-cis-N-(substituted benzyI)-N-normetazotines, benzomorphan derivatives synthesized as potential SPECT and PET diagnostic agents (23). In this latter study the binding affinity of N-(benzyl)-N-normetazocine for the u-l receptor was 0.67 nM, and this value actually increased to 1.45 nM (98.8%) and 0.97 nM (27.9%), respectively, upon ortho- and para-fluorine substitution on the benzyl ring. In contrast, with the 4-halobenzamides used in the present study, we found the binding affinity enhanced in going from the non-fluoro-substituted 4-I-henzamide (1.70 nM), to the ortho (0.38 nM (77.6%)) and to the para

2 -c -+ 2 ? k

=

nM)

of

a-2 (Rat liver) [3H]DTG + Dex Ki WW

a-1 (Guinea pig brain) [3H]( +)Pentazocine Ki(nW 0.89 0.38 1.70 0.84 0.60 0.98

(I$

0.11 0.03 0.44 0.09 0.07 0.05

3.77 ? 0.13 20.30 ? 0.65 25.20 2 1.28 4.02 ? 0.87 22.80 ? 1.09 Not determined

(0.89 nM (47.6%)) fluoro-substituted N-benzylpiperidin analogs. A less dramatic but still similarly enhanced binding affinity trend was observed with the N-benzylpiperidin 4Br-benzamides in which the Ki value for the non-fluoro-substituted analog (0.98 nM) decreased to 0.60 nM (38.8%) for the ortho fluoro-substituted and to 0.84 nM (14.3%) for the para fluoro-substituted analog (Table 1). In both cases (2b and 2d), the ortho fluoro-substituted analogs showed the best binding affinities. Therefore, based on their affinity constants, these novel fluoro-substituted benzamides may be potentially more sensitive ligands to probe sigma receptors in viwo.

Radiochemical

Synthesis

The radiochemical syntheses (Fig. 2) of the intermediates 2- and 4-[18F]FBI were performed in one step by the use of diiodosilane (DIS) as described previously (18). We obtained better results (better control of exothermicity; also, any undissolved iodide was left out of the reaction) when the freshly prepared DIS reagent euus added to the test tube containing the purified [‘sF]benzaldehyde. We also synthesized [‘“Flbenzyl iodides with the more conventional two-step, lithium aluminum hydride/hydriodic acid (LAH/HI) reduction/iodination of the intermediate [‘“Flbenzaldehyde (6). The overall radiochemical yield starting from the fluoro-for-nitro substitution was 32-65% (EOS) for the DIS one-step synthesis, compared to 25-39% (EOS) for the two-step LAH/HI reaction. In our experience, a serious problem encountered with either method to produce the [‘“Flfluorobenzyl-iodinated intermediates (6, 18, 20) has been the purification step. Attempts to purify the crude mixture with either silica gel columns or C-18 Sep-Pak@ cartridges were time-consuming and resulted in great loses of radioactivity or in products that were still visibly contaminated with traces of colored material (iodine and iodine by-products). The products obtained were, in general, less reactive in the ensuing alkylation reactions. To circumvent these difficulties a new solid-phase purification medium was developed that consisted of silica bound-sodium bicarbonate and silica bound-sodium thiosulfate. Two grams of each phase added to a small column containing 0.5 g of plain silica proved very efficient in removing colored material, acidic byproducts, and excess reagents from the DIS reaction mixture. We were also successful in purifying with a similar column an LAH/HI reaction mixture. In both instances the desired [‘“FIFBI was eluted

338

C. S. Dence et al.

Table 2. Rat Biodistribution Haloperidol at 1 h

of

Z[‘*F](N-Fl

uorobenzyl-piperidin-4.yJ)-4*Iodobenzamide

30 min Tissue Blood Lung Liver Kidney Heart Brain Spleen Muscle Fat Adrenals Bone Ratios Brain/Blood Heart/Blood

0.033 1.947 5.098 1.666 0.459 1.230 2.202 0.160 0.581 5.552 0.503

? k ? 2 + + f + + ? 2

0.004 0.211 1.295 0.229 0.083 0.100 0.162 0.032 0.141 1.216 0.070

37.98 2 7.18 14.52 2 3.84

Ih co-injected

Ih 0.023 2.036 7.005 1.656 0.478 1.313 2.274 0.162 0.744 6.385 0.531

? ? 2 ? k 2 ? k 2 ? ?

and

0.002 0.554 0.721 0.197 0.071 0.165 0.255 0.033 0.201 0.945 0.058

57.74 2 13.03 20.96 2 4.57

0.033 0.805 8.897 0.905 0.252 0.809 1.489 0.124 1.267 6.260 0.333

2 ” -c t ? 2 2 ? + ? ?

