Syntheses of carbon-11 labeled piperidine esters as potential in vivo substrates for acetylcholinesterase

ISSN 0969-8051/98/$19.00 1 0.00 PII S0969-8051(98)00072-9

Nuclear Medicine & Biology, Vol. 25, pp. 761–768, 1998 Copyright © 1998 Elsevier Science Inc.

Syntheses of Carbon-11 Labeled Piperidine Esters as Potential in Vivo Substrates for Acetylcholinesterase Thinh B. Nguyen, Scott E. Snyder and Michael R. Kilbourn DIVISION OF NUCLEAR MEDICINE, DEPARTMENT OF INTERNAL MEDICINE, UNIVERSITY OF MICHIGAN SCHOOL OF MEDICINE, ANN ARBOR, MI 48109, USA

ABSTRACT. A series of carbon-11 labeled N-methylpiperidinyl esters were prepared as potential in vivo substrates for acetylcholinesterase (AChE). Target compounds were designed based on the structure of N-[11C]methylpiperidin-4-yl propionate, an ester currently used to measure AChE enzymatic activity in the human brain, to examine the structure–activity relationship for in vivo enzymatic hydrolysis. Changes in steric bulk and in the ester order (“reverse” esters) were made. Addition of methyl groups was made to both the acid side chain (synthesis of N-[11C]methylmethylpiperidin-4-yl isobutyrate) and to the piperidine ring (syntheses of N-[11C]methyl-4-methylpiperidin-4-yl propionate, N-[11C]methyl-4-methylpiperidin-4-yl acetate, and N-[11C]methyl-3-methylpiperidin-4-yl propionate). Alterations of the order of the ester heteroatoms was accomplished through syntheses of the N-[11C]methyl-2,3- and 4-piperidinecarboxylic acid ethyl esters. Finally, an additional piperidine-based ester (N-[11C]methylpiperidin-2-yl)methyl propionate was also prepared. All carbon-11-labeled esters were prepared by N-[11C]methylation reactions, using the desmethyl precursors and no-carrier-added [11C]methyltriflate, and were obtained in decay-corrected yields (not optimized) of 10 – 40% and high specific activities. NUCL MED BIOL 25;8:761–768, 1998. © 1998 Elsevier Science Inc. KEY WORDS. Carbon-11, Tomography (emission computed), Acetylcholinesterase

INTRODUCTION The primary function of acetylcholinesterase (AChE) in the central and peripheral nervous systems is the rapid hydrolysis of acetylcholine, thus terminating cholinergic neurotransmission. Proper functioning of the cholinergic system is proposed to be an integral part of the memory function of the brain, with decrements in this system evidenced in such conditions as Alzheimer’s disease (AD) (3, 19, 21). Our knowledge of the dysfunction of the cholinergic system in disease comes largely from postmortem studies of AD patients, or from indirect evidence gathered from pharmacological studies in animals and humans. Direct methods to evaluate the cholinergic system in vivo in the living human brain would prove most valuable in defining the involvement of this neurotransmitter system in the inception and progression of disease. Perhaps more importantly, such in vivo methods might provide a better method for evaluation of existing or emerging therapies intended to arrest or reverse the degenerative process. In vivo imaging of the biochemical components of the cholinergic system, including the enzymes of acetylcholine synthesis (choline acetyltransferase) and degradation (AChE), transport proteins (high affinity choline transporter and vesicular acetylcholine transporter), and receptors (muscarinic and nicotinic cholinergic receptors) would form a valuable approach to measurement of cholinergic function in health and disease. Although most of the effort in development of in vivo radioligands for this system has been in the area of receptors (17) and vesicular acetylcholine transporters (11), recent studies have focused on the enzyme AChE. Two different Address correspondence to: Dr. Michael R. Kilbourn, Division of Nuclear Medicine, Department of Internal Medicine, University of Michigan Medical Center, B1G 412 University Hospital, Ann Arbor, MI 48109-0028, USA; e-mail: [email protected] Received 14 March 1998. Accepted 5 June 1998.

