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Synthesis of 11C-labeled desipramine and its metabolite 2-hydroxydesipramine: Potential radiotracers for pet studies of the norepinephrine transporter
Nuclear Copyright
Medicine &. Biology, Vol. 0 1997 Elsevier Science
24, pp. 707-71 Inc.
I, I997
ISSN
0969~8051/97/$17.00 + 0.00 PII SO969-8051(97)00109-1
ELSEVIER
Synthesis of “C-Labeled Desipramine and Its Metabolite 2-Hydroxydesipramine: Potential Radiotracers for PET Studies of the Norepinephrine Transporter Marcian E. Van DOG, * Jae-Hoon Kim, f Louis Tluczek DIVISION
OF NUCLEAR
MEDICINE,
and
LJEPARTMENT OF INTERNAL MEDICINE, UNIVERSITY ANN ARBOR, Ml 48109-0552, USA
Donald M. Wiehnd
OF MICHIGAN
MEDICAL
SCHOOL,
and its principal metabolite 2shydroxydesipramine ABSTRACT. The antidepressant desipramine (DMI) (HDMI) have been radiolabeled with “C for PET studies. The normethyl precursors of DMI and HDMI were synthesized from iminodibenzyl in 35% and 11% overall yield, respectively. Direct methylation of the normethyl precursor with [ “C]CH,I, f o 11owed by HPLC purification, provided [“CIDMI and [“CIHDMI in 18-30% and 15-23% decay-corrected radiochemical yields, respectively, in a 45 min synthesis time from end of bombardment. The specific activities of the two radiotracers were > I459 Ci/mmol at the end of synthesis. [ “C]DMI and [“CIHDMI h ave potential utility as PET radiotracers for the norepinephrine transporter. NUCL MED BIOL 24;8:707-711, 1997. 0 1997 Elsevier Science Inc. KEY WORDS. Desipramine, transporter, Positron emission
2-Hydroxydesipramine, tomography
INTRODUCTION The neuronal membrane-bound norepinephrine transporter (NET) plays a critical role in the regulation of synaptic concentrations of norepinephrine (NE) in the central and peripheral nervous system (3, 13). Abnormalities of the NE reuptake system have been implicated in clinical depression (6) and in cardiac failure (2). More recently, Ungerer et al. (14) have reported upregulation of NET in a perfused, ischemic rat heart model. A radiotracer specific for NET could therefore be potentially useful for studying the role of the NE reuptake system in these disease states. Desipramine (DMI), a well-known tricyclic antidepressant, is a selective and high-affinity inhibitor of NE reuptake (K, = 2.4 nM; Fig. 1) (8). Specific, high-affinity binding sites for [‘H]DMI in rat have been demonstrated in tissues receiving dense noradrenergic input, such as heart, submaxillary gland, cortex and hypothalamus (10). 2-Hydroxydesipramine (HDMI), a metabolite of DMI, also retains the excellent NET selectivity profile of DMI, albeit with a slightly lower inhibition of NE reuptake (K, = 7.8 nM; Fig. 1) (8). Furthermore, the increased hydrophilicity of HDMI over DMI could result in a lower in viva nonspecific binding for the former derivative, which would be an important advantage in imaging studies. These two candidates were therefore chosen as lead com* Address correspondence to: Marcian E. Van Don, Ph.D., Division of Nuclear Medicine, 3480 Kresge 111 Building, University of Michigan Medical School, Ann Arbor, MI 48109-0552. Portions of this work have been reported previously in abstract form: Van Don M. E., Tluczek L., Sherman I’. S., Raffel D. M. and Wieland D. M. (1995) Synthesis of carbon-l 1 labeled desipramine: A potential radiotracer for the cardiac norepinephrme transporter. J. Label. Cmpd. Radiopham. 37, 251-252; Van Don M. E., Kim J. H., Sh erman I’. S., Tluczek L. and Wieland D. M. (1996) Carbon- 11 labeled 2-hydroxydesipramine: A potential radiotracer for the cardiac norepinephrine transporter. J. Nucl. Med. 37, 50 I’. t Present address: Department of Polymer Science and Engineering, Kum-Oh National University of Technology, 188 Shinpyung, Kumi, Kyunghuk 7 30-070, Korea. Received 2 February 1997. Accepted 5 May 1997.
Carbon-
11, Radiotracer,
Antidepressant,
Norepinephrine
pounds for initial NET radiotracer development. In this paper we describe the synthesis of “C-labeled DMI and its metabolite HDMI for in ho positron emission tomography (PET) studies.
MATERIALS
AND
METHODS
Melting points were determined in open capillary tubes using a Thomas Hoover melting point apparatus and are uncorrected. ‘H nuclear magnetic resonance (NMR) spectra were obtained on a Bruker WM 300 MHz spectrometer with tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained on a Finnigan 4021 GCMS/DS (low resolution) or a UG70-250-S (high resolution) instrument. Desipramine hydrochloride was purchased from Sigma Chemical Company (St. Louis, MO). All other chemical reagents were purchased from Aldrich Chemical Company (Milwaukee, WI). Thin-layer chromatography (TLC) was performed on Analtech silica gel GF Uniplates (250 km). Following development, TLC plates were visualized under ultraviolet (UV) light and by spraying with ethanolic phosphomolybdic acid reagent with subsequent heating (130-l 50°C). Flash chromatography (12) was performed using Merck silica gel 60 (230-400 mesh).
HPLC
and Radio-HPLC
Analyses
High performance liquid chromatography (HPLC) purification of the radiolabeled product was performed on a Phenomenex Selectosil NH, column (5 p,rn particle size, 250 X 10 mm I.D.) plus guard (50 X 10 mm I.D.) eluted with a mixture of hexane:isopropanol: methanol:diethylamine in either of the following volume-to-volume ratios: 580:10:10:0.6 (system A) or 600:80:80:0.7 (system B). UV absorbance (254 nm) and radioactivity were monitored by an in-line UV absorbance and y-detector, respectively. With a flow rate of 5 mL/min (system A), the retention time of [“CIDMI was 10.2 min and that of the desmethyl precursor 3 was 18.7 min. [“CIHDMI was purified using system B at a flow rate of 7 mL/min.
708
M.
E. Van
generated calibration curve. Radioactivity measurements tained with a Capintec CRC-12 radioisotope calibrator.
5-(3-Chloropropyljiminodibenzyl
R=H;
DMI
R=OH;
HDMI
NE
5-HT
DMI
2.4
100
6000
HDMI
7.0
180
9400
COMPOUND
FIG. 1. Inhibition constants of DMI and HDMI for monoamines. Asterisk indicates data from Javaid et al. (8).
The retention times for HDMI (8) and its desmethyl precursor 6 were 11.7 and 16.9 min, respectively, under these conditions. Radio-HPLC analyses for radiochemical purity and specific activity determination were conducted using a Phenomenex Ultremex 5 CN column (5 pm particle size, 50 X 4.6 mm I.D.) with CH,CN:CH,OH:lO mM KzHPO, (pH = 7.0) (60:15:25) as the mobile phase. A flow rate of 0.5 mL/min was utilized with UV detection at 254 nm. The retention times of 3, DMI, 6 and HDMI were 6.9, 10.4, 6.2 and 8.7 min, respectively, under these conditions.
Radio-TLC
Analyses
Radio-TLC analyses were performed on Analtech Spice Plates (Unibond NH,) using a mixture of hexane:isopropanol:methanol: diethylamine in either of the following ratios: 93:3.5:3.5:1 (system C) or 90:5:5:1 (system D). The labeled compound was co-spotted with the authentic unlabeled compound prior to development. TLC plates were scanned for radioactivity using a Berthold Model LB 2832 TLC-Linear Analyzer equipped with a model LB 500 Data Acquisition System. The Ri values for the desmethyl derivative 3 and DMI were 0.13 and 0.30, respectively, using system C, and the values for 6 and HDMI were 0.17 and 0.38, respectively (system D).
