Synthesis of radioiodinated analogs of 2-(4-Phenylpiperidino)cyclohexanol (vesamicol) as vesamicol-like agent

Vol. 22. No. 2. DD.205-210. 1995 Copyright Q 1995 El&&r Science Ltd Printed in Great Britain. All rights reserved

Nucl. Med. Bd.

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0969-8051(94)00093-X

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Synthesis of Radioiodinated Analogs of 2-(4-Phenylpiperidino)cyclohexanol (Vesamicol) as Vesamicol-like Agent K. SHIBA’*, H. MORI’, H. MATSUDA’, S. TSUJ13, I. KUJ13, H. SUMIYA’, K. KINUYA3, N. TONAM13, K. HISADA and T. SUMIYOS14 ‘Radioisotope Center and ‘Department of Nuclear Medicine, 4Department of Neuropsychiatry, School of Medicine. Kanazawa University, Kanazawa, Japan and ‘National Center Hospital of Neurology and Psychiatry, Tokyo, Japan (Accepted 7 July 1994) Three iodovesamicol analogs, iodinated at the ortho, meta, and para positions of the 4-phenylpiperidine moiety, were synthesized and labeled with ‘*‘I by isotopic exchange reaction. Their potencies as a vesamicol-like drug were evaluated with competitive inhibition studies using ( - )[‘H]vesamicol. The radiochemical yields were 40-85%, the radiochemical purities exceeded 95% and their specific activities were 370-740 GBq/mmol. The descending order of binding affinity of the tested compounds against the vesamicol receptor was m-iodovesamicol > o-iodovesamicol >p-iodovesamicol. The receptor binding affinity of m-iodovesamicol (IC,, = 133 nM) was comparable with that of vesamicol (IC,,, = 109 nM). Therefore, the meta position of the 4-phenylpiperidinyl fragment of vesamicol was the optimum site for iodination, and radioiodinated m-iodovesamicol may serve as a useful radiopharmaceutical for in oitro and in uitlo studies of presynaptic cholinergic neurons in rats.

Introduction

vesamicol, and competitive inhibition studies on these compounds to ascertain the optimum site for iodination were investigated.

Vesamicol was originally discovered as a neuromuscular blocking agent with an unusual mode of action (Marshall, 1970). Several investigators have demonstrated that the drug inhibits the storage of acetylcholine (AcCh) in the nerve terminals of synaptic vesicles (Bahr and Parsons, 1986; Marshall and Parsons, 1987). Marien et al. (1987) and Alter and Marien (1988) have demonstrated potential use of the vesamicol receptor as a presynaptic cholinergic marker by their preliminary characterization of [3H]vesamicol binding in the rodent brain. Vesamicol labeled with a y-emitter, such as I- 123, could serve as a potential presynaptic cholinergic marker for singlephoton emission computed tomography (SPECT). In fact, several radioiodinated vesamicol analogs [IBVM (1) (Jung et al., 1990) and 4-HIPP (2) (Efange et al., 1992)] were synthesized and subsequently evaluated as potential radiopharmaceuticals for SPECT. These vesamicol analogs were labeled with radioiodine in the phenyl fragment of the cyclohexyl moiety (Fig. 1). In this study, we synthesized three vesamicol, 3-5, analogs, iodinated at the ortho (3), meta (4) and para (5) positions of the 4-phenylpiperidine moiety of *Author

Materials

and Methods

General

NMR spectra were recorded on a Jeol JNMGSXSOO (taken in deuterated chloroform with tetramethylsilane as the internal standard). MS recorded on a Hitachi M-80 spectrometer coincided well with the proposed structures. Elemental analyses were performed on a Yanagimoto CHN-Corder MT-3; all values were within f0.3% of theoretical values. High performance liquid chromatography (HPLC) was performed on a Zorbax ODS reverse phase column (4.6 mm x 25 cm) eluted with 0.05% triethylamine acetate (pH 4.O)/acetonitrile (1: 1, v/v). The eluent was monitored with a radioisotope detector (Aloka RGC-212). Preparation hexanol (1’)

of

2-(4-(4-nitrophenyl)piperidino)cy&-

2-(4-Phenylpiperidino)cyclohexanol (5.18 g, 20mmol), was added to 9.0 mL of an ice-cold HNO, /concentrated H, SO, mixture (equivalent to 60 mmol of HNO, and 80mmol of H,SO,) by dripping over a period of 5 min. The solution was

for correspondence. 205

i(.