approaches have been taken: (a) synthesis of high-affinity antagonists of AChE (16, 18) and (b) synthesis of radiolabeled substrates for the enzyme (2, 7, 8, 13). These approaches, although quite different, will be complementary as success in both areas would provide in vivo measures of both numbers and functions of this important enzyme in the human brain. For measurements of AChE enzymatic activity, we (2, 13) and others (7–9) have prepared radiolabeled labeled esters of 1-methyl4-hydroxypiperidine. In vivo, these lipophilic esters penetrate the blood– brain barrier easily; in the brain, they are cleaved by AChE to a hydrophilic metabolite, 1-[11C]methyl-4-hydroxypiperidine, which is efficiently trapped in the tissues in proportion to the enzymatic activity. A limited series of these esters have been prepared in radiolabeled form, and they exhibit different reactivities toward AChE in vitro (7, 8) and different regional brain distributions as measured in vivo in animals (7, 8, 12). Based on the results of animal studies, two research groups have chosen different esters, 4-N-[11C]methylpiperidinyl acetate (4-[11C]AMP) and 4-N-[11C] methylpiperidinyl propionate (4-[11C]PMP) for human studies using positron emission tomography (PET) (9, 14). With the possibility that neither 4-[11C]AMP or 4-[11C]PMP may be the optimal radiotracer for measurement of AChE throughout the entire brain (12), given the large regional differences between various brain regions, further studies of the structure– activity relationship for AChE-mediated hydrolysis of piperidinyl esters seem warranted. We report here the chemical and radiochemical syntheses of a series of structurally related N-methylpiperidine esters. In vivo biological studies with the radiolabeled compounds will be reported separately. MATERIALS AND METHODS 1

H nuclear magnetic resonance (NMR) spectra were obtained using Brucker AM-200 MHz and AM-360 MHz spectrometers in the

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Department of Chemistry, and Brucker DPX-300 MHz and DRX500 MHz spectrometers in the College of Pharmacy, University of Michigan. All NMR spectra were taken in CDCl3 using tetramethylsilane as an internal standard. Electron impact ionization (EI), chemical ionization (CI with NH3), and high resolution mass spectral (HRMS) analyses were done at the Department of Chemistry of the University of Michigan. EI and CI mass spectra are reported as mass (relative intensity). 4-Hydroxypiperidine, 4-hydroxy-1-methylpiperidine, ethyl 2-piperidinecarboxylate, ethyl 3-piperidinecarboxylate, ethyl 4-piperidinecarboxylate, piperidin-4one, and 2-piperidinemethanol were obtained from Aldrich Chemical Co. (Milwaukee, WI). All reagents were also purchased from Aldrich Chemical Co. and used without further purification unless noted. Tetrahydrofuran (THF) was dried over KOH and then distilled over sodium and benzophenone. All reactions were carried out under nitrogen atmosphere unless specifically stated. Reactions were monitored by thin layer chromatography (TLC) using silica gel plates (Merck Type 60H) and visualization by iodine or by dipping them in a solution (0.5 mL of sulfuric acid, 9 mL of ethanol, and 0.5 mL of anisaldehyde) followed by heating at ; 120 –150°C. Flash chromatography was done using silica gel (230 – 400 mesh) from Aldrich Chem. Co.

Radiochemical Syntheses No-carrier-added [ C]CO2 was prepared by proton irradiation of a nitrogen gas target [14N(p,a)11C] and converted sequentially to [11C]methyl iodide and [11C]methyl triflate. The [11C]methyl triflate was bubbled through a small vessel containing a solution of 1–2 mg of a piperidinyl ester in 100 mL of dimethyl sulfoxide (DMSO). After complete transfer of the radioactive precursor, the reaction was stopped by addition of 500 mL of a 0.1-M NH4OAC/ethanol (9/1) solution. Purification of products were done as follows: the crude reaction mixture was injected onto a reversed-phase C8 high performance liquid chromatography (HPLC)-column (Phenomenex Ultremex, 5 m, 250 3 4.6 mm) and eluted with a 0.1-M NH4OAC/ethanol (9/1) using flow rates of 1.0 –1.3 mL/min. For quality control (QC), the products were analyzed for purity using a reversed-phase C8 column (30 3 4.6 mm), using ultraviolet (UV; 220 nm) and radioactivity flow detectors. The mobile phase for HPLC was acetonitrile/0.05M NH4CHO2 (25/75)with flow rates of 1.5–1.6 mL/min. All [11C]esters were obtained in .95% chemical and radiochemical purities. Radiochemical yields ranged from 10 to 50% (corrected for decay) with overall synthesis and isolation times of 30 – 40 min from end-of-bombardment; synthesis times and yields were not optimized. Isolated amounts of final products ranged from 10 to 200 mCi. With the exception of [11C]2, which had an apparent specific activity of 150 Ci/mmol, all other [11C]esters were obtained in specific activities of 1,000 –3,000 Ci/mmol. GENERAL PROCEDURE FOR [11C]METHYLATIONS.