Production
of [“C]CHJ
[“C]CO, was produced with a biomedical cyclotron (CS-30 accelerator; Cyclotron Corporation) by the i4N(p,a)“C reaction using high-purity nitrogen gas in an aluminum target. [“C]CO, was converted to [“C]CH,OH by LiAIH, reduction and treated with aqueous HI at reflux (4) to generate [“C]CH,I. The initial activity of the [“C]CO, produced at bombardment was estimated from a previously determined calibration curve of ‘iC activity produced versus irradiation times in the same target. Estimates of initial target activity from this curve were used to determine overall radiochemical yields.
Specific The peak quantitated
Activity area by
Determinations corresponding UV absorbance
to
the radiolabeled at 254 nm using
product was a previously
Dort
et al.
were
ob-
(I)
The title compound was synthesized by a modification of the published procedure (7) as follows. A stirred solution of iminodibenzyl (6.0 g, 30.7 mmol) and I-bromo-3chloropropane (6.3 g, 40.0 mmol) in dry benzene (240 mL) was warmed under an argon atmosphere to 55°C and treated portionwise over a 30 min period with lithium amide (0.77 g, 33.7 mmol). Following reflux for 24 h, the cooled benzene solution was washed with Hz0 (3 X 100 mL) and dried (Na,S04). Removal of volatiles gave an oil that was dissolved in hexane and filtered to remove unreacted iminodibenzyl. Flash chromatography (silica gel; hexane:EtOAc; 22:l) gave 5.4 g (65%) of 1 as a colorless oil: ‘H NMR (CDCl,) 6 7.16-7.02 (m, 6H), 6.93 (t, lH, J = 7.2 Hz), 6.92 (t, lH, J = 7.1 Hz), 3.90 (t, 2H, J = 6.5 Hz), 3.56 (t, 2H, J = 6.4 Hz), 3.16 (s, 4H), 2.04 (quintet, 2H, J = 6.4 Hz); HRMS m/e 271.1119 (C,zH,,CIN requires 271.1128); EIMS (70 eV) m/e (relative intensity) 273 (13.2), 271 (28.5), 209 (23), 208 (base), 194 (19.9), 193 (43), 192 (lo), 178 (6.4), 165 (6.7) 91 (6.9).
5-[3-{Bis(phenylmethyl)aminoJpropyl]iminodibenzy1
(2)
A mixture of 1 (4.8 g, 17.7 mmol) and dibenzylamine (10.5 g, 53.2 mmol) was heated at 140°C for 72 h under an argon atmosphere. The cooled mixture was diluted with anhydrous Et,0 (250 mL) and filtered to remove crystalline dibenzylamine hydrochloride. Flash chromatography of the crude product (silica gel; hexane:EtOAc; 35:l) provided 4.6 g (60%) of 2 as a colorless oil that gave white crystals on standing: mp 69-71°C; ‘H NMR (CDCL,) 6 7.29-7.16 (m, lOH), 7.12-6.99 (m, 6H), 6.88 (t, lH, J = 7.2 Hz), 6.87 (t, lH, J = 7.2 Hz), 3.70 (t, 2H, J = 6.6 Hz), 3.48 (s, 4H), 2.94 (s, 4H), 2.44 (t, 2H, J = 6.9 Hz), 1.73 (quintet, 2H, J = 6.8 Hz); HRMS m/e 432.2561 (C,,H,,N, requires 432.2565); EIMS (70 eV) m/e (relative intensity) 432 (1.7), 341 (32.8), 236 (20.7), 235 (77.5), 234 (46.2) 222 (56.3) 210 (21.5) 208 (30.1) 193 (30.2), 146 (42.3), 91 (base).
5-(3.Aminopropyl)iminodibenzyl
(3)
A suspension of 2 (0.29 g, 0.67 mmol), ammonium formate (0.17 g, 2.7 mmol) and 0.05 g of 20% Pd(OH), on carbon (Pearlman’s catalyst) in CH,OH (3 mL) was refluxed for 2 h. The catalyst was removed by filtration and the crude product flash chromatographed (silica gel; CHCl,:CH,OH:NH,OH; 9:l:O.l) to afford 151 mg (89.5%) of 3 as a yellow oil (7): ‘H NMR (CDCl,) 6 7.14-7.06 (m, 6H), 6.91 (t, lH, J = 7.1 Hz), 6.90 (t, lH, J = 7.3 Hz), 3.79 (t, 2H, J = 6.7 Hz), 3.15 (s, 4H), 2.71 (t, 2H, J = 7.0 Hz), 1.70 (quintet, 2H, J = 6.9 Hz), 1.13 (br s, 2H); HRMS m/e 252.1626 (C,zH,,N, requires 252.1626); EIMS (70 eV) m/e (relative intensity) 252 (39), 235 (45.3), 234 (36.5), 209 (26), 208 (base), 195 (43.1), 194 (44), 193 (67.1), 192 (23.2), 179 (13.8), 178 (14.6), 165 (14.9), 130 (14.5), 91 (15.2).
2-Formyl-5-[3-{bis(phenylmethyl)amino)ppropyl]iminodibenql (4) Phosphorous oxychloride (2.3 mL, 24.7 mmol) was added dropwise to dimethylformamide (DMF) (4.6 mL, 59.4 mmol) at 0°C under argon and the resulting mixture was allowed to warm to room
Synthesis
of ’ ‘C-Labeled
Desipramine
709
temperature over a 10 min period. The dibenzylamine derivative 2 (4.1 g, 9.5 mmol) was then added and the mixture heated at 95°C for 4 h. The reaction was quenched with ice-water (60 mL), made basic (pH = 12) with aqueous 2N NaOH, extracted with CHCl, (4 X 100 mL) and the combined organic layers dried (Na,S04). Flash chromatography (silica gel; hexane:EtOAc; 9:l) gave 4.25 g (97%) as a light yellow oil: ‘H NMR (CDCL,) 6 9.84 (s, lH), 7.64 (dd, lH, J = 8.4 Hz, 2.1 Hz), 7.54 (d, lH, J = 2.0 Hz), 7.36-7.01 (m, liH), 3.83 (t, 2H, J = 6.5 Hz), 3.51 (s, 4H), 3.04 (m, 2H), 2.87 (m, 2H), 2.45 (t, 2H, J = 6.7 Hz), 1.75 (quintet, 2H, J = 6.6 Hz); HRMS (Cl with CH, and NH,) m/e 461.2606 (C,ZH3,Nz0 requires 461.2593); CIMS (CH, and NH,) m/e (relative intensity) 463 (12.2), 462 (44.9), 461 (base), 370 (11.2), 369 (28.5), 263 (16.2), 250 (20.2), 238 (13.1), 224 (20.3), 107 (14.3), 105 (14.4), 93 (28.5), 91 (37.2), 79 (14.5).