Shibd

i’f

rri

&qy---+ 1. IEIVM

Vesamico?

3 $ 5

I

&% - I

RI= I, Rz,Rs= H Rt,Rs= H, Rz= I R,,R,= H, Ra= I

&

4-HIPP

Fig. 1. Vesamicol and iodovesamicol analogs.

then stirred for 2.5 h at room temperature prior to adding to 100mL ice-cold water. The solution was made alkaline with 2N NaOH, then extracted with 5% MeOH in CH,Cl,. The organic solution, dried over Na,SO,, was evaporated to leave a crystalline solid, which was recrystallized from ethanol to yield 1’ (4.37 g, 71.9%). The residue, composed of 2-(4-(2-nitrophenyl)piperidino)cyclohexanol (1) and 2-(4-(4-nitrophen(l’), was used for yl)piperidino)cyclohexanol the preparation of 2-(4-~2-aminophenyl}pipe~dino)cyclohexanol (2) without further pu~fication. Compound I: Mp 152-154°C. NMR(CDC1,): 6 1.23 (4H, m), 1.59-1.89 (8H, m), 2.16 (lH, m), 2.26 (2H, m), 2.62 (lH, m), 2.77 (2H, nz), 2.97 (lH, m), 3.40 (lH, m), 7.38 (2H, d, J = 8.70 Hz), 8.17 (2H, d, J = 8.70 Hz). Mass spectrum(m/e): 304 [Ml+. Elemental analysts C,,H,,N,O,; theory C: 67.08, H: 7.95, N: 9.20; found C: 66.97, H: 8.07, N: 9.12. Preparation cyclohexanol

of (2)

2-(4-(2-aminophenyllgiperidino)-

The residue containing both 1 and 1’ (1.52 g, 5 mmol) was added with iron (sponge) (1.70 g, 30 mmol) to 15mL of 50% ethanol. The mixture was heated to boiling on a water bath before a solution of 0.9 mL (10.8 mmol) of concentrated hydrochloric acid in 3 mL of 50% ethanol was added slowly. The mixture was refluxed for 30min, then made alkaline with 2 N NaOH. After removing the iron by filtration, the alkaline solution was then extracted with 5% MeOH in CH, Cl,. The organic solution was dried over MgSO, and evaporated to leave a brown oil behind. This residue was purified by preparative thin layer chromatography on silica gel using 5%

MeOH in CH,Cl, to yield 2 (592 mg, 43.2%) and 2’ (502 mg, 36.7%). Compound 2. Mp 138-141°C. NMR(CDC1,):

6 1.18-1.28 (4H, m), 1.62-1.92 (8H, m), 2.14 (lH, m). 2.27 (2H, m), 2.47 (lH, m), 2.77 (2H, m), 2.97 (IH, m), 3.40 (IH, m), 3.63 (2H, m), 6.69 (lH, dd, J = 7.81 Hz, J = 1.46 Hz), 6.80 (lH, ddd, J = 7.81 Hz, J = 7.32 Hz, J = 0.98 Hz), 7.03 (lH, ddd, J = 7.81 Hz, J = 7.32Hz, J = 1.46Hz), 7.13 (lH, dd%J = 7.81 Hz, f = 0.98 Hz). Mass spectrum (m/e): 274 [Ml+. Elemental analysis C,,H,,N,O; theory C: 74.41, H: 9.55, N: 10.21; found C: 74.30, H: 9.66, N: 9.97. Preparation hexanol (2’)

qf 2-~4-~4-a~~~nuphenyZ~piper~d~n~~~y~,~o-

The same procedure used to prepare 2 was followed, starting from 2-(4-(4nitrophenyl)piperidino)cyclohexanol (1’) (3.04g, 10mmol). Compound 2’ was obtained in 83.5% yield. Compound 2: Mp 144-145’C. NMR (CDCI,): 6 1.16-1.29 (4H, m), 1.51&1.82 (8H, nr), 2.12-2.27 (4H, m), 2.32-2.43 (IH, m), 2.72 (2H, m), 2.91 (lH, m). 3.38 (IH, m). 3.56 (2H, brs), 6.64 (2H, d, J = 8.42 Hz), 7.01 (2H, d, J = 8.42 Hz). Mass spectrum (m/e): 274 [Ml+. Elemental analysis C17H26N20;theory C: 74.41, H: 9.55, N: 10.21;found C: 74.20, H: 9.56, N: 10.14. Preparation hexanol (3)

yf

2-(4-(2-iodophenyl)piperidino)cyclo-

Firstly, 2- (4- (2-aminophenyl)piperidino)cyclo hexanol (2) (1.37 g, 5 mmol) was dissolved in 30 mL of 2 N HCI, at O”C, then sodium nitrite (414mg, 6 mmol) in water (3 mL) was added. The mixture was stirred for 15min before adding the potassium iodide