11

Yield 27%; Prep. HPLC: 1.0 mL/min; Anal. HPLC: Rt 5 7.42 min at 1.5 mL/min. ETHYL N-[11C]METHYLPIPERIDIN-4-YL CARBOXYLATE ([11C]1).

Yield 10%; Prep. HPLC: 1.0 mL/min; Anal. HPLC: Rt 5 6.37 min at 1.5 mL/min. ETHYL N-[11C]METHYLPIPERIDIN-3-YL CARBOXYLATE ([11C]2).

Yield 30%; Prep. HPLC: 1.0 mL/min; Anal. HPLC: 7.98 min at 1.5 mL/min. ETHYL N-[11C]METHYLPIPERIDIN-2-YL CARBOXYLATE ([11C]3).

N-[11C]METHYLPIPERIDIN-4-YL ISOBUTYRATE ([11C]4). Yield 40%; Prep. HPLC. 1.3 mL/min; Anal. HPLC: 11.11 min at 1.6 mL/min.

Yield 52%; Prep. HPLC: 1.0 mL/min; Anal. HPLC: 2.89 min at 1.6 mL/min. 4-METHYL-N-[11C]METHYLPIPERIDIN-4-YL

ACETATE

([11C]5).

4-METHYL-N-[11C]METHYLPIPERIDIN-4-YL PROPIONATE ([11C]6). Yield 43%; Prep. HPLC: 1.0 ml/min; Anal. HPLC: 5.24 min at 1.5 mL/min. 3-METHYL-N-[11C]METHYLPIPERIDIN-4-YL PROPIONATE ([11C]7). Yield 29%; Prep. HPLC: 1.3 mL/min; Anal. HPLC: 8.24 min at 2.0 mL/min.

Yield 22%; Prep. HPLC: 1.0 mL/min; Anal. HPLC: 3.74 min at 1.5 mL/min. (N-[11C]METHYLPIPERIDIN-2-YL)METHYL PROPIONATE ([11C]8).

Syntheses of Precursors and Standards As the synthetic routes employed four standard reactions (protection of piperidine nitrogens as the N-benzoylcarbamates; esterification of alcohols with acid chlorides; removal of N-benzoylcarbamates by hydrogenation; and N-alkylation of piperidine nitrogens with methyl iodide), representative experimental methods are provided for each of these reactions. Unless otherwise noted, the same reaction conditions were used in subsequent syntheses. EXAMPLE OF N-BENZYLCARBAMOYLATION OF PIPERIDINE ANALOGS.

To a solution of 4-hydroxypiperidine (2.02 g, 20.0 mmol) in H2O (50 mL) was added Na2CO3 (15 g, 94 mmol) and benzyl chloroformate (3.42 g, 2.86 mL, 20.0 mmol). The reaction mixture was stirred overnight, then diluted with water and extracted with CH2Cl2. The organic extracts were combined, dried over MgSO4, and concentrated under reduced pressure to give N-(benzylcarbamoyl)piperidin-4-yl alcohol in 94% yield (4.4 g). 1H NMR d 1.42–1.54 (m, 2H), 1.65 (br s, 1H), 3.14 (dd, t, 2H), 3.81–3.96 (m, 4H), 5.12 (s, 2H), 7.27–7.40 (m, 5H). N-(BENZYLCARBAMOYL)PIPERIDIN-4-YL ALCOHOL (10).

EXAMPLE OF ESTERIFICATION OF PIPERIDINYL ALCOHOLS. SYNTHESIS

To a solution of 4-hydroxy-N-methylpiperidine (169 mg, 1.47 mmol) in CH2Cl2 (5 mL) was added isobutyryl chloride (159 mg, 0.157 mL, 1.5 mmol) at room temperature. After 1 h, it was quenched with aqueous NaHCO3, extracted with additional CH2Cl2, and the organic extracts dried over MgSO4 and concentrated. Flash chromatography (CH2Cl2/CH3OH, 9/1) provided 156 mg of N-methylpiperidin-4-yl isobutyrate (57% yield): 1H NMR d 1.16 (t, J 5 10 Hz, 6H), 1.83–1.90 (m, 2H), 2.0 –2.1 (m, 2H), 2.47 (s, 3H), 2.50 –2.84 (m, 5H), 4.89 (t, 1H). Mass spec (EI) 185 (51, M1), 114 [38, M1-OCCH(CH3)2], 98 [100, M1-OOCCH(CH3)2]. HRMS calculated for C10H19NO2 185.1416, found 185.1414. OF N-METHYLPIPERIDIN-4-YL ISOBUTYRATE (4).

