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Applied Radiation and Isotopes 62 (2005) 737–743

Synthesis, radiosynthesis and in vivo evaluation in mice of

[123I]-(4-fluorophenyl) {1-[2-(4-iodophenyl)ethyl]piperidin-

4-yl}methanone for visualization of the 5-HT2A receptor

with SPECT

P. Blanckaerta,Ã, I. Burvenicha, L. Staelensa, R.A. Dierckxb, G. Slegersa

aLaboratory for Radiopharmacy, Ghent University, Harelbekestraat 72, B-9000 Gent, BelgiumbDivision of Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium

Received 2 June 2004; received in revised form 7 October 2004; accepted 29 October 2004

Abstract

This work reports the synthesis, radiolabelling and preliminary in vivo evaluation of [ 123I]-(4-fluorophenyl){1-[2-(4-

iodophenyl)ethyl]piperidin-4-yl}methanone. The tributylstannylprecursor was synthesized with a yield of 30%.

Radiolabelling was performed using an electrophilic iododestannylation. Tracer yield was 80%, radiochemical purity

was495% and specific activity was at least 55 Ci/mmol. Log P was 1.5. The tracer showed uptake in mice brain

(2.72% ID/g tissue at 5 min p.i.) and therefore will be evaluated further by regional brain biodistribution and

displacement studies in rabbits.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Serotonin; 5-HT2A receptor; SPECT; Radiotracer

1. Introduction

Serotonin (5-hydroxytryptamine, 5-HT) mediates a

number of neuronal processes both in central nervous

system and peripheral tissues. Multiple 5-HT receptor

subtypes have been characterized and grouped in seven

classes (5HT1 –5HT7) (Saxena, 1995). Except for the 5-

HT3 receptor, which is a ligand-gated ion channel, 5-HT

receptors belong to the G-protein-coupled receptor

(GPCR) superfamily. The 5-HT2 class includes the

subtypes 5-HT2A, 5-HT2B and 5-HT2C which are

grouped together considering their high degree of

transmembrane sequence homology and second messen-

ger coupling system (Claudi et al., 1999).

The 5-HT2A subtype is present in the brain (cortical

regions) (Pazos et al., 1987b) and periphery (gastro-

intestinal tract, cardiovascular system) (Peroutka and

Snyder, 1979); the 5-HT2B subtype is expressed in rat

stomach fundus and in the human brain and the 5-HT2C

subtype is widely distributed in the brain (Pazos et al.,

1987a).

It has been suggested that the 5-HT2A receptor may

be implicated in the pathology of several mental

illnesses like schizophrenia and depression (Pralong

et al., 2000). Different research groups found a

decreased 5-HT2A receptor density in cortical brain

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E-mail address: [email protected] (P. Blanckaert).

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tribromide (8.12 g, 2.8 ml, 30 mmol) was added drop-

wise. The reaction mixture was stirred at room

temperature for 20 h. The mixture was poured onto

crushed ice (100g), saturated NaHCO3-solution

(100 ml) was added, and the mixture was stirred for

30 min. The mixture was extracted with chloroform

(3Â 100 ml) and the combined extracts washed once

with saturated NaHCO3-solution (100 ml) and once

with brine (100ml). The organic phase was dried

over anhydrous Na2SO4, filtered and evaporated

under reduced pressure. A yellowish oil was obtained

(6 g, 91%).1H–NMR (d6 –DMSO, d): 7.40–7.15 (q, 4H, Br–ArH),

3.63 ( t, 2H, Br–CH2), 3.01 (t, 2H, Ar–CH2 –R).

2.1.3. (4-Fluorophenyl){ 1-[2-(4-

bromophenyl)ethyl]piperidin-4-yl }methanone (5)

To a solution of 4-(4-fluorobenzoyl)piperidine (4)

(1.9 g, 9.6 mmol) in dry dimethylformamide (DMF)

(50 ml) under a nitrogen atmosphere was added 3

(3.3 g, 12.5 mmol), followed by K2CO3 (5.5 g, 40 mmol).