0.008 0.090 1.122 0.034 0.032 0.064 0.197 0.010 0.416 1.838 0.037

26.08 t 8.77 7.93 2 1.76

Blocking

2h 0.016 2.119 7.660 1.540 0.493 1.306 2.289 0.146 0.604 5.161 0.473

? ? k ? ? ? k ? ? ? ?

Experiment

with

4h 0.002 0.463 0.591 0.368 0.081 0.187 0.234 0.034 0.092 0.159 0.097

83.06 ? 14.74 31.17 ? 4.85

0.013 1.980 7.767 1.565 0.493 1.360 2.323 0.160 0.706 3.094 0.463

? ? ? ? ? ? t? ? ? t

0.001 0.378 1.693 0.152 0.025 0.100 0.181 0.021 0.201 0.504 0.021

105.67 f 17.43 38.29 5 5.40

Female Sprague-Dawley rats (7 to 8 weeks old, n = 4) were injected into the tail vein with 14-20 pCi of the labeled compound, or co-injected with haloperidol (5 &animal). Tissue distribution values were determined at different times and are expressed as XID/g 2 SD. with 3 mL of dichloromethane. Under the experimental conditions described for the DIS synthesis, the intermediate [‘aF]FBl was obtained with a radiochemical yield of 63-80% (EOS) starting from the [lsF]benzaldehyde within 10 mm of preparation time, and the purified product in dichloromethane was used directly for the final alkylation reaction. We assessed the radiochemical purity of the [‘“F]FBl on a C-18 analytical-size column, using as the mobile phase an acetonitrile/O.Ol M KHzPO, mixture at pH 4.5. We encountered no degradation problems for the iodides due to acetonitrile such as have been reported for the case of [‘sF]F-4,5 dimethoxybenzyl iodide (18). The overall synthesis for the [‘sF]benzamides is illustrated in Fig. 2. The desired substrate (la) was obtained with a 77% yield, by debenzylation of the starting material [I]N-(N-benzylpiperidin4-yl)-4-iodobenzamide (4-IBP) (12) by treatment with l-chloroethyl chloroformate in chloroform followed by reflux with ethanolic HCl and without isolation of the intermediate carbamate. The hydrochloride salt of the substrate was then rebenzylated with the purified [‘“FIFBI in the presence of DMF and triethylamine. The non-optimized total synthesis time was about 2.5 h. The overall radiochemical yields were 3-10% (EOS), the radiochemical purity >99%, and the specific activity between 800-1200 Ci/mmol (29.6-44.4 TBq/mmol).

Animal

Biodistribution

Study

2-[‘sF](N-Fluorobenzylpiperidin-4-yl)4-iodobenzamide, which had the highest affinity for the a-l receptor (Table 1, 2b), was chosen for in viva evaluation in mature female Sprague-Dawley rats, 7 to 8 weeks old. The results of the biodistribution at 0.5, 1, 2, and 4 h are presented in Table 2. We also show in this table the results of a blocking experiment at 1 h upon co-injection of haloperidol (5 p,g/animal) with the [‘“F]-labeled tracer. As seen in Table 2, the compound is taken up by the peripheral organs known to contain (T receptors such as lungs, kidneys, heart, brain, and spleen. The uptake in these organs ranged from 0.462.2O%ID/g, and the activity remains constant for up to 4 h. At times as early as 30 min the uptake is within ?lO% of the values found at 4 h. The high uptake found in the liver (7%lD/g at 1 h) suggests