EXAMPLE OF HYDROGENATION OF PIPERIDINE ANALOGS. SYNTHESIS

To a solution of (N-benzylcarbamoyl)piperidin-4-yl isobutyrate (11) (3.6 g, 10.0 mmol) in diethyl ether (20 mL) was added 10% Pd/C (0.72 g). The reaction flask was purged with H2 three times, and the hydrogen pressure was maintained at 1 atm. The reaction mixture was stirred vigorously for 2 h. TLC indicated that all starting material was converted into product. The catalyst was removed by filtration and the organic solvent to provide piperidin-4-yl isobutyrate in 97% yield: 1H NMR d 1.70 (d, J 5 7 Hz, 6H), 1.95–2.11 (m, 2H), 2.18 –2.28 (m, 2H), OF PIPERIDIN-4-YL ISOBUTYRATE (12).

Synthesis of in Vivo Acetylcholinesterase Substrates

2.47–2.56 (m, 1H), 3.18 –3.32 (m, 4H), 5.05–5.15 (m, 1H). HRMS calculated for C9H17NO2 171.1259, found 171.1260. EXAMPLE OF N-METHYLATION OF PIPERIDINE ANALOGS. ETHYL

To a solution of ethyl 2-piperidinecarboxylate (0.786 g, 0.78 mL, 5.0 mmol) in dry THF (5 mL) was added methyliodide (0.738 g, 0.324 mL, 5.2 mmol) dropwise. The reaction mixture was stirred for 3 h at room temperature. It was then quenched with aqueous NaHCO3, extracted with ether, and the organic extract dried over MgSO4 and concentrated under reduced pressure. Flash chromatography (CH2Cl2/CH3OH, 9/1) provided 0.553 g (65%) of ethyl N-methylpiperidin-2-yl carboxylate: 1H NMR d 1.28 (t, J 5 7 Hz, 3H), 1.60 –1.76 (m, 5H), 1.82–1.86 (m, 1H), 2.06 (t, J 5 7 Hz, 1H), 2.26 (s, 3H), 2.70 (d, J 5 7 Hz, 1H), 2.92–2.97 (m, 1H), 4.21 (q, J 5 15 Hz, 2H). Mass spec (EI) 171 (63, M1), 170 (66), 156 (28, M1-CH3), 142 (100, M1-CH2CH3), 126 (63), 98 (70, M1COOCH2CH3). HRMS calculated for C9H17NO2 171.1259, found 171.1337. N-METHYLPIPERIDIN-2-YL CARBOXYLATE (3).