The mixture was heated at 90 1C for 22 h. After cooling

to room temperature, the mixture was filtered, and the

precipitate was washed with DMF (20 Ml). The solvent

was evaporated under reduced pressure, and the residue

was purified by column chromatography with 20:80:10

EtOAc/hexane/Et3N to give 5 as a yellow solid (2.32 g,

60%). ESI-MS: 390/392 (MH+).1H–NMR (d6 –DMSO, d): 7.43–7.33–7.18 (m, 8H,

2x Ar–H), 3.35 (m, 1H, R–CH–R), 2.93–2.69–2.50

(m, 12H, 6x R–CH2 –R), 2.10(m, 2H, Ar–CH2 –R),

1.74 (d, 2H, R–CH2 –CH–CO–Ar), 1.53 (m, 2H,

R–CH2 –CH–CO–Ar).

2.1.4. (4-Fluorophenyl){ 1-[2-(4-

tributylstannylphenyl)ethyl]piperidin-4-yl }methanone

(6 )

To a solution of 5 (146 mg, 0.375 mmol) in anhydrous

toluene (10 ml) under nitrogen was added a catalytic

amount of tetrakistriphenylphosphinepalladium (10 mg)and hexabutylditin (0.5 ml, 1 mmol). The mixture was

heated at 120 1C for 15 h in the dark. The mixture was

cooled to room temperature, filtered, and the solvent

was removed under reduced pressure. The residue was

purified by preparative thin layer chromatography

(TLC), using methanol:dichloromethane:triethylamine

(9:1:1) as eluent. After extraction of the silica with

methanol and removal of the solvent under reduced

pressure, pure 6 (121 mg, 0.2mmol, 54% yield) was

obtained. ESI-MS: 602.1 (MH+).1H–NMR (d6 –DMSO, d): 8.04–7.33–7.17 (m, 8H, 2x

Ar–R), 2.94 (q, 1H, R2 –CH –CO), 2.68 (2H, t,

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Fig. 2. Synthesis of the tributylstannylprecursor 6.

P. Blanckaert et al. / Applied Radiation and Isotopes 62 (2005) 737–743 739



Ar–CH2 –CH2 –N–R2), 2.65 (2H, t, Ar–CH2 –CH2 –R),

2.29–2.19 (m, 4H, R1 –N–(CH2 –R2)2), 1.80–1.50

(m, 4H, R1 –CO–CH–(CH2 –R2)2), 0.8–1 (m, 27H,

tributylstannyl).

2.1.5. (4-Fluorophenyl){ 1-[2-(4-iodophenyl)ethyl]piperidin-4-yl }methanone (7b)

6 (120 mg, 0.2mmol) was dissolved in chloroform

(20 ml). At room temperature, iodine in chloroform

(0.53 ml, 1 M solution) was slowly added. The

reaction mixture was stirred at room temperature for

15 min. The reaction was quenched by the addition of

sodiummetabisulphite (3.2 ml; 5% solution in water)

and potassium fluoride (3.2 ml; 1 M solution in metha-

nol). The mixture was stirred for 5 min, and the phases

were separated.

The water phase was extracted with chloroform

(3Â 100 ml), and the combined organic phases were

dried over sodium sulphate. After filtration, the solvent

was removed under reduced pressure, and the residue

was purified by preparative TLC with methanol:dichlor-

omethane (12:88) as eluent. The silica was extracted with

methanol, the methanol was removed under reduced

pressure, and pure 7b (30 mg, 0.07 mmol, 35% yield) was

obtained.

ESI-MS: 438 (MH+).1H–NMR (d6 –DMSO, d): 7.90 (q, 2H, F–ArH), 7.65

(m, 2H, I–ArH), 7.03 (m, 2H, F–ArH), 6.93–6.77 (m,

2H, I–ArH), 3.30–3.20 (m, 4H), 3.10 (t, 1H), 2.90

(t, 2H, Ar–CH2 –CH2), 2.60 (t, 2H, Ar–CH2 ), 2.10–1.92

(m, 4H).