(0.32 g, 3.0 mmol) and benzyl chloroformate (0.31 g, 1.8 mmol) and stirred at room temperature for 24 h. The organic layer was removed, the aqueous layer extracted with CHzCl, (2 X 50 mL) and the combined organic layers washed with saturated brine (50 mL), Hz0 (50 mL) and dried (Na,SO,). The crude product was flash chromatographed (silica gel; CH,Cl,:EtOAc; 9:l) to give 0.43 g (71%) as a cream amorphous solid: ‘H NMR (CDCl,) 6 7.38-7.24 (m, 5H), 7.11-6.98 (m, 3H), 6.89-6.84 (m, 2H), 6.59-6.54 (m, 2H), 5.52 (s, lH), 5.05 (s, 2H), 4.82 (br t, lH), 3.67 (t, 2H, J = 6.5 Hz), 3.20 (quartet, 2H, J = 6.4 Hz), 3.06 (quartet, 4H, J = 4.4 Hz), 1.79-1.72 (m, 2H); HRMS m/e 402.1925 (C,,H,,N,O, requires 402.1943); EIMS (70 eV) m/e (relative intensity) 402 (21.0), 294 (9.7), 225 (20.7), 224 (base), 211 (13.6), 210 (23.4), 209 (27.6), 107 (10.4), 91 (49.0), 79 (11.4), 77 (10.9), 65 (9.0).
2-Hydroxy-5-[3-(bis[phenylmethyl)amino)propyl]-
2-Hydroxy-5-{3-(N-methylamino]propyl)iminodiben~l
iminodibenml
A vigorously stirred suspension of lithium aluminum hydride (0.23 g, 6.0 mmolj in dry tetrahydrofuran (THF) (5 mL) at 5_“C (ice bath) under argon was treated dropwise with a solution of 7 (0.40 g, 1.0 mmol) in dry THF (5 mL) over a period of 15 min. The mixture was allowed to warm to room temperature and refluxed for 3 h. The reaction mixture was cooled to 5”C, treated dropwise with 5 mL of saturated (NH,),SO,, filtered, and the solid residue washed with hot EtOAc (25 mL). Removal of volatiles gave a light brown oil that was flash chromatographed (silica gel; CHCl,:CH,OH: NH,OH; 75:25:1) to give 0.23 g (83%) as a white amorphous solid (1): ‘H NMR (CD,OD) S 7.04 (m, 2H), 7.01 (d, lH, J = 7.6 Hz), 6.93 (d, lH, J = 7.9 Hz), 6.81 (br dt, lH), 6.56-6.54 (m, 2H), 4.98 (s, 2H), 3.69 (t, 2H, J = 6.5 Hz), 3.06 (s, 4H), 2.57 (t, 2H, J = 7.3 Hz), 2.28 (s, 3H), 1.73 (quintet, ZH, J = 7.0 Hz); HRMS m/e 282.1731 (C,,H,,N,O requires 282.1732); EIMS (70 eV) m/e (relative intensity) 284 (2.7), 283 (24.1), 282 (92.0), 252 (19.4), 251 (82.9), 250 (81.9), 236 (21.4), 225 (25.5), 224 (99.8), 212 (21.0), 211 (base), 210 (52.9), 209 (59.8), 130 (16), 86 (30.0), 84 (43.6), 71 (38.7), 51 (18.6).
(5)
A solution of 4 (4.0 g, 8.7 mmol) in CH,Cl, (60 mL) at 0°C under argon was treated dropwise with trifluoroacetic acid (2.4 mL, 31.2 mmol) followed by a solution of m-chloroperbenzoic acid (4.9 g, 14.10 mmol) in CH,Cl, (95 mL). The mixture was then stirred for 2 h at 5”C, 2 h at room temperature and quenched with aqueous 1M NaHSO, (80 mL). The reaction mixture was treated with Hz0 (250 mL), the pH adjusted to 8 with 2N NaOH, extracted with CHCl, (3 X 100 mL) and dried (Na,S04). The intermediate formate ester was dissolved in CH,OH (60 mL) and hydrolyzed by stirring for 2 h at room temperature with concentrated HCl (85 mL). The mixture was then diluted with Hz0 (500 mL), the pH adjusted to 8 with aqueous 2N NaOH, extracted with CHCl, (4 X 250 mL) and dried (NazSO,). Removal of volatiles and flash chromatography of the crude product (hexane:EtOAc; 85:15) provided 2.54 g (65%) of 5 as a brown viscous oil: ‘H NMR (CDCl,) 6 7.28-7.19 (m, lOH), 7.08 (t, lH, J = 8.3 Hz), 7.00 (t, 2H, J = 6.6 Hz), 6.89-6.82 (m, 2H), 6.57 (t, lH, J = 2.9 Hz), 6.54 (s, lH), 3.64 (t, 2H, J = 6.6 Hz), 3.48 (s, 4H), 2.96-2.92 (m, 2H), 2.88-2.84 (m, 2H), 2.44 (t, 2H, J = 6.8 Hz), 1.72 (quintet, 2H, J = 6.7 Hz), 1.58 (br s, 1H); HRMS m/e 448.6142 (C,,H,,N,O requires
448.6135).
2-Hydroxy-5-[3-aminopropyljiminodibenzyl
(6)
A suspension of 5 (2.54 g, 5.7 mmol), ammonium formate (1.43 g, 22.6 mmol) and Pearlman’s catalyst (0.5 g) in CH,OH (25 mL) was refluxed for 4 h. The catalyst was removed by filtration and the crude product flash chromatographed (silica gel; CHCl,:CH,OH: NH,OH; 40:10:0.2) to afford 1.21 g (80%) of 6 as a cream amorphous solid: ‘H NMR (CD,OD): 6 7.06 (m, 2H), 7.02 (d, lH, J = 7.1 Hz), 6.95 (d, lH, J = 8.2 Hz), 1.97 (br dt, lH), 6.57-6.55 (m, 2H), 4.94 (s, 3H), 3.72 (t, 2H, J = 6.6 Hz), 3.07 (s, 4H), 2.69 (t, 2H, J = 7.0 Hz), 1.72 (quintet, 2H, J = 6.4 Hz); HRMS m/e 268.1577 (C,TH,,N,O requires 268.1576); EIMS (70 eV) m/e (relative intensity) 269 (12.9), 268 (50.7), 251 (25.6), 250 (26.9), 225 (22.4), 224 (base), 211 (31.3), 210 (31.3), 209 (39.9), 180 (12.3), 91 (11.7), 77 (12.5).
(8)
5-/3-(N-[“C]Methylamino)propyl/iminodiben~l ([“C]DMI) [’ ‘C]Methyl a reaction
iodide was carried by a nitrogen stream and trapped vial containing a solution of 3 (0.8 mg, 3.2 p,mol)
*
1;
q
a
1;
*
Tb
Cl
c
(ip
in in
1:
& WBN, 2
\
NH2
3
2-Hydroxy-S-[3-(N-(benzyloxycarbonyl)amino)propyl]iminodibenzyl
(7)
A solution CH,Cl,:H,O
of 6 (0.40 (10 mL:lO
g, 1.5 mmol) in a two-phase mixture of mL) was treated successively with NazCO,
FIG. 2. Synthesis of desmethyldesipramine conditions: (a) 1 -bromo-3-chloropropane, reflux; (b) dibenzylamine, 140°C; (c) bon (Pearlman’s catalyst), HCOONH,,
(3). Reagents and LiNH,, benzene, 20% Pd(OH), on carCH,OH, reflux.
710
M.
E. Van
Dort
et al.