ETHYL N-METHYLPIPERIDIN-4-YL CARBOXYLATE (1). This compound was prepared in 35% yield by alkylation of ethyl 4-piperidinecarboxylate with methyl iodide: 1H NMR d 1.25 (t, J 5 8 Hz, 3H), 1.7–2.1 (m, 6H), 2.67 (s, 3H), 2.55 (s, 1H), 2.82 (d, J 5 13 Hz, 2H), 4.12 (d, J 5 15 Hz, 2H); Mass spec (CI) 172 (100, M1 1 H), 158 (M1-CH2), 98 (85, M1-COOCH2CH3). HRMS calculated for C9H17NO2 171.1259, found 171.1257. ETHYL N-METHYLPIPERIDIN-3-YL CARBOXYLATE (2). This compound was prepared in 55% yield by alkylation of ethyl 3-piperidinecarboxylate with methyl iodide: 1H NMR d 1.26 (t, J 5 7 Hz, 3H), 1.37–1.47 (m, 1H), 1.6 –1.8 (m, 2H), 1.9 –2.1 (m, 2H), 2.15 (t, J 5 11 Hz, 1H), 2.31 (s, 3H), 2.5–2.7 (m, 1H), 2.7–2.8(m, 1H), 2.9 –3.1 (m, 1H), 4.13 (q, J 5 15 Hz, 2H). Mass spec (EI) 171 (54, M1), 156 (21, M1-CH3), 142 (100, M1-CH2CH3), 126 (45), 98 (58, M1-COOCH2CH3). HRMS calculated for C9H17NO2 171.1259, found 171.1252. N-METHYLPIPERIDIN-4-YL ISOBUTYRATE (4). This ester was prepared by two independent routes. Acylation of N-methyl-4-piperidinol with isobutyryl chloride (see example procedure above) gave the authentic material in good yield. The same ester was prepared by a second route, involving formation of the ester before N-methylation, as follows. The reaction of isobutyryl chloride (1.59 g, 1.56 mL, 15 mmol) with N-(benzylcarbamoyl)piperidin-4-ol (10) (1.56 g, 6.6 mmol) produced, after flash chromatography (hexane/ethyl acetate, 3/1), N-benzylcarbamoyl)piperidin-4-yl isobutyrate (11) in 85% yield: (1.71 g). 1H NMR d 1.16 (d, J 5 7 Hz, 6H), 1.64 (s, br, 2H), 1.83 (s, br, 2H), 2.49 –2.58 (m, 1H), 3.34 –3.42 (m, 2H), 3.68 –3.76 (m, 2H), 4.90 – 4.96 (m, 1H), 5.13 (s, 2H), 7.26 –7.39 (m, 5H). This compound was used without further purification. Hydrogenolysis of the N-protecting group was done as described above, to yield piperidin-4-yl isobutyrate (12) in 97% yield: 1H NMR d 1.70 (d, J 5 7 Hz, 6H), 1.99 –2.06 (m, 2H), 2.17–2.27 (m, 2H), 2.52–2.59 (m, 1H), 3.22–3.30 (m, 4H), 5.04 –5.06 (m, 1H). HRMS calculated for C9H17NO2 171.1259, found 171.1260. Finally, alkylation of piperidin-4-yl isobutyrate with methyl iodide provided a sample of the N-methyl compound identical by TLC and HPLC with the material prepared by acylation of the N-methylpiperidinol. 4,N-DIMETHYLPIPERIDIN-4-YL ACETATE (5). N-(Benzylcarbamoyl) piperidin-4-one (13) was prepared by reaction of piperidin-4-one with benzyl chloroformate: Yield 93%; 1H NMR d 2.45 (s (br) 4H),