2.2. Radiosynthesis

The precursor 6 (400mg, 0.6 mmol) was dissolved in

ethanol (50mL)). n.c.a. [123I]NaI in sodium hydroxide

solution (15mL 0.01 M), chloramine T (CT) (282mg,

1mmol) and glacial acetic acid (GAA) (5mL) were

added. The mixture was stirred and left to react for

10 min at room temperature. The reaction mixture was

quenched with sodium metabisulphite (285 mg, 1.5 mmol

in 15 mL water) and injected onto an HPLC column for

purification (Alltech Alltima RPC18, 4.6Â250 mm),with 55/45 acetonitrile/phosphate buffer (0.02 M, pH9)

as eluent at 2 ml/min. The desired radiolabelled product

7a (Rt ¼ 19:6 min) was collected, and diluted with water

to bring the acetonitrile concentration below 10%. The

mixture was passed through a Sep-Pak cartridge (Waters

Sep-Pak Light tC18), and rinsed with 5 ml saline. The

cartridge was previously activated with 1 ml methanol

and rinsed with 1 ml saline. The tracer was eluted with

1 ml ethanol. An aliquot was reinjected onto the same

HPLC system for quality control and stability testing.

For biodistribution studies, the tracer was formulated in

an ethanol/saline solution (containing less then 10%

ethanol), and filtered through a 0.2mm filter (Schlei-

cher&Schuell, FP 013/AS).

2.3. Determination of specific acitvity

Since no UV-signal was obtained from 1 mCiof the radiolabelled tracer 7a, specific activity was

calculated by determination of the detection limit

of the UV-detector, using a calibration curve with

cold 7b.

2.4. Calculation of log P

Determination of the partition coefficient was per-

formed according to published literature (Azizian et al.,

1981; Meyer et al., 1999; Wilson et al., 2001). About

10mL (0.1 mCi) of the radioligand 7a was added to a

separatory funnel containingn

-octanol (100 ml) and

phosphate buffer (0.02 M, pH 7.4, 100 ml). The mixture

was shaken manually for 3 min and the layers were

separated. The aqueous layer was discarded, to remove

any hydrophilic impurities present. The n-octanol layer

(100 ml) was transferred to a second separatory funnel

containing phosphate buffer (100 ml). The mixture was

shaken for 3 min, and the layers were separated. A 5 ml

aliquot of both layers was counted for radioactivity. The

aqueous layer was discarded. Once again, the n-octanol

layer (95 ml) was transferred to a new separatory funnel

containing phosphate buffer (95 ml), the funnel shaken

for 3 min, the layers separated, and a 5 ml aliquot of

both layers was taken and counted for radioactivity.This process was repeated once more. The radioactivity

counts were decay-corrected, and the partition coeffi-

cient was calculated: P ¼ counts in n-octanol/counts in

buffer. Reported log P value represents the mean of 3

determinations.

2.5. Biodistribution study in NMRI mice

A biodistribution study of the radiotracer 7a was

performed in NMRI mice. Adult white male NMRI

mice weighing 20–25 g were each injected with 1–2mCi

of 7a in the tail vein. The mice were sacrificed atselected time points after injection (n ¼ 3 per time

point). Blood and organs (brain, heart, lung, liver,

kidney, e.a.) were rapidly removed and weighed. Radio-

activity of the samples was measured in an automated

g-counter (Cobra, Packard Canberra). Tissue radio-

activity concentrations were expressed as percent

of injected dose per gram of tissue (% ID/g tissue).

All experiments were conducted following the

principles of laboratory animal care and the Belgian

Law on the protection of animals. Our research

protocol was approved by the local ethical committee

(ECP 03/22).