FIG. 3. Synthesis of HDMI (8). Reagents and conditions: (a) POCl,, DMF, 95°C; (b) (1) me chloroperbenzoic acid, (2) concentrated HCL, room temperature; (c) 20% Pd(OH),, HCOONH,, CH,OH,reflux; (d) PhCHzOCOCY Na,CO,, H,O:CH,CI, (1:l); (e) LiAIH,, THF, reflux. NHCOOCH2Ph 4
z
anhydrous DMF (0.15 mL) at -30°C to -40°C. The reaction mixture was heated at 85°C for 5 min, then purged with nitrogen for 15 set to remove unreacted [“C]CH,I. The reaction mixture was then diluted with water (0.5 mL) and transferred by remote handling onto a short reversed-phase column (300 mg C-18; Fisher Scientific) that had been prewashed with CH,OH (5 mL) followed by water (10 mL). The extraction column was rinsed with water (1 .O mL) and dried by purging it with nitrogen at 80 psi for 2 min. The crude-labeled product was then eluted off the C-18 column with the HI’LC mobile phase (system A) onto the semipreparative column for purification. The radioactive peak corresponding to [“CIDMI was collected in a sterile vial and the solvent removed by evaporation at 40°C using a nitrogen flow. The residue was formulated in normal saline:EtOH (95:5) (pH = 6.0) and filtered through a 0.2 p,rn alumina filter (Anotop) into a sterile 10 mL multidose vial. The radiochemical and chemical purity was >95%. The decay-corrected radiochemical yield ranged from 18-30% (n = 3), and the specific activity was >1610 Ci/mmol at end of synthesis. The average synthesis time was 45 min (end of bombardment).
transfer hydrogenation conditions (9) to provide the desired analog 3 in 35% overall yield. The normethyl precursor (6) of HDMI was prepared as shown in Figure 3 using methods similar to that described for the synthesis of 2-hydroxyimipramine (1). Formylation of the dibenzylamine analog 2 with POCl,/DMF provided the 2-formyl analog 4 in 98% yield. Conversion of 4 to the phenol 5 was accomplished using a modified Baeyer-Villiger oxidation procedure followed by acid hydrolysis of the formate intermediate. Catalytic debenzylation of 5 was conducted as previously described to give the desmethyl precursor 6 in 11% overall yield. A portion of the desmethyl analog 6 was converted to the desired target compound HDMI (8) for use as a chromatographic standard. Thus, treatment of 6 with benzyl chloroformate gave the carbamate 7, which on subsequent reduction with lithium aluminum hydride afforded HDMI in 59% overall yield. Reversed-phase conditions for chromatographic purification of the radiotracers in a directly injectable solution (using 0.1 M NaH,PO,:EtOH mixtures as the mobile phase) were initially evaluated using several different bonded phases (C-8, C-18, cyano and cation exchange). However, both DMI and HDMI displayed extensive peak tailing as well as poor baseline resolution with respect to their normethyl precursors under these chromatographic conditions. This may have been due to the high lipophilicity or strong basicity (or both) of these compounds. These shortcomings were subsequently overcome by use of an amino column under normal phase conditions (5). [“CIDMI and [“CIHDMI were synthesized by direct N-[“Clmethylation of their respective normethyl precursors with [’ ‘C]CH,I in a 45 min synthesis time from end of bombardment (Fig. 4). The identity of the radiolabeled product was confirmed by radio-TLC and radio-HPLC analysis. The radiotracers were obtained in moderate radiochemical yields (15-30%; end of bombardment and decay corrected) and in high specific activity (>1459 Ci/mmol; end of synthesis) following purification by HPLC. Radiochemical and chemical purities of the tracers were >95%.
2-Hydroxy-5-(3-(N-[‘1C~methylamino)propy1)iminodibenzyt ([“C]HDMI) In a similar manner, [“Clmethylation of 6 (0.8 mg, 3.0 p,mol) followed by HPLC purification (system B) afforded [’ ‘CIHDMI in 15-23% (n = 3) decay-corrected radiochemical yield and specific activity >1459 Ci/mmol (end of synthesis). The average synthesis time was 45 min (end of bombardment).
RESULTS
AND
4 (HDMI)
DISCUSSION
The normethyl precursor (3) of DMI was synthesized from commercially available iminodibenzyl as shown in Figure 2. Treatment of the lithium salt of iminodibenzyl with I-bromo-3chloropropane afforded 1 in 65% yield (7). C on d ensation of 1 with dibenzylamine gave 2, which was deprotected by catalytic debenzylation under
“CH31 DMF,
FIG.
4. Synthesis
of [“CIDMI
and
85 ‘C,
5 min
[“CIHDMI. R=H;
3
R = H ;
[“C]DMI
R=OH;
fi
R = OH ; [“C]HDMI
Synthesis
of r ‘C-Labeled
Desipramine
711
In conclusion, the antidepressant DMI and its principal metabolite HDMI have been radiolabeled with “C with radiochemical yields and specific activities suitable for in viva PET studies. This development would also allow investigation of the short-term pharmacokinetics and in viva NET occupancy of these compounds in humans that could help improve our understanding of the mechanism of action of antidepressant drugs (11). We thank the staff of the Medical Cyclotron Michigan for the production of ’ ‘C and the tory for the use of their facilities. This work Instirutes of Health grants HL 27555, HL
Facility at the University of Phoenix Memorial Laborawas supported by National 47543 and NS 25656.
References M., Chen Y.-Y. and Fishpaugh J. R. (1991) A facile synthetic 1. Adamczyk route to hydroxy metaholites of tricyclic antidepressants. Org. Prep. Proc. Jnt. 23, 365-372. 2. Bohm M., La Resee K., Schwinger R. H. G. and Erdmann E. (1995) Evtdence for reductton of norepinephrme uptake sites in the failing human heart. .I. Am. Call. Cardiol. 25, 1466153. transporter of the 3. Bonisch H. and Bruss M. (1994) Th e noradrenaline neuronal plasma membrane. Ann. N. Y. Acad. Sci. 733, 193-202. C., Langstrom B., Pike V. W. and Coenen H. H. (1987) 4. Crouzel Recommendations for a practical production of [“Clmethyl iodide. Appl. R&at. Jsot. 36, 601-603. H., Goethals I’., Cattoir H., Bogaert M., Vandewalle T., 5. Denutte V
production in high yield of N’-(4-[“Clmethyl)-imipramine. J. Nucl. Med. 24, 1185-1187. R. W. (1981) Enhancement of monoaminergic transmisston by 6. Fuller antidepressant drugs. In Antidepressants: Nemochemical, Behavioral, and Clinical Perspectives (Edited by Enna S. J., Mallick J. B. and Richelson E.), pp. 1-13. Raven Press, New York. 7. Geigy J. R. (1963) 5Substituted 5H-dibenz[b,flazepines and their lO,ll-dihydro derivatives. Chem. Abstr. 59, 10011a. 8 Javaid J. I., Perel J. M. and D avis J. M. (1979) Inhibition of biogenic amines uptake by imipramine, desipramine, 2-OH-imipramine and 2-OH-desipramme in rat brain. Life Sci. 24, 21-28. Purchase C. F. II and Gael 0. P. (1991) A new synthesis of primary aliphatic amines by N,N-didebenzylation: Synthesis of a ptrmenol ((X-845) metabolite J. Org. Chem. 56, 4577459. Raisman R., Sette M., Pimoule C., Briley M. and Langer S. Z. (1982) Hugh affinity [sH]desipramine binding in the peripheral and central nervous system: A specific site associated with the neuronal uptake of noradrenaline. Eur. J. Pharmacol. 78, 345-351.
Stern
S. L., Cooper
T. B., Nelson
L. D., Johnson
M. H. and Suckow
R. F. (1996) Z-Hydroxydesipramine and desipramine plasma levels: How are they related to antidepressant response? Int. Clin. Psychopharmad. 11, 219-227. Still W. C., Kahn M. and Mitra A. (1978) Rapid chromatographic technique for preparative separations with moderate resolution. .J. Org. Chem. 43, 2923-2925. Trendelenberg U. (1991) The TIPS lecture: Functional aspects of the neuronal uptake of noradrenaline. Trends Pharmacol. Set. 12, 334-337. 14. Ungerer M., Chlisralla A. and Richardt G. (1996) Upregulation <>f cardiac uptake 1 carrier m ischemic and nonischemic rat heart. Circ. Res. 78, 1037-1043.