Synthesis of radioiodinated

solution (3.31 g, 20 mmol). The reaction mixture was stirred for 2 h at room temperature, then made alkaline with 1 N NaOH prior to extraction with 5% MeOH in CH,Cl,. The organic solution was dried over Na,SO, and evaporated to leave a crystalline solid, which was purified by column chromatography on silica gel using ether/hexane (1: 1, v/v) as the eluent to yield 3 (967 mg, 50.2%). Compound 3. Mp 151-153°C. NMR (CDCI,): ii 1.24 (4H, m), 1.81 (8H, m), 2.14 (lH, m), 2.28 (2H, m), 2.78 (2H, m), 2.96 (IH, m), 3.40 (lH, m), 6.90 (lH, ddd, J = 7.81 Hz, J = 7.81 Hz, J = 1.5 Hz), 7.23 (lH, dd, J = 7.81 Hz, J = 1.5 Hz), 7.32 (lH, ddd, J = 7.81, J = 7.81, J = l.OHz), 7.83 (lH, dd, J = 7.81 Hz, J = 1.0 Hz). Mass spectrum (m/e): 385 [Ml‘. Elemental analysis C,,H,,NOI; theory C: 53.00. H: 6.28, N: 3.64; found C: 53.25, H: 6.39, N: 3.47. Preparation of2-(4-(3-iodo-4-aminophenyl)piperidino)cyclohexanol (4’) To a solution of 2-(4-(4-aminophenyl)piperidino)cyclohexanol (1.37 g, 5 mmol) in glacial AcOH (15 mL), ICI (893 mg, 5.5 mmol) was dropped at room temperature for 3 h under N?. After removing the solvent, the residue was diluted with water, and then made alkaline with NH40H. The mixture was extracted with 5% MeOH in CH2C1,. After drying over MgSO,, the organic solution was evaporated to leave an oily product, which was purified by column chromatography on silica gel using ether/hexane (1: 1, v/v) as the eluent to yield 4’ (990 mg, 49.5%). Compound 4’. Mp 184187°C. NMR (CDCl,): 6 1.16.-1.28 (4H, m), 1.53-1.83 (8H, m). 2.12-2.25 (3H, m). 2.31l2.37 (lH, m), 2.67-2.75 (2H, m), 2.92 (1H. m), 3.39 (lH, m), 3.98 (2H, s), 6.70 (lH, d, J = 8.25 Hz). 7.00(lH, dd. J = 8.25 Hz, J = 2.29 Hz),

Fig. 2. Synthesis of iodovesamicol NMB:2,2--o

vesamicol analogs

207

7.49 (lH, d, J = 2.29 Hz). Mass spectrum (m/e): 400 [Ml’. Elemental analysis C,,H,,N,OI; theory C: 51.01, H: 6.29, N: 7.00; found C: 50.73, H: 6.34, N: 6.71. Preparation hexanol (4)

of

2-(4-(3-iodophenyl)piperidino)cyclo

-

Aqueous NaNO, (152 mg in 2 mL; 2.2 mmol) was added to 4’ (800 mg, 2 mmol) in 3 N HCl (5 ml) at 0°C. After stirring for 20min, CuSO, (500 mg, 2 mmol) in 95% ethanol (5 ml) was added and the mixture was stirred for an additional 30 min at 70 C. The reaction mixture was made alkaline with NaOH and extracted with 5% MeOH in CH,Cl,. After drying over MgSO,, the organic solution was evaporated to an oily product. This was purified by column chromatography on silica gel using ether/hexane (1: 1. v/v) as the eluent to yield 4 (490mg, 63.7%). Compound 4. Mp 112-114C. NMR (CDCI,): (5 1.23 (4H, m), 1.58-1.87 (8H, m), 2.13 (lH, m), 2.22 (2H, m), 2.43 (lH, m), 2.74 (2H, m), 2.94 (lH, m), 3.39 (lH, m), 7.03 (IH, t. J=7.82Hz), 7.18 (lH, d. J = 7.82 Hz), 7.53 (lH, dd, J = 7.82 Hz, J = 1.5 Hz). 7.57 (lH, d, J = 1.5 Hz). Mass spectrum (m/e): 385 [Ml+. Elemental analysis C,,H2,NOI; theory C: 53.00, H: 6.28, N: 3.64; found C: 52.74, H: 6.15. N: 3.56. Prepuration hexanol (5)