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3. Results and discussion

3.1. Synthesis and radiosynthesis

Synthesis of the tributylstannylprecursor 6 is shown in

Fig. 2. 4-Bromophenylacetic acid 1 was reduced to the

alcohol 2 with BH3.THF, after which the hydroxyl

group was replaced by bromine using phosphorous

tribromide, thus creating a much better leaving group.

This ethylbromide 3 was then coupled with 4-(4-

fluorobenzoyl)piperidine 4 by nucleophilic substitution

(Fu et al., 2002a). The bromine-atom of 5 was replaced

by a tributylstannyl-group by reaction with hexabutyl-

ditin. The cold iodinated product 7b was obtained by

reacting the tributylstannylderivative 6 with iodine in

chloroform (Fig. 3). The overall yield was about 30%.

The radiolabelling was conducted by an electrophilic

iododestannylation on the tributylstannylprecursor (Fig.

3). A radiochemical yield of 80%75% was obtained.

The radiochemical purity of the tracer was495%.

Identification of the collected tracer was performed by

comparing retention times on HPLC between the

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Fig. 3. Radiosynthesis and cold iodination.

Table 1

Tissue concentrations of radioactivity in NMRI mice at various times following intravenous administration of 7a

Time (min)

Tissue 0.3 0.6 1 1.5 2 3

Blood 4.070.7 2.670.8 2.370.3 1.370.3 1.570.1 1.070.1Brain 2.570.2 2.570.6 2.370.7 1.170.3 1.470.3 2.070.4

Heart 6.770.7 5.370.8 5.370.5 2.770.4 3.270.5 2.370.8

Lungs 25.776.7 21.677.1 16.275.2 9.873.1 15.273.8 12.973.1

Stomach 0.570.1 2.570.1 1.670.1 1.170.3 1.370.3 2.570.5

Spleen 1.270.0 4.170.2 2.770.4 2.370.8 2.570.8 4.171.1

Liver 3.470.2 9.070.3 5.170.7 5.670.5 8.670.8 14.670.6

Kidneys 4.770.5 10.470.3 7.570.7 4.770.8 6.271.0 8.270.9

Sm. Int. 0.770.1 2.170.4 1.370.3 1.170.6 1.470.3 1.570.3

L. Int. 0.570.1 0.470.1 0.570.1 0.370.1 0.670.3 0.870.2

Bladder 0.970.2 1.970.6 0.670.3 0.770.2 0.870.3 1.370.2

Fat 0.670.1 1.670.1 1.170.2 1.370.3 1.970.7 0.870.2

5 10 20 40 60 120

Blood 1.170.2 0.970.1 1.170.1 0.970.1 0.970.0 1.570.1

Brain 2.770.5 2.670.2 2.570.2 2.270.5 2.170.4 1.870.6

Heart 2.171.0 1.170.4 0.970.1 1.270.1 1.170.1 1.070.4

Lungs 8.172.4 7.272.4 4.071.6 3.970.4 3.970.3 3.670.4

Stomach 2.170.8 3.770.7 5.570.1 4.270.2 3.571.1 4.671.6

Spleen 4.970.8 4.370.7 3.370.7 3.671.2 2.970.5 2.370.1

Liver 13.771.1 21.270.1 12.270.6 14.171.8 11.871.5 13.871.7

Kidneys 7.070.4 4.970.3 4.770.5 4.070.9 3.270.2 2.770.6

Sm. Int. 1.970.6 2.170.3 2.570.5 1.970.5 2.170.5 2.770.6

L. Int. 1.170.2 1.170.2 1.170.1 1.070.6 1.470.5 2.470.8

Bladder 1.970.6 1.370.4 2.270.6 2.170.7 3.770.8 3.570.8

Fat 2.370.5 1.170.6 1.970.3 2.670.8 3.070.7 5.970.2

Units are % injected dose/g tissue. Reported are the 95% confidence intervals.




radioactive labelled product 7a and the unlabelled

iodinated molecule 7b.