that this organ is the site of metabolism of the tracer; the liver uptake remains constant for up to 4 h. Likewise, the uptake by adrenals at 30 min was high, 5.6%lD/g, but after 4 h, it dropped by 44% to a value of 3.09%lD/g, indicating perhaps a less specific binding to u receptors. Table 2 also presents data at 1 h co-injection with the nonselective a-l ligand haloperidol. In the presence of haloperidol the uptake was blocked by 34.5% for the spleen, 38.4% for the brain, 45.4% for the kidneys, 47.3% for the heart, and 60.5% for the lungs. No blocking was observed in the liver (suggested metabolic site) and adrenals (non-specific-binding site). At 1 h, the brain-to-blood ratio was 57.7 and the heart-to-blood ratio was 21, and after 4 h these ratios increased to 106 and 38, respectively. In the blocking experiment at 1 h, the brain-to-blood ratio decreased to 26.08 (53.6%) and the heart-to-blood ratio decreased to 7.93 (62%). No increase in bone uptake with time was observed, indicating that no extensive in viva defluorination was taking place. In another similarly run biodistribution experiment (data not shown) we found the uptake at 1 h by the uterus (1.54%lD/g) and the ovaries (3.13%lD/g) (33) was reduced by about 30% with co-injection with haloperidol; we also found the uptake in the cerebellum (1.29%lD/g), cortex (1.24%lD/g), and striatum Even though (l.O7%lD/g) was blocked by 30 ‘0/ with haloperidol. these brain uptake values are lower than those reported (25) using (+)-[C-l I]-cis-N-benzyl-normetazocine (est. 1 h: 5.7-4%lD/g), similarly higher uptake for the cerebellum and cortex vs. the striatum was observed in both studies. In general, the % blocking found in our biodistribution studies may be partially dependent on the dose of haloperidol used as blocking agent, about 30 pg/kg. However, this assumed dose-dependence with haloperidol must be verified as it was in the case with (+)-pentazocine, in which an 80% degree of blocking in the brain was reduced to 50% when the dose of (+)-pentazocine was decreased from 1000 kg/kg to 100 k&g

(25).

SUMMARY Four new tracers derived from the (N-benzylpiperidin-4-yl)4-halobenzamide moiety substituted with iodo or bromo and containing

Evaluation

of [‘“F] Labeled

fluorine at the 2- or 4-benzyl position have been synthesized, and their a-l and u-2 in vitro affinity constants have been determined. Two of the four fluorine-containing analogs were then synthesized with ‘“F in a synthesis time of about 2.5 h. In each case the overall radiochemical yield was 3-10% (EOS), the radiochemical purity >99%, and the specific activity was between 800-1200 Ci/mmol (29.6-44.4 TBq/mmol). Rat biodistribution and blocking studies were then performed with 2-[‘sF](N-fluorobenzylpiperidin-4-yl)4iodobenzamide, the analog with the best K, value (0.38 nM). The compound had significant uptake in the lungs, kidney, heart, spleen, and brain (0.46-2.20%ID/g). This uptake remained constant for up to 4 h, and it was blocked at 1 h (35560%) by co-injected haloperidol. The high liver uptake (7%ID/g) was not blocked by haloperidol, perhaps reflecting the site of metabolism. Similarly, a high uptake in the adrenals was observed at 1 h (6.4%ID/g), but after 4 h it dropped by more than 50% (3.09%ID/g). This adrenal uptake was not blocked by haloperidol. Brain-to-blood and heart-to-blood ratios of 57.7 and 21, respectively, at 1 h were decreased by co-injection with haloperidol to 53.6% and 62%, respectively. The results presented for the in oiwo and in vitro studies suggest that this family of compounds may be useful to map a-sites in viwo with PET. However, the high liver uptake and slow clearance from this organ must be taken into consideration for clinical application.

The authors wish to thank Elizabeth Sherman and Mary Stephensonfor the animal biodistribution work, W. Margenau for the production of the isotope, T. McCarthy for helpful d’lscussions , and Joseph B. Dence for reading the manuscript. This work was supported by NIH grants and CA58496 (George WashP01HL1385 I (Washington University) ington

339

Benzamides

University).

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Dence