qf 2-(4-(4-iodophenyl)piperidino)gclo

-

The same procedure used to prepare 3 was followed, starting from 2-(4-(4-aminophenyl)piperidino)cyclohexanol. 5 was obtained in 51.5% yield. Compound 5. Mp 162-163°C. NMR (CDCI,): (5 1.23 (4H, m), 1.60-1.85 (8H, m), 2.13 (lH, m), 2.22 (2H, m), 2.44 (lH, m), 2.74 (2H, m), 2.93 (lH, m). 3.39 (lH, m), 6.97 (2H, d, J =8.30Hz), 7.61 (2H, d, J = 8.30 Hz). Mass spectrum (m/e): 385 [Ml+.

analogues (3-5). (a) HNO,, HzS04/Fe, HCI, (b) ICI, (c) NaNO,. (d) NaNO,, CuSO,.

KI.

Elemental analysis C,,H1qNOI; theory C: 53.00, II: 6.28, N: 3.64; found C: 53.00. H: 6.41, N: 3.37. Cortical membranes of rats were prepared as follows. The cortex from male Sprague-Dawley rats (200-220g) was homogenized in 10 volumes of ice-cold 0.32 M sucrose using a Teflon-glass homogenizer. The homogenate was centrifuged at IOOOgfor IOmin at 4°C. The supernatant obtained was further centrifuged at 10,000g for 20 min at 4°C. The pellet was resuspended in ice-cold water at 5.S-fold the volume of pellet. The material was then dispersed with a glass homogenizer. Tris-maleate (1: 40 v/v, 400 mM; pH 6.5) was added to the homogenate before centrifugation at 55,000g for 60 min at 4‘C. The final pellet obtained was resuspended in an isosmotic buffer (0.7 M glycine, 0.10 M HEPES, 1 mM EDTA, and 1mM EGTA, pH 7.8). Cortical membranes of rats (0.4 mL; 0.2 mg protein) were incubated in quintuplicate with (-)[‘H]vesamicol (10 mM, 1.83TBq/mmol) and various concentrations of unlabeled vesamicol, o-iodovesamicol(3), ~-iodovesamicoi (4) and ~-iodovesamicol(5) (from IO-” to IO-‘M) at 23‘C for 30 min. The incubated samples were quickly diluted with 5mL of ice-cold isosmotic buffer prior to filtration with glass filters (Whatman GF/F) precoated with 0.5% polyethylenimine (Bruns et al., 1983).The filters were

washed twice with 5ml of buf‘er before furthct incubation for 30 mm in scintiliation vials containing IO mL of Aquasol- (DuPont-New, England Nuclear-. Boston, MA. IJ.S.A.) and 0.5 mL of water. Radioactivity retained on the filters was counted with a liquid scintillation counter (Aloka, LX-1000). Radioiodination Iodovesamicol analogs (3-5) were labeled with “‘I as follows. To a solution of 5 pg of 3-5 in acetic acid (IOO~~L), 0.5 mg of Cu(I)Cl and I mg of ascorbic acid followed by 3.7-18.5 MBq of [“‘I]NaI (carrier free) were added. After heating for 2 h at 120°C. the reaction mixture was neutralized with NH,OH and purified by HPLC using 0.1% triethylamine acetate (pH 4,0)/acetonitrile (1: I, v/v). Results Chemistry Preparation of o- and p-iodovesamicol (3,5) and m-iodovesamicol (4) from vesamicol (Rogers et al., 1989) via three- and four-step reactions produced respective overall yields of 4.6, 31.6 and 19.3% (Fig. 2). The mixture of o- and ~-nitrove~micol (1,l’) was prepared by the nitration of vesamicol. Isolation of o-nitrovesamicol (1) from the mixture was very difficult. After p-nitrovesamicol (1’) was isolated by recrystallization, the remaining mixture

100 (%I

A ---y)

A A 1\

\

“\\\

\

.

o-o

vesamicol

5-5

o-I-vesami

colf3)

0-e

m-I-vesami

co1 (4)

A-A

p-I-vesami

co1 (5) -

50

-9

-8

-7

-6

-5

-4

-Log[Drugl(Mohr)

Fig. 3. Inhibition of [3H]vesamicolbinding by vesamicolanalogs.