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3.77–3.81 (m, 4H), 5.18 (s, 2H), 7.32–7.39 (m, 5H). This ketone was then used for the methylation step without further purification. To a suspension of CeCl3 z 7H2O [3.75 g, 10.0 mmol; dried under reduced pressure (0.05 mmHg) at 100°C for 3 h] in THF (50 mL) was added methyllithium (7.5 mL, 10.0 mmol in THF) slowly at 278°C. After stirring for 1 h, N-(benzylcarbamoyl)piperidin-4-one (2.33 g, 10.0 mmol) in THF (10 mL) was added dropwise to the reaction mixture. It was stirred for 3 h, then quenched with aqueous NaHCO3, extracted with ethyl acetate, and the extracts dried over MgSO4 and concentrated to give of N-(benzylcarbamoyl)-4-hydroxy-4-methylpiperidine (14) (2.18 g, 87% yield). Reaction of N-(benzylcarbamoyl)-4-hydroxy-4-methylpiperidine (771 mg, 3.13 mmol) and acetyl chloride (0.47 g, 0.43 mL, 6.0 mmol) gave N-(benzylcarbamoyl)-4-methylpiperidin-4-yl acetate (15a) in 63% yield (571 mg): 1H NMR d 1.52 (s, 3H), 1.51–1.57 (m, 2H), 2.02 (s, 2H), 2.18 (s, 1H), 2.21 (s, 1H), 3.12 (t, J 5 8 Hz), 2H), 3.89 (s, br, 2H), 5.17 (s, 2H), 7.26 –7.39 (m, 5H). The N-protecting group was then removed by hydrogenolysis to give (4-methylpiperidin)-4-yl acetate (16a) in 87% yield: 1H NMR d 1.29 (s, 3H), 1.50 –1.64 (m, 3H), 2.10 (s, 3H), 3.10 –3.17 (m, 2H), 3.42–3.53 (m, 2H), 4.16 – 4.24 (m, 1H). HRMS calculated for C8H15NO2 157.1103, found 157.1102. Finally, 4-methylpiperidin-4-yl acetate was alkylated with methyl iodide to give 4,N-dimethylpiperidin-4-yl acetate (5) in 53% yield: 1 H NMR d 1.52 (s, 3H), 1.56 –1.73 (m, 2H), 2.03 (s, 3H), 2.22–2.34 (m, 3H), 2.31 (s, 3H), 2.56 –2.65 (m, 2H). Mass spec (CI) 172 (100, M1 1 H), 158 (15), 112 (M1-OOCCH3). HRMS (CI) calculated for C9H17NO2 [(M1H)1] 171.1259, found 172.1337. 4,N-DIMETHYLPIPERIDIN-4-YL PROPIONATE (6). This compound was prepared in a manner analagous to the acetate ester. Acylation of N-(benzylcarbamoyl)-4-hydroxy-4-methylpiperidine (14) with propionyl chloride gave N-(benzylcarbamoyl)-4-methylpiperidin-4-yl propionate (15b) in 60% yield: 1H NMR d 1.12 (t, J 5 7 Hz, 3H), 1.52 (s, 3H), 1.51–1.56 (m, 2H), 2.18 (d, J 5 9 Hz, 2H), 2.29 (q, J 5 15 Hz, 2 H), 3.10 ( t, J 5 12 Hz, 2H), 3.90 (s, 2H), 5.13 (s, 2H), 7.273–7.35 (m, 5H). Hydrogenolysis gave (4-methylpiperidin)-4-yl propionate (16b) in 97% yield: 1H NMR d 1.14 (t, J 5 7 Hz, 3H), 1.51 (s, 3H), 1.53–1.72 (m, 3H), 2.15 [d (br), J 5 13 Hz, 2H], 2.27 (q, J 5 7 Hz, 2H), 3.11–3.14 (m, 1H), 3.43–3.54 (m, 2H), 4.18 – 4.23 (m, 1H). HRMS calculated for C9H17NO2 171.1259, found 171.1260. As a last step, 4-methylpiperidin-4-yl propionate was alkylated with methyl iodide to give 4,N-dimethylpiperidin-4-yl propionate (6) in 43% yield: 1H NMR d 1.15 (t, J 5 10 Hz, 3H), 1.58 (s, 3H), 2.12–2.18 (m, 2H), 2.29 –2.46 (m, 2H), 2.73 (s, 3H), 2.67–2.82 (m, 2H) 3.23 (d (br), J 5 8 Hz, 2H), 4.04 (q, J 5 7 Hz, 2H). Mass spec (CI) 186 (100, M1 1 H), 172 (15), 112 (M1-OOCCH2CH3). HRMS (CI) calculated for C10H20NO2 [(M1H)1] 186.1494, found 186.1494. 3,N-DIMETHYLPIPERIDIN-4-YL PROPIONATE (7). This ester was prepared in five steps from the N-protected piperidinone, as follows. To a solution of LDA [lithium di-isopropylamide; freshly prepared from di-isopropylamine (2.12 g, 2.94 mL, 21.0 mmol) and n-butyllithium (8.94 mL, 21.0 mmol) in THF (40 mL) at 278°C] was added N-benzylcarbamoylpiperidin-4-one (4.66 g, 20.0 mmol) in THF (20 mL). After stirring for 1 h at this temperature, methyliodide (3.55 g, 1.56 mL, 25 mmol) was added to the reaction mixture and stirring continued for 3 h. The reaction was quenched with aqueous NaHCO3, extracted with ethyl acetate, and the organic solution dried over MgSO4 and concentrated. The crude product (17) was dissolved in a 1/1 solution of CH3OH/CH2Cl2. NaBH4 was added

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8 Hz, 2H), 2.69 ( d, J 5 10 Hz, 2 H), 2.9 –3.1 (m, 2H), 4.46 – 4.54 (m, 1H). N-alkylation of 3-methylpiperidin-4-yl propionate with methyl iodide gave 3,N-dimethylpiperidin-4-yl propionate (7) in 59% yield: 1H NMR d 1.01 (t, J 5 17 Hz, 3H), 1.15 (t, J 5 7 Hz, 3H), 1.69 –1.81 (m, 1H), 1.88 [s (br), 1H], 2.05 [s (br), 1H], 2.15 (s, 3H), 2.28 (s, 3H), 2.35 (q, J 5 8 Hz, 2H), 2.64 [s (br), 1H], 4.54 – 4.58 (m, 1H). Mass spec (CI) 186 (100, M1 1 H), 172 (14), 112 (M1-OOCCH2CH3). HRMS (CI) calculated for C10H20NO2 [(M1H)1] 186.1494, found 186.1487.