Tracer stability at room temperature in phosphate

buffer pH 7.4 was tested by reinjecting the tracer into the

HPLC system; the radiochemical purity was 95,3% at

12 h after synthesis.

3.2. Specific activity and log P determination

Specific activity was calculated to be at least 55 Ci/

mmol. This is an excellent value for performing brain

SPECT studies with this tracer, and for in vitro work. A

log P value of 1.4370.02 was obtained; for optimal

brain penetration the log P value should be between 2

and 3. Nevertheless, biodistribution results have shown

adequate brain uptake of the tracer in vivo.

3.3. Biodistribution results

Results of the biodistribution study for different

organs are shown in Table 1.

Uptake of the tracer in mouse brain was demon-

strated. A maximum value of 2.72% ID/g tissue in brain

was obtained 5 min post injection. The compound was

cleared out of the bloodstream quite rapidly (with less

than 1% ID/g in the blood after 3 min), and blood

activity remained lower than brain activity. This

indicates the possible usefulness of the tracer for

visualizing brain structures.

Other organs with high uptake of the tracer were the

liver, which indicates possible metabolism or degrada-

tion, and the lungs and kidneys (data not shown).

4. Conclusion

This work reported the synthesis and radiolabelling of

[123I]-(4-fluorophenyl){1-[2-(4-iodophenyl)ethyl]piperi-

din-4-yl}methanone 7a, a potential radiotracer for in

vivo visualization of the 5-HT2A receptor with SPECT,

and its biodistribution in NMRI mice. The tributyl-

stannylprecursor 6 was synthesized in an overall yield of

30%. The tracer was labelled in good yield (80%). The

specific activity was at least 55 Ci/mmol, an excellentvalue for future SPECT and in vitro studies. Log P was

1.5. Although the partition coefficient was not optimal,

the tracer showed good brain uptake in mice (2.72% ID/

g tissue at 5 min p.i.). Regional biodistribution and

displacement studies in rabbit brain are further required

to demonstrate specific binding in brain regions expres-

sing 5-HT2A receptors.

Acknowledgements

We thank the FWO Belgium for financial support.

References

Audenaert, K., Van Laere, K., Dumont, F., Slegers, G.,

Mertens, J., Van Heeringen, C., Dierckx, R.A., 2001.

Decreased frontal serotonin 5-HT2a receptor binding index

in deliberate self-harm patients. Euro. J. Nucl. Med. 28 (2),

175–182.Azizian, H., Eaborn, C., Pidcock, A., 1981. Synthesis of

organotrialkylstannanes—the reaction between organic

halides and hexaalkyldistannanes in the presence of

palladium complexes. J. Organomet. Chem. 215 (1), 49–58.

Claudi, F., Scoccia, L., Giorgioni, G., Accorroni, B., Marucci,

G., Gessi, S., Siniscalchi, A., Borea, P.A., 1999. 4-(4-

fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]pi-

peridine and its derivatives: synthesis and affinity at 5-

HT2A, 5-HT2B and 5-HT2C serotonin receptors. Euro. J.

Med. Chem. 34 (10), 843–852.

Dean, B., Hayes, W., Hill, C., Copolov, D., 1998. Decreased

Serotonin(2A) receptors in Brodmann’s area 9 from

schizophrenic subjects—a pathological or pharmacological

phenomenon? Mol. Chem. Neuropathol. 34 (2–3), 133–145.

Egan, C.T., Herrick-Davis, K., Miller, K., Glennon, R.A.,

Teitler, M., 1998. Agonist activity of LSD and lisuride at

cloned 5HT(2A) and 5HT(2C) receptors. Psychopharma-

cology 136 (4), 409–414.

Fu, X., Tan, P.Z., Kula, N.S., Baldessarini, R., Tamagnan, G.,

Innis, R.B., Baldwin, R.M., 2002a. Synthesis, receptor

potency, and selectivity of halogenated diphenylpiperidines

as serotonin 5-HT2A ligands for PET or SPECT brain

imaging. J. Med. Chem. 45 (11), 2319–2324.

