performing in oivo exploration of the dopamine uptake system by SPECT. We describe here the synthesis of halogenated analogues of the diphenyl piperazine GBR as potential ligands for studying the presynaptic dopamine carrier by SPECT. According to the comparative study of different GBR analogues carried out by Van der Zee et al. (1980), we decided to develop unsaturated compounds because of their enhanced dopamine uptake inhibiting activities. Moreover, Bonnet and Costentin (1986) showed that unsaturated triatiated GBR 12783 is one of the most selective dopamine uptake inhibitors described to date.

Nucl.Med. Bid.Vol.19, No. 5,pp.597-500,1992 hf.J. Radiat. Appl. Instrum. Part B 0 Pergamon Press Ltd 1992. Printed in Great Britain 0883-2897192 $5.00 + 0.00

Synthesis and In Vitro Binding Properties of Halogenated Analogues of GBR as New Dopamine Uptake Carrier Ligands

Materials and Methods Synthesis

C. FOULON’, L. GARREAU2, S. CHALON’, G. DESPLANCHES’, Y. FRANGIN’, J.-C. BESNARD’, J. L. BAULIEU’ and D. GUILLOTEAU’” ‘INSERM U 316, 2, Bd Tonnelle, 37044 Tours Cedex, 2Laboratoire de Biophysique Pharmaceutique, 2b Bd Tonnellt, 37032 Tours Cedex and ‘Compagnie Cis Bio International, BP 32 91192, Gif-stir-Yvette Cedex, France (Received 10 September

1991)

We present the original synthesis of two halogenated analogues of the diphenyl piperazine GBR, bromo-GBR and iodo-GBR, as new dopamine uptake carrier ligands. The derivatives were purified by HPLC and chemically characterized. Bromo-GBR and iodo-GBR are potent inhibitors of [‘HIGBR 12935 binding to rat striatal membrane, with K, values of 116 and 113 nM, respectively. We prepared iodoGBR labeled with iodide-125 from the brominated derivative and concluded that [rz31]iodo-GBR could be a potential tool to explore the in vivo dopamine uptake carrier.

Introduction In uiuo studies of the dopamine uptake system in humans would be of great interest in the understanding of neurological and psychiatrical diseases such as Parkinson’s disease and schizophrenia involving central dopaminergic systems. For this purpose, a small number of ligands were recently developed for positron emission tomography (PET) explorations. such as I’Clnomifensine (Aauilonius et al., 1987; Ulin et’&, 1989)8nd ‘*F-GBR 13119*(Kilbourn and’Haka; 1988). [“ClNomifensine was first used in an experimental model of hemi-parkinsonism induced in the primate and was shown to be decreased by 80-90% in the lesioned striata thus indicating a loss of dopamine striatal nerve terminals (Leenders et al., 1988). Moreover, in humans suffering from Parkinson’s disease a mean 40% decrease of in vioo binding of [“Clnomifensine was observed (Salmon et al., 1990). Recent data showed that i8F-GBR 13119 binds specifically to the dopamine uptake system in vivo in the monkey (Kilbourn et al., 1989). Binding of this ligand is also markedly decreased in the rat after destruction of the nigro-striatal dopaminergic pathway (Ciliax ef nl., 1990). All of these results show the potential usefulness of such ligands to investigate the in uivo striatal dopaminergic function in pathological situations by’ PET. However. for widespread clinical appication, single photon emission computed‘tomography (SPECT) exploration would be more suitable than PET. Until now, there have been no available ligands for

Flash-chromatography was carried out using silica gel (230-400 mesh) ASTM and TLC were conducted in silica gel plates 60 F,,, , 0.2 mm from Merck. HPLC analyses were performed on a Beckman 331 isocratic liquid chromatograph fitted with a U.V. system, Zorbax TMS 5 pm column with 1% AcCN/phosphate buffer, pH 7.5 (65/35), as eluent. ‘H-NMR spectra were performed on a Briicker AM 300, 200 MHz and i.r. spectra were recorded on a Perkin-Elmer. Phenylmagnesium bromide 3 M in diethyl ether, lithium aluminium hydride 1 M in diethyl ether, 4-bromobenzophenone (lb) and thionyl chloride were obtained from Aldrich Chemical. 4-Cyano- 1-iodobenzene was purchased from Kodak. N-(3-phenylpropenyl)-N-hydroxy ethylpiperazine (3) was prepared according to a previously published method (Van der Zee er a/., 1980) and purified by distillation at 180°C under 4 torr. 4-Iodobenrophenone (la). Under N, atmosphere, 50 mmol of phenylbromide magnesium 3 M in Et,0 (15 mL) and 20 mmol 4-cyano- 1-iodobenzene (4.6 g) were mixed in a coated glass vessel at room temperature for 15 h, and then heated to 40°C for 1 h. Hydrolysis was carried out by adding 50 g of frozen water and 100 mL HCl 12 N: a yellow solid appeared (imine) which was evacuated by filtration on Biichner. The final product was obtained as a yellow solid after extraction in CHCl,. 4-Halogenobenrhydrol (A/2&). 1 mmol of ketone (la/lb) and 2.5 mmol LAH 1 M in Et,0 were mixed in a coated glass vessel under N, atmosphere. The vessel was placed in an ice-water bath (4°C) during introduction of the hydride. Then the mixture was agitated for 10 min and then returned to the cold bath in order to carry out the hydrolysis: 10 mL H,SO, 4N are added dropwise and the product was extracted in Et,O, dried over Na*SO, and evaporated to dryness. These products were added to the piperazine (3) without further purification. X-GBR (4a/4b). 0.50 mmol alcohol (2a/Zb), 1 mmol piperazine (3) and 0.50 mmol SOCl, were heated in a coated glass vessel at 120°C for 1 h. Hydrolysis was carried out by introducing 15 mL H,O and 30 mL NaOH 1 N. The product was extracted in Et,O, dried over Na,SO, and evaporated to dryness. Flash-chromatography was performed in silica gel with absolute MeOH as eluent. Preparation of [“‘Ijiodo GBR