Synthesis of radioiodinated vesamicol analogs Table

I. Inhibition of

[3H]vesamicol vesamicol analogs

Vesamicol

analogs

Vesamicol o-iodovesamicol PI-iodovesamicol

binding by

IC,, @Ml*

(3) (4)

109?6 197 * 21 133*7 1102 * 93

p-iodovesamicol(5) *ICjo representsthe concentrationof analogs that induced50% inhibition of the specific binding.Values are the means F SEM of five experrments.

was used for the synthesis of o-aminovesamicol (2). The separation of o-aminovesamicol (2) from the mixture of u- and p-aminovesamicol (2,2’) was rendered possible with preparative thin layer chromatography (p-TLC) on silica gel with 5% MeOH in CH,CI,. The end products, o-iodovesamicol (3) and p-iodovesamicol (5), were derived from 2and 4-aminovesamicol (2,2’) by diazotization and reaction with potassium iodide, respectively. The compound, m-iodovesamicol (4), was prepared by iodinating 4-aminovesamicol (2’) with iodine monochloride followed by deamination with NaNO, and CuSO,. Each iodovesamicol(3-5) was characterized by NMR, mass spectroscopy and elemental analysis, accordingly. The analytical data were consistent with the assigned structures. Competitiae

inhibition

studies

The inhibition of ( -)[3H]vesamicol binding to cortical membranes of the rodent brain in the presence of the iodovesamicol analogs (3-5) and cold vesamicol revealed the affinity order of these compounds for the synaptosomal membranes as vesamicol > m -iodovesamicol (4) > o -iodovesamicol (3) > p-iodovesamicol(5) (Fig. 3). The IC,, values are summarized in Table 1, The binding affinity of 4 against the receptor (IC,, = 133 nM) was comparable to that of vesamicol (IC,, = 109 nM).

20’)

important that the radioiodine incorporated into the vesamicol analogs should not affect vesamicollike inhibitory activity. By employing a phenyl fragment in the cyclohexyl moiety, 5-iodobenzovesamicol (IBVM, 1) and 2-hydroxy-3-(4-iodophenyl)-l-(4phenylpiperidinyl)propane (CHIPP, 2) were radioiodinated. The radioiodinated IBVM (1) and 4-HIPP (2) were vesamicol-sensitive, and indicated high accumulation levels with long retention times in the rodent brain. However, a different regional distribution of [‘I511 1 from that of [“‘I] 2 has been reported (Jung et al., 1990; Efange et al., 1992). Furthermore, structure-activity studies have indicated that the drug potency can be enhanced by substituting a methyl group in the ortho position of the phenyl ring of 4-phenylpiperazinyl group of vesamicol analogs (Rogers et al., 1989). As such, vesamicol analogs in our study were synthesized by incorporating iodine at the ortho, meta and para positions of the phenylpiperidinyl fragment of vesamicol (3-5). Relatively high radiochemical yields (40-85%) for 3-5 by isotopic exchange were achieved with the use of [1251]NaI (range: 3.7-18.5 MBq). The radioiodinated vesamicol analogs ([12’1] 3-5) purified by HPLC, concentrated under a stream of N, and dissolved in 0.05% triethylamine (the pH was adjusted to 6.0 with acetic acid) at 4 C in the dark, showed less than 1% deiodination for up to 4 weeks at a radioactive concentration of 37 MBq/ml. This revealed the high stability achieved by [“‘I] 3-5.

Radioiodinatiorz

The labeling of iodovesamicol analogs (3-5) with lZiI was based upon a Cu(I)Cl-assisted isotopic exchange reaction, which produced a regiospecific radioiodination with a radiochemical yield of 40-85% (Abbas et ui., 1990). The radiochemical purity of [“‘I] 3-5 following HPLC purification exceeded 95% when determined by radio-HPLC (Aloka, RLC-700) (Fig. 4). The specific activities of [“‘I] 3-5, estimated using a UV spectrophotometer (Shimazu, UV-2100), were 370-740 GBq/mmol.