Fu, X., Tan, P.Z., Kula, N.S., Baldessarini, R., Tamagnan, G.,

Innis, R.B., Baldwin, R.M., 2002b. Synthesis, receptor

potency, and selectivity of halogenated diphenylpiperidines

as serotonin 5-HT2A ligands for PET or SPECT brainimaging. J. Med. Chem. 45 (11), 2319–2324.

Hashimoto, T., Kitamura, N., Kajimoto, Y., Shirai, Y.,

Shirakawa, O., Mita, T., Nishino, N., Tanaka, C., 1993.

Differential changes in serotonin 5-Ht(1A) and 5-Ht(2)

receptor-binding in patients with chronic-schizophrenia.

Psychopharmacology 112 (1), S35–S39.

Laruelle, M., Abidargham, A., Casanova, M.F., Toti, R.,

Weinberger, D.R., Kleinman, J.E., 1993. Selective abnorm-

alities of prefrontal serotonergic receptors in schizophre-

nia—a postmortem study. Arch. Gen. Psychiatry 50 (10),

810–818.

Meyer, J.H., Kapur, S., Houle, S., DaSilva, J., Owczarek, B.,

Brown, G.M., Wilson, A.A., Kennedy, S.H., 1999. Pre-

frontal cortex 5-HT2 receptors in depression: an [F-

18]setoperone PET imaging study. Am. J. Psychiatry 156

(7), 1029–1034.

Oh, S.J., Ha, H.J., Chi, D.Y., Lee, H.K., 2001. Serotonin

receptor and transporter ligands—current status. Curr.

Med. Chem. 8 (9), 999–1034.

Okubo, Y., Suhara, T., Suzuki, K., Kobayashi, K., Inoue, O.,

Terasaki, O., Someya, Y., Sassa, T., Sudo, Y., Matsushima,

E., Iyo, M., Tateno, Y., Toru, M., 2000. Serotonin 5-HT2

receptors in schizophrenic patients studied by positron

emission tomography. Life Sci 66 (25), 2455–2464.

Pazos, A., Probst, A., Palacios, J.M., 1987a. Serotonin

receptors in the human-brain. 4. autoradiographic mapping

of Serotonin-2 receptors. Neuroscience 21 (1), 123–139.

ARTICLE IN PRESS

P. Blanckaert et al. / Applied Radiation and Isotopes 62 (2005) 737–743742



Pazos, A., Probst, A., Palacios, J.M., 1987b. Serotonin

receptors in the human-brain 4. autoradiographic

mapping of Serotonin-2 receptors. Neuroscience 21 (1),

123–139.

Peroutka, S.J., Snyder, S.H., 1979. Multiple serotonin recep-

tors—differential binding of [5-Hydroxytryptamine-H-3,

[Lysergic-H-3 Acid Diethylamide and [H-3]Spiroperidol.Mol. Pharmacol. 16 (3), 687–699.

Pralong, D., Tomaskovic-Crook, E., Opeskin, K., Copolov, D.,

Dean, B., 2000. Serotonin(2A) receptors are reduced in the

planum temporale from subjects with schizophrenia. Schi-

zophr. Res. 44 (1), 35–45.

Saxena, P.R., 1995. Serotonin receptors—subtypes, functional-

responses and therapeutic relevance. Pharmacol. Ther. 66

(2), 339–368.

Ullrich, T., Rice, K.C., 2000. Practical synthesis of the

serotonin 5-HT2A receptor antagonist MDL 100907 and

MDL 105725, its precursor for [C-11]labeled PET ligands,

based on optical resolution via formation of diastereomericsalts. Abstr. Pap. Am. Chem. Soc. 220, U556–U556.

Wilson, A.A., Jin, L., Garcia, A., DaSilva, J.N., Houle, S.,

2001. An admonition when measuring the lipophilicity of

radiotracers using counting techniques. Appl. Radiat.

Isotopes 54 (2), 203–208.

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