Medicine &. Biology, Vol. 0 1997 Elsevier Science
24, pp. 707-71 Inc.
I, I997
ISSN
0969~8051/97/$17.00 + 0.00 PII SO969-8051(97)00109-1
ELSEVIER
Synthesis of “C-Labeled Desipramine and Its Metabolite 2-Hydroxydesipramine: Potential Radiotracers for PET Studies of the Norepinephrine Transporter Marcian E. Van DOG, * Jae-Hoon Kim, f Louis Tluczek DIVISION
OF NUCLEAR
MEDICINE,
and
LJEPARTMENT OF INTERNAL MEDICINE, UNIVERSITY ANN ARBOR, Ml 48109-0552, USA
Donald M. Wiehnd
OF MICHIGAN
MEDICAL
SCHOOL,
and its principal metabolite 2shydroxydesipramine ABSTRACT. The antidepressant desipramine (DMI) (HDMI) have been radiolabeled with “C for PET studies. The normethyl precursors of DMI and HDMI were synthesized from iminodibenzyl in 35% and 11% overall yield, respectively. Direct methylation of the normethyl precursor with [ “C]CH,I, f o 11owed by HPLC purification, provided [“CIDMI and [“CIHDMI in 18-30% and 15-23% decay-corrected radiochemical yields, respectively, in a 45 min synthesis time from end of bombardment. The specific activities of the two radiotracers were > I459 Ci/mmol at the end of synthesis. [ “C]DMI and [“CIHDMI h ave potential utility as PET radiotracers for the norepinephrine transporter. NUCL MED BIOL 24;8:707-711, 1997. 0 1997 Elsevier Science Inc. KEY WORDS. Desipramine, transporter, Positron emission
2-Hydroxydesipramine, tomography
INTRODUCTION The neuronal membrane-bound norepinephrine transporter (NET) plays a critical role in the regulation of synaptic concentrations of norepinephrine (NE) in the central and peripheral nervous system (3, 13). Abnormalities of the NE reuptake system have been implicated in clinical depression (6) and in cardiac failure (2). More recently, Ungerer et al. (14) have reported upregulation of NET in a perfused, ischemic rat heart model. A radiotracer specific for NET could therefore be potentially useful for studying the role of the NE reuptake system in these disease states. Desipramine (DMI), a well-known tricyclic antidepressant, is a selective and high-affinity inhibitor of NE reuptake (K, = 2.4 nM; Fig. 1) (8). Specific, high-affinity binding sites for [‘H]DMI in rat have been demonstrated in tissues receiving dense noradrenergic input, such as heart, submaxillary gland, cortex and hypothalamus (10). 2-Hydroxydesipramine (HDMI), a metabolite of DMI, also retains the excellent NET selectivity profile of DMI, albeit with a slightly lower inhibition of NE reuptake (K, = 7.8 nM; Fig. 1) (8). Furthermore, the increased hydrophilicity of HDMI over DMI could result in a lower in viva nonspecific binding for the former derivative, which would be an important advantage in imaging studies. These two candidates were therefore chosen as lead com* Address correspondence to: Marcian E. Van Don, Ph.D., Division of Nuclear Medicine, 3480 Kresge 111 Building, University of Michigan Medical School, Ann Arbor, MI 48109-0552. Portions of this work have been reported previously in abstract form: Van Don M. E., Tluczek L., Sherman I’. S., Raffel D. M. and Wieland D. M. (1995) Synthesis of carbon-l 1 labeled desipramine: A potential radiotracer for the cardiac norepinephrme transporter. J. Label. Cmpd. Radiopham. 37, 251-252; Van Don M. E., Kim J. H., Sh erman I’. S., Tluczek L. and Wieland D. M. (1996) Carbon- 11 labeled 2-hydroxydesipramine: A potential radiotracer for the cardiac norepinephrine transporter. J. Nucl. Med. 37, 50 I’. t Present address: Department of Polymer Science and Engineering, Kum-Oh National University of Technology, 188 Shinpyung, Kumi, Kyunghuk 7 30-070, Korea. Received 2 February 1997. Accepted 5 May 1997.
Carbon-
11, Radiotracer,
Antidepressant,
Norepinephrine
pounds for initial NET radiotracer development. In this paper we describe the synthesis of “C-labeled DMI and its metabolite HDMI for in ho positron emission tomography (PET) studies.
MATERIALS
AND
METHODS
Melting points were determined in open capillary tubes using a Thomas Hoover melting point apparatus and are uncorrected. ‘H nuclear magnetic resonance (NMR) spectra were obtained on a Bruker WM 300 MHz spectrometer with tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained on a Finnigan 4021 GCMS/DS (low resolution) or a UG70-250-S (high resolution) instrument. Desipramine hydrochloride was purchased from Sigma Chemical Company (St. Louis, MO). All other chemical reagents were purchased from Aldrich Chemical Company (Milwaukee, WI). Thin-layer chromatography (TLC) was performed on Analtech silica gel GF Uniplates (250 km). Following development, TLC plates were visualized under ultraviolet (UV) light and by spraying with ethanolic phosphomolybdic acid reagent with subsequent heating (130-l 50°C). Flash chromatography (12) was performed using Merck silica gel 60 (230-400 mesh).
HPLC
and Radio-HPLC
Analyses
High performance liquid chromatography (HPLC) purification of the radiolabeled product was performed on a Phenomenex Selectosil NH, column (5 p,rn particle size, 250 X 10 mm I.D.) plus guard (50 X 10 mm I.D.) eluted with a mixture of hexane:isopropanol: methanol:diethylamine in either of the following volume-to-volume ratios: 580:10:10:0.6 (system A) or 600:80:80:0.7 (system B). UV absorbance (254 nm) and radioactivity were monitored by an in-line UV absorbance and y-detector, respectively. With a flow rate of 5 mL/min (system A), the retention time of [“CIDMI was 10.2 min and that of the desmethyl precursor 3 was 18.7 min. [“CIHDMI was purified using system B at a flow rate of 7 mL/min.
708
M.
E. Van
generated calibration curve. Radioactivity measurements tained with a Capintec CRC-12 radioisotope calibrator.
5-(3-Chloropropyljiminodibenzyl
R=H;
DMI
R=OH;
HDMI
NE
5-HT
DMI
2.4
100
6000
HDMI
7.0
180
9400
COMPOUND
FIG. 1. Inhibition constants of DMI and HDMI for monoamines. Asterisk indicates data from Javaid et al. (8).
The retention times for HDMI (8) and its desmethyl precursor 6 were 11.7 and 16.9 min, respectively, under these conditions. Radio-HPLC analyses for radiochemical purity and specific activity determination were conducted using a Phenomenex Ultremex 5 CN column (5 pm particle size, 50 X 4.6 mm I.D.) with CH,CN:CH,OH:lO mM KzHPO, (pH = 7.0) (60:15:25) as the mobile phase. A flow rate of 0.5 mL/min was utilized with UV detection at 254 nm. The retention times of 3, DMI, 6 and HDMI were 6.9, 10.4, 6.2 and 8.7 min, respectively, under these conditions.
Radio-TLC
Analyses
Radio-TLC analyses were performed on Analtech Spice Plates (Unibond NH,) using a mixture of hexane:isopropanol:methanol: diethylamine in either of the following ratios: 93:3.5:3.5:1 (system C) or 90:5:5:1 (system D). The labeled compound was co-spotted with the authentic unlabeled compound prior to development. TLC plates were scanned for radioactivity using a Berthold Model LB 2832 TLC-Linear Analyzer equipped with a model LB 500 Data Acquisition System. The Ri values for the desmethyl derivative 3 and DMI were 0.13 and 0.30, respectively, using system C, and the values for 6 and HDMI were 0.17 and 0.38, respectively (system D).