[‘251]IodoGBR was obtained from the brominated derivative, bromo-GBR, by an exchange reaction between bromide and iodide-125 according to Mertens et al. (1987). In vitro binding experiments

Nomifensine maleate was purchased from R.B.I. Bioblock, t3H]GBR 12935 from NEN (spec. act.: 902.8 GBq/ mmol) and mazindol was kindly donated by Sandoz.

597

Technical Note

598

Binding experiments were performed according to Bonnet et al. (1990) with some modifications. Striatal membranes were prepared from brains of Wistar rats. Tissues were homogenized in 10 ~010.32 M sucrose using an Ultra-Turrax (Ultra-Turrax T25). After a 1000~ centrifugation for 10 min at 2°C the pellet was collected,-homogenized and centrifuged as described above. Supernatants were pooled and centrifuged at 17,500g for 30min at 2°C. Pellets were homogenized, suspended in 20~01 of a pH 7.5 bicarbonate buffer and centrifuged at 50,OOOgfor 10min at 2°C. Final pellets were suspended in a small volume of buffer and the protein concentration was determined using bovine serum albumin as standard (Bradford, 1976). Membranes were incubated in the presence of 0.01% bovine serum albumin. Binding assays were run in duplicate in silicone coated test tubes, in a final volume of 4 mL containing 2.4 mL of incubation buffer, 0.4mL of [‘H]GBR 12935, 0.2mL of 0.5% ascorbate or drugs and 1 mL of membrane suspension containing 0.1 mg protein. For the saturation assays, [3H]GBR 12935 was used at 0.1-5 nM concentrations and for the competition assays [‘H]GBR 12935 was used at 1 nM concentration. Samples were incubated at 25°C for 45 min and filtered under reduced pressure on glass fiber filters GF/B (Whatman). Filters were washed 3 times with 4 mL of ice-cold buffer; radioactivity remaining on the filters was measured after addition of a scintillator (Optiphase Highsafe II, LKB) using a beta counter (LKB Rack Beta 1215). Specific binding was calculated by subtracting the non-specific binding, determined in the presence of 10e5 M mazindol, from the total binding.

0/

MgQ +

RC2SUltS Synthesis

The synthesis scheme is shown in Fig. 1. 4-Iodobenzophenone (la). The product is obtained with 43% yield and has a melting point of 78°C. Infra-red wavelengths (cm-‘): 3020 (w. ~c_u arom.); 1600 (s, v-). R, (TLC) in MeOH = 0.60. 4-Halogenobenzhydrol @u/Z&). (2a) is a yellow oil obtained with 67% yield whereas (2b) is a whitish solid, m.p. of 72°C obtained with 68% vield. Infra-red wavelennths in cm-’ (Za): 3400 (1, vou); 3020 (w, vc_n arom.); 2920 (f, rc_u aliph.). No band at 16OOcm-‘. (Zb): 3400 (1, van); 3030 (w, vc_n arom.); 2900 (f, vc_n aliph.). No band at 1600 cm-‘. NMR data (Za): 2.76 (d, lH, J,,,, = 2 Hz); 5.70 (d, lH, J,..,_.. nH= 2 Hz); 7.30-7.90 (m, 9H, arom.); (2b): 2.69 (6 1I-LJa.0~ = 2 Hzj; 5.63 (d, II-I, J u,ou = 2 Hz); 7.13-7.41 (m. 9H. arom.). R,(TLCj in MeOH: (2~) = 0.68: (2bf = 0.54. ~ X-GBR (ii&j: Yield (4a) = 10”/,‘(4b) = kiti. NMR data (4a): 2.50-2.65 (m. lOH, piperazine, N-CH,--CH,-); 3.14 (d, 2H, NXH*-CH= )J,,, = 7.0Hz); 3.53 (t, 2H, G-CH, 3J,,,,, = 5.8 Hz); 5.23 (s, IH, CHa); 6.18 (dt, lH, CH,-CH=CH ‘J,,, = 6.9 Hz, ‘J,,, = 16.0 Hz); 6.47 (d, IH, CH,