2

4

6

8

Time

(min)

10

12

14

10.58

‘A

cold

~ 4

Discussion The relationship between the structure of vesamico1 analogs and AcCh transport inhibition and receptor binding has been extensively reported (Kaufman et al., 1989; Rogers et ctl., 1989; Efange et al., 1991). To develop a radioiodinated vesamicol analog as a potential radiopharmaceutical for use in SPECT, it is

:/__ 2

4

6 Time

8

10

12

14

(min)

Fig. 4. Chromatograms obtained by HPLC analysis of [“51]m-iodovesamicol ([“51]4) and cold m-iodovesamicol (4).

K. Shiba Y( u/.

210

Competitive inhibition studies showed that substituting an iodine atom in either the orrho or tneta positions of the 4-phenylpiperidinyl fragment of vesamicol hardly affected the binding affinity against the vesamicol receptor, whereas the vesamicol analog incorporated iodine at the para position manifested a dramatic decrease in the binding affinity. The decreasing order of binding affinity of the iodinated compounds against the vesamicol receptor was m -iodovesamicol (4) > o -iodovesamicol (3) > p iodovesamicol (5). These results suggest that the rneta position of the 4-phenylpiperidinyl fragment of vesamicol is the optimum site for iodination of vesamicol analogs. The radioiodinated m-iodovesamicol(4) may serve as a useful radiopharmaceutical for in vitro and in vivo studies of presynaptic cholinergic neurons in rats.

References Abbas H. G., Hankes L. V. and Feinendegen L. E. (1990) Synthesis of 2-(5-(4-[‘231/‘3’I]iodophenyl)pentyl)oxirane2-carboxylic acid. J. Label. Comp. Radiopharm. 28, 955-966. Altar C. A. and Marien M. R. (1988) [‘H]Vesamicol binding in brain: autoradiographic distribution, pharmacology, and effects of cholinergic lesions. Synapse 2, 486493. Bahr B. A. and Parsons S. M. (1986) Acetylcholine transport and drug inhibition kinetics in torpedo synaptic vesicles. J. Neurochem. 46, 1214-1218. Bruns R. F., Lawson-Wendling K. and Pugsley T. A. (1983) A rapid filtration assay for soluble receptors using

polyethylenimine-treated filters. .&u/. Hio&r?~ 132. 74-81. Efange S. M. N.. Michelson R. H., Dutta A. K. and Parsons S. M. (1991) Acyclic analogues of 2-(4-phenylpiperrdino)cyclohexano(vesamicol): conformationallv mobile inhibitors of vesicular acetylcholine transport .I. Map//. Chem. 64, 2638-2643.

Efange S. M. N., Dutta A. K.. Michelson R. H.. Kung I-1. F.. Thomas J. R.. Billings J. and Boudreau R. J. (1992) Radioiodinated 2-hydroxy-3-(4-iodophenyl)-1-(4-phenyipiperidinyl)propane: Potential radiotracer for mapptng central cholinergic innervation in W’IW. Vucl. .Mrd. Biol. 19, 337-348. Jung Y. W., Van Dort M. E., Gildersleeve D. L. and Wieland D. M. (1990) A radiotracer for mapping cholinergic neurons of the brain, J. Med. Chem. 33, 2065-2068. Kaufman R., Rogers G. A., Fehlman C. and Parsons S. M. (1989) Fractional vesamicol receptor occupancy and acetylcholine active transport inhibition in synaptic vesicles. Molec. Pharmacol. 36, 452458. Marien M. R., Persons S. M. and Altar C. A. (1987) Quantitative autoradiography of brain binding sites for the vesicular acetylcholine transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH5183). Proc. Nafl. Acad. Sci L’SA. 84, 8766880. Marshall 1. G. (1970) Studies on the blocking action of 2(4-phenylpiperidino)cyclohexanol (AH5183). Br. J. Phurmacol. 38, 5033516. Marshall I. G. and Parsons S. M. (1987) The vesicular acetylcholine transport system. Trends Neurosci. 10, 174m~l77. Rogers G. A., Parsons S. M., Anderson D. C., Nilsson L. M., Bahr B. A., Kornreich W. D., Kaufman R., Jacobs R. S. and Kirtman B. (1989) Synthesis, in oitro acetylcholine storage-blocking activities, and biological properties of derivatives and analogues of trans-2(4.phenylpiperidino)cyclohexanol (vesamicol). J. Med. Chem. 32, 1217~1230.