Production
of [“C]CHJ
[“C]CO, was produced with a biomedical cyclotron (CS-30 accelerator; Cyclotron Corporation) by the i4N(p,a)“C reaction using high-purity nitrogen gas in an aluminum target. [“C]CO, was converted to [“C]CH,OH by LiAIH, reduction and treated with aqueous HI at reflux (4) to generate [“C]CH,I. The initial activity of the [“C]CO, produced at bombardment was estimated from a previously determined calibration curve of ‘iC activity produced versus irradiation times in the same target. Estimates of initial target activity from this curve were used to determine overall radiochemical yields.
Specific The peak quantitated
Activity area by
Determinations corresponding UV absorbance
to
the radiolabeled at 254 nm using
product was a previously
Dort
et al.
were
ob-
(I)
The title compound was synthesized by a modification of the published procedure (7) as follows. A stirred solution of iminodibenzyl (6.0 g, 30.7 mmol) and I-bromo-3chloropropane (6.3 g, 40.0 mmol) in dry benzene (240 mL) was warmed under an argon atmosphere to 55°C and treated portionwise over a 30 min period with lithium amide (0.77 g, 33.7 mmol). Following reflux for 24 h, the cooled benzene solution was washed with Hz0 (3 X 100 mL) and dried (Na,S04). Removal of volatiles gave an oil that was dissolved in hexane and filtered to remove unreacted iminodibenzyl. Flash chromatography (silica gel; hexane:EtOAc; 22:l) gave 5.4 g (65%) of 1 as a colorless oil: ‘H NMR (CDCl,) 6 7.16-7.02 (m, 6H), 6.93 (t, lH, J = 7.2 Hz), 6.92 (t, lH, J = 7.1 Hz), 3.90 (t, 2H, J = 6.5 Hz), 3.56 (t, 2H, J = 6.4 Hz), 3.16 (s, 4H), 2.04 (quintet, 2H, J = 6.4 Hz); HRMS m/e 271.1119 (C,zH,,CIN requires 271.1128); EIMS (70 eV) m/e (relative intensity) 273 (13.2), 271 (28.5), 209 (23), 208 (base), 194 (19.9), 193 (43), 192 (lo), 178 (6.4), 165 (6.7) 91 (6.9).
5-[3-{Bis(phenylmethyl)aminoJpropyl]iminodibenzy1
(2)
A mixture of 1 (4.8 g, 17.7 mmol) and dibenzylamine (10.5 g, 53.2 mmol) was heated at 140°C for 72 h under an argon atmosphere. The cooled mixture was diluted with anhydrous Et,0 (250 mL) and filtered to remove crystalline dibenzylamine hydrochloride. Flash chromatography of the crude product (silica gel; hexane:EtOAc; 35:l) provided 4.6 g (60%) of 2 as a colorless oil that gave white crystals on standing: mp 69-71°C; ‘H NMR (CDCL,) 6 7.29-7.16 (m, lOH), 7.12-6.99 (m, 6H), 6.88 (t, lH, J = 7.2 Hz), 6.87 (t, lH, J = 7.2 Hz), 3.70 (t, 2H, J = 6.6 Hz), 3.48 (s, 4H), 2.94 (s, 4H), 2.44 (t, 2H, J = 6.9 Hz), 1.73 (quintet, 2H, J = 6.8 Hz); HRMS m/e 432.2561 (C,,H,,N, requires 432.2565); EIMS (70 eV) m/e (relative intensity) 432 (1.7), 341 (32.8), 236 (20.7), 235 (77.5), 234 (46.2) 222 (56.3) 210 (21.5) 208 (30.1) 193 (30.2), 146 (42.3), 91 (base).
5-(3.Aminopropyl)iminodibenzyl
(3)
A suspension of 2 (0.29 g, 0.67 mmol), ammonium formate (0.17 g, 2.7 mmol) and 0.05 g of 20% Pd(OH), on carbon (Pearlman’s catalyst) in CH,OH (3 mL) was refluxed for 2 h. The catalyst was removed by filtration and the crude product flash chromatographed (silica gel; CHCl,:CH,OH:NH,OH; 9:l:O.l) to afford 151 mg (89.5%) of 3 as a yellow oil (7): ‘H NMR (CDCl,) 6 7.14-7.06 (m, 6H), 6.91 (t, lH, J = 7.1 Hz), 6.90 (t, lH, J = 7.3 Hz), 3.79 (t, 2H, J = 6.7 Hz), 3.15 (s, 4H), 2.71 (t, 2H, J = 7.0 Hz), 1.70 (quintet, 2H, J = 6.9 Hz), 1.13 (br s, 2H); HRMS m/e 252.1626 (C,zH,,N, requires 252.1626); EIMS (70 eV) m/e (relative intensity) 252 (39), 235 (45.3), 234 (36.5), 209 (26), 208 (base), 195 (43.1), 194 (44), 193 (67.1), 192 (23.2), 179 (13.8), 178 (14.6), 165 (14.9), 130 (14.5), 91 (15.2).
2-Formyl-5-[3-{bis(phenylmethyl)amino)ppropyl]iminodibenql (4) Phosphorous oxychloride (2.3 mL, 24.7 mmol) was added dropwise to dimethylformamide (DMF) (4.6 mL, 59.4 mmol) at 0°C under argon and the resulting mixture was allowed to warm to room
Synthesis
of ’ ‘C-Labeled
Desipramine
709
temperature over a 10 min period. The dibenzylamine derivative 2 (4.1 g, 9.5 mmol) was then added and the mixture heated at 95°C for 4 h. The reaction was quenched with ice-water (60 mL), made basic (pH = 12) with aqueous 2N NaOH, extracted with CHCl, (4 X 100 mL) and the combined organic layers dried (Na,S04). Flash chromatography (silica gel; hexane:EtOAc; 9:l) gave 4.25 g (97%) as a light yellow oil: ‘H NMR (CDCL,) 6 9.84 (s, lH), 7.64 (dd, lH, J = 8.4 Hz, 2.1 Hz), 7.54 (d, lH, J = 2.0 Hz), 7.36-7.01 (m, liH), 3.83 (t, 2H, J = 6.5 Hz), 3.51 (s, 4H), 3.04 (m, 2H), 2.87 (m, 2H), 2.45 (t, 2H, J = 6.7 Hz), 1.75 (quintet, 2H, J = 6.6 Hz); HRMS (Cl with CH, and NH,) m/e 461.2606 (C,ZH3,Nz0 requires 461.2593); CIMS (CH, and NH,) m/e (relative intensity) 463 (12.2), 462 (44.9), 461 (base), 370 (11.2), 369 (28.5), 263 (16.2), 250 (20.2), 238 (13.1), 224 (20.3), 107 (14.3), 105 (14.4), 93 (28.5), 91 (37.2), 79 (14.5).
(0.32 g, 3.0 mmol) and benzyl chloroformate (0.31 g, 1.8 mmol) and stirred at room temperature for 24 h. The organic layer was removed, the aqueous layer extracted with CHzCl, (2 X 50 mL) and the combined organic layers washed with saturated brine (50 mL), Hz0 (50 mL) and dried (Na,SO,). The crude product was flash chromatographed (silica gel; CH,Cl,:EtOAc; 9:l) to give 0.43 g (71%) as a cream amorphous solid: ‘H NMR (CDCl,) 6 7.38-7.24 (m, 5H), 7.11-6.98 (m, 3H), 6.89-6.84 (m, 2H), 6.59-6.54 (m, 2H), 5.52 (s, lH), 5.05 (s, 2H), 4.82 (br t, lH), 3.67 (t, 2H, J = 6.5 Hz), 3.20 (quartet, 2H, J = 6.4 Hz), 3.06 (quartet, 4H, J = 4.4 Hz), 1.79-1.72 (m, 2H); HRMS m/e 402.1925 (C,,H,,N,O, requires 402.1943); EIMS (70 eV) m/e (relative intensity) 402 (21.0), 294 (9.7), 225 (20.7), 224 (base), 211 (13.6), 210 (23.4), 209 (27.6), 107 (10.4), 91 (49.0), 79 (11.4), 77 (10.9), 65 (9.0).