FIG. 1. Syntheses of ethyl N-methylpiperidin-4-yl carboxylate, ethyl N-methylpiperidin-3-yl carboxylate, and ethyl N-methylpiperidin-2-yl carboxylate. in small portions at room temperature, and the reaction mixture then stirred for 5 h. It was concentrated and water added, extracted with ethyl acetate, and the organic extracts dried over MgSO4 and concentrated. The crude product (18) was used in the next step without further purification. Esterification was done using propionyl chloride, by methods described above. The N-(benzylcarbamoyl)3-methylpiperidin-4-yl propionate (19) was obtained in 40% yield for three steps. 1H NMR d 0.91 (d, J 5 6 Hz, 3H), 1.15 ( t, J 5 7 Hz, 3H), 1.40 –1.72 (m, 3H), 2.35 (q, J 5 7 Hz, 1H), 3.24 [s (br), 1H], 3.75 [s (br), 1H], 3.67– 4.06 (m, 2H), 4.56 (t, 1H), 5.13 (s, 2H), 7.26 –7.38 (m, 5H). Hydrogenolysis under standard conditions gave (3-methylpiperidin)-4-yl propionate (20) in 95% yield: 1H NMR d 0.87 (d, J 5 6 Hz, 3H), 1.15 ( t, J 5 7 Hz, 3H), 1.59 –1.71 (m, 4H), 2.34 ( q, J 5

(N-METHYLPIPERIDIN-2-YL)METHYL PROPIONATE (8). Reaction of 2-piperidinemethanol with benzyl chloroformate gave N-(benzylcarbamoylpiperidin-2-yl)methanol (21) in 99% yield: 1H NMR d 1.40 –1.86 (m, 6H), 2.3–2.5 [s (br), 1H], 2.90 (t, J 5 7 Hz, 1H), 3.62–3.75 (m, 1H), 3.82 (t, J 5 10 Hz, 1H), 3.9-4.0 (m, 1H), 4.3– 4.4 (m, 1H), 5.13 (s, 2H), 7.2–7.4 (m, 5H). Acylation of the N-protected alcohol (1.75 g, 7.03 mmol) with propionyl chloride (1.39 g, 1.3 mL, 15.0 mmol) yielded [N-(benzylcarbamoyl)piperidin-2-yl]methyl propionate (22) in 83% yield (1.77 g): 1H NMR d 1.06 (t, J 5 7Hz, 3H), 1.41–1.51 (m, 2H), 1.52–1.66 (m, 4H), 2.21 (q, J 5 14 Hz, 2H), 2.90 (t, J 5 7 Hz, 1H), 4.10 – 4.13 (m, 2H), 4.31 (t, J 5 9 Hz, 1H), 5.58 [s (br), 1H], 5.13 (s, 2H), 7.29 –7.36 (m, 5H). Hydrogenolysis of 1-[(N-benzyl)piperidin-2-yl]methyl propionate (0.584 g) in methanol (10 mL) using H2 and 10% Pd/C (0.12 g) required 2 days for complete reaction, yielding 1-(piperidin-2yl)methyl propionate (23) in quantitative yield. The reaction was also done using 4.4% formic acid in methanol as a H2 source (5), with the reaction complete in 1 h. 1H NMR d 1.07 (t, J 5 7 Hz, 3H), 1.52–1.91 (m, 6H), 2.40 (q, J 5 7 Hz, 2H), 2.68 –2.76 (m,

FIG. 2. Synthesis of N-methylpiperidin-4-yl isobutyrate.

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FIG. 3. Syntheses of 4,N-dimethylpiperidin-4-yl acetate and propionate. 1H), 2.99 –3.03 (m, 1H), 3.75–3.81 (m, 1H), 3.98 – 4.06 (m, 1H), 4.41– 4.52 (m, 1H). As a final step, 1-(piperidin-2-yl)methyl propionate was alkylated with methyl iodide to provide the desired final product, (Nmethylpiperidin-2-yl)methyl propionate (8) in 95% yield: 1H