2-Hydroxy-5-[3-(bis[phenylmethyl)amino)propyl]-
2-Hydroxy-5-{3-(N-methylamino]propyl)iminodiben~l
iminodibenml
A vigorously stirred suspension of lithium aluminum hydride (0.23 g, 6.0 mmolj in dry tetrahydrofuran (THF) (5 mL) at 5_“C (ice bath) under argon was treated dropwise with a solution of 7 (0.40 g, 1.0 mmol) in dry THF (5 mL) over a period of 15 min. The mixture was allowed to warm to room temperature and refluxed for 3 h. The reaction mixture was cooled to 5”C, treated dropwise with 5 mL of saturated (NH,),SO,, filtered, and the solid residue washed with hot EtOAc (25 mL). Removal of volatiles gave a light brown oil that was flash chromatographed (silica gel; CHCl,:CH,OH: NH,OH; 75:25:1) to give 0.23 g (83%) as a white amorphous solid (1): ‘H NMR (CD,OD) S 7.04 (m, 2H), 7.01 (d, lH, J = 7.6 Hz), 6.93 (d, lH, J = 7.9 Hz), 6.81 (br dt, lH), 6.56-6.54 (m, 2H), 4.98 (s, 2H), 3.69 (t, 2H, J = 6.5 Hz), 3.06 (s, 4H), 2.57 (t, 2H, J = 7.3 Hz), 2.28 (s, 3H), 1.73 (quintet, ZH, J = 7.0 Hz); HRMS m/e 282.1731 (C,,H,,N,O requires 282.1732); EIMS (70 eV) m/e (relative intensity) 284 (2.7), 283 (24.1), 282 (92.0), 252 (19.4), 251 (82.9), 250 (81.9), 236 (21.4), 225 (25.5), 224 (99.8), 212 (21.0), 211 (base), 210 (52.9), 209 (59.8), 130 (16), 86 (30.0), 84 (43.6), 71 (38.7), 51 (18.6).
(5)
A solution of 4 (4.0 g, 8.7 mmol) in CH,Cl, (60 mL) at 0°C under argon was treated dropwise with trifluoroacetic acid (2.4 mL, 31.2 mmol) followed by a solution of m-chloroperbenzoic acid (4.9 g, 14.10 mmol) in CH,Cl, (95 mL). The mixture was then stirred for 2 h at 5”C, 2 h at room temperature and quenched with aqueous 1M NaHSO, (80 mL). The reaction mixture was treated with Hz0 (250 mL), the pH adjusted to 8 with 2N NaOH, extracted with CHCl, (3 X 100 mL) and dried (Na,S04). The intermediate formate ester was dissolved in CH,OH (60 mL) and hydrolyzed by stirring for 2 h at room temperature with concentrated HCl (85 mL). The mixture was then diluted with Hz0 (500 mL), the pH adjusted to 8 with aqueous 2N NaOH, extracted with CHCl, (4 X 250 mL) and dried (NazSO,). Removal of volatiles and flash chromatography of the crude product (hexane:EtOAc; 85:15) provided 2.54 g (65%) of 5 as a brown viscous oil: ‘H NMR (CDCl,) 6 7.28-7.19 (m, lOH), 7.08 (t, lH, J = 8.3 Hz), 7.00 (t, 2H, J = 6.6 Hz), 6.89-6.82 (m, 2H), 6.57 (t, lH, J = 2.9 Hz), 6.54 (s, lH), 3.64 (t, 2H, J = 6.6 Hz), 3.48 (s, 4H), 2.96-2.92 (m, 2H), 2.88-2.84 (m, 2H), 2.44 (t, 2H, J = 6.8 Hz), 1.72 (quintet, 2H, J = 6.7 Hz), 1.58 (br s, 1H); HRMS m/e 448.6142 (C,,H,,N,O requires
448.6135).
2-Hydroxy-5-[3-aminopropyljiminodibenzyl
(6)
A suspension of 5 (2.54 g, 5.7 mmol), ammonium formate (1.43 g, 22.6 mmol) and Pearlman’s catalyst (0.5 g) in CH,OH (25 mL) was refluxed for 4 h. The catalyst was removed by filtration and the crude product flash chromatographed (silica gel; CHCl,:CH,OH: NH,OH; 40:10:0.2) to afford 1.21 g (80%) of 6 as a cream amorphous solid: ‘H NMR (CD,OD): 6 7.06 (m, 2H), 7.02 (d, lH, J = 7.1 Hz), 6.95 (d, lH, J = 8.2 Hz), 1.97 (br dt, lH), 6.57-6.55 (m, 2H), 4.94 (s, 3H), 3.72 (t, 2H, J = 6.6 Hz), 3.07 (s, 4H), 2.69 (t, 2H, J = 7.0 Hz), 1.72 (quintet, 2H, J = 6.4 Hz); HRMS m/e 268.1577 (C,TH,,N,O requires 268.1576); EIMS (70 eV) m/e (relative intensity) 269 (12.9), 268 (50.7), 251 (25.6), 250 (26.9), 225 (22.4), 224 (base), 211 (31.3), 210 (31.3), 209 (39.9), 180 (12.3), 91 (11.7), 77 (12.5).
(8)
5-/3-(N-[“C]Methylamino)propyl/iminodiben~l ([“C]DMI) [’ ‘C]Methyl a reaction
iodide was carried by a nitrogen stream and trapped vial containing a solution of 3 (0.8 mg, 3.2 p,mol)
*
1;
q
a
1;
*
Tb
Cl
c
(ip
in in
1:
& WBN, 2
\
NH2
3
2-Hydroxy-S-[3-(N-(benzyloxycarbonyl)amino)propyl]iminodibenzyl
(7)
A solution CH,Cl,:H,O
of 6 (0.40 (10 mL:lO
g, 1.5 mmol) in a two-phase mixture of mL) was treated successively with NazCO,
FIG. 2. Synthesis of desmethyldesipramine conditions: (a) 1 -bromo-3-chloropropane, reflux; (b) dibenzylamine, 140°C; (c) bon (Pearlman’s catalyst), HCOONH,,
(3). Reagents and LiNH,, benzene, 20% Pd(OH), on carCH,OH, reflux.
710
M.
E. Van
Dort
et al.
FIG. 3. Synthesis of HDMI (8). Reagents and conditions: (a) POCl,, DMF, 95°C; (b) (1) me chloroperbenzoic acid, (2) concentrated HCL, room temperature; (c) 20% Pd(OH),, HCOONH,, CH,OH,reflux; (d) PhCHzOCOCY Na,CO,, H,O:CH,CI, (1:l); (e) LiAIH,, THF, reflux. NHCOOCH2Ph 4
z
anhydrous DMF (0.15 mL) at -30°C to -40°C. The reaction mixture was heated at 85°C for 5 min, then purged with nitrogen for 15 set to remove unreacted [“C]CH,I. The reaction mixture was then diluted with water (0.5 mL) and transferred by remote handling onto a short reversed-phase column (300 mg C-18; Fisher Scientific) that had been prewashed with CH,OH (5 mL) followed by water (10 mL). The extraction column was rinsed with water (1 .O mL) and dried by purging it with nitrogen at 80 psi for 2 min. The crude-labeled product was then eluted off the C-18 column with the HI’LC mobile phase (system A) onto the semipreparative column for purification. The radioactive peak corresponding to [“CIDMI was collected in a sterile vial and the solvent removed by evaporation at 40°C using a nitrogen flow. The residue was formulated in normal saline:EtOH (95:5) (pH = 6.0) and filtered through a 0.2 p,rn alumina filter (Anotop) into a sterile 10 mL multidose vial. The radiochemical and chemical purity was >95%. The decay-corrected radiochemical yield ranged from 18-30% (n = 3), and the specific activity was >1610 Ci/mmol at end of synthesis. The average synthesis time was 45 min (end of bombardment).
transfer hydrogenation conditions (9) to provide the desired analog 3 in 35% overall yield. The normethyl precursor (6) of HDMI was prepared as shown in Figure 3 using methods similar to that described for the synthesis of 2-hydroxyimipramine (1). Formylation of the dibenzylamine analog 2 with POCl,/DMF provided the 2-formyl analog 4 in 98% yield. Conversion of 4 to the phenol 5 was accomplished using a modified Baeyer-Villiger oxidation procedure followed by acid hydrolysis of the formate intermediate. Catalytic debenzylation of 5 was conducted as previously described to give the desmethyl precursor 6 in 11% overall yield. A portion of the desmethyl analog 6 was converted to the desired target compound HDMI (8) for use as a chromatographic standard. Thus, treatment of 6 with benzyl chloroformate gave the carbamate 7, which on subsequent reduction with lithium aluminum hydride afforded HDMI in 59% overall yield. Reversed-phase conditions for chromatographic purification of the radiotracers in a directly injectable solution (using 0.1 M NaH,PO,:EtOH mixtures as the mobile phase) were initially evaluated using several different bonded phases (C-8, C-18, cyano and cation exchange). However, both DMI and HDMI displayed extensive peak tailing as well as poor baseline resolution with respect to their normethyl precursors under these chromatographic conditions. This may have been due to the high lipophilicity or strong basicity (or both) of these compounds. These shortcomings were subsequently overcome by use of an amino column under normal phase conditions (5). [“CIDMI and [“CIHDMI were synthesized by direct N-[“Clmethylation of their respective normethyl precursors with [’ ‘C]CH,I in a 45 min synthesis time from end of bombardment (Fig. 4). The identity of the radiolabeled product was confirmed by radio-TLC and radio-HPLC analysis. The radiotracers were obtained in moderate radiochemical yields (15-30%; end of bombardment and decay corrected) and in high specific activity (>1459 Ci/mmol; end of synthesis) following purification by HPLC. Radiochemical and chemical purities of the tracers were >95%.
2-Hydroxy-5-(3-(N-[‘1C~methylamino)propy1)iminodibenzyt ([“C]HDMI) In a similar manner, [“Clmethylation of 6 (0.8 mg, 3.0 p,mol) followed by HPLC purification (system B) afforded [’ ‘CIHDMI in 15-23% (n = 3) decay-corrected radiochemical yield and specific activity >1459 Ci/mmol (end of synthesis). The average synthesis time was 45 min (end of bombardment).
RESULTS
AND
4 (HDMI)
DISCUSSION
The normethyl precursor (3) of DMI was synthesized from commercially available iminodibenzyl as shown in Figure 2. Treatment of the lithium salt of iminodibenzyl with I-bromo-3chloropropane afforded 1 in 65% yield (7). C on d ensation of 1 with dibenzylamine gave 2, which was deprotected by catalytic debenzylation under
“CH31 DMF,
FIG.
4. Synthesis
of [“CIDMI
and
85 ‘C,
5 min
[“CIHDMI. R=H;
3
R = H ;
[“C]DMI
R=OH;
fi
R = OH ; [“C]HDMI
Synthesis
of r ‘C-Labeled
Desipramine
711
In conclusion, the antidepressant DMI and its principal metabolite HDMI have been radiolabeled with “C with radiochemical yields and specific activities suitable for in viva PET studies. This development would also allow investigation of the short-term pharmacokinetics and in viva NET occupancy of these compounds in humans that could help improve our understanding of the mechanism of action of antidepressant drugs (11). We thank the staff of the Medical Cyclotron Michigan for the production of ’ ‘C and the tory for the use of their facilities. This work Instirutes of Health grants HL 27555, HL
Facility at the University of Phoenix Memorial Laborawas supported by National 47543 and NS 25656.
References M., Chen Y.-Y. and Fishpaugh J. R. (1991) A facile synthetic 1. Adamczyk route to hydroxy metaholites of tricyclic antidepressants. Org. Prep. Proc. Jnt. 23, 365-372. 2. Bohm M., La Resee K., Schwinger R. H. G. and Erdmann E. (1995) Evtdence for reductton of norepinephrme uptake sites in the failing human heart. .I. Am. Call. Cardiol. 25, 1466153. transporter of the 3. Bonisch H. and Bruss M. (1994) Th e noradrenaline neuronal plasma membrane. Ann. N. Y. Acad. Sci. 733, 193-202. C., Langstrom B., Pike V. W. and Coenen H. H. (1987) 4. Crouzel Recommendations for a practical production of [“Clmethyl iodide. Appl. R&at. Jsot. 36, 601-603. H., Goethals I’., Cattoir H., Bogaert M., Vandewalle T., 5. Denutte V
production in high yield of N’-(4-[“Clmethyl)-imipramine. J. Nucl. Med. 24, 1185-1187. R. W. (1981) Enhancement of monoaminergic transmisston by 6. Fuller antidepressant drugs. In Antidepressants: Nemochemical, Behavioral, and Clinical Perspectives (Edited by Enna S. J., Mallick J. B. and Richelson E.), pp. 1-13. Raven Press, New York. 7. Geigy J. R. (1963) 5Substituted 5H-dibenz[b,flazepines and their lO,ll-dihydro derivatives. Chem. Abstr. 59, 10011a. 8 Javaid J. I., Perel J. M. and D avis J. M. (1979) Inhibition of biogenic amines uptake by imipramine, desipramine, 2-OH-imipramine and 2-OH-desipramme in rat brain. Life Sci. 24, 21-28. Purchase C. F. II and Gael 0. P. (1991) A new synthesis of primary aliphatic amines by N,N-didebenzylation: Synthesis of a ptrmenol ((X-845) metabolite J. Org. Chem. 56, 4577459. Raisman R., Sette M., Pimoule C., Briley M. and Langer S. Z. (1982) Hugh affinity [sH]desipramine binding in the peripheral and central nervous system: A specific site associated with the neuronal uptake of noradrenaline. Eur. J. Pharmacol. 78, 345-351.
Stern
S. L., Cooper
T. B., Nelson
L. D., Johnson
M. H. and Suckow
R. F. (1996) Z-Hydroxydesipramine and desipramine plasma levels: How are they related to antidepressant response? Int. Clin. Psychopharmad. 11, 219-227. Still W. C., Kahn M. and Mitra A. (1978) Rapid chromatographic technique for preparative separations with moderate resolution. .J. Org. Chem. 43, 2923-2925. Trendelenberg U. (1991) The TIPS lecture: Functional aspects of the neuronal uptake of noradrenaline. Trends Pharmacol. Set. 12, 334-337. 14. Ungerer M., Chlisralla A. and Richardt G. (1996) Upregulation <>f cardiac uptake 1 carrier m ischemic and nonischemic rat heart. Circ. Res. 78, 1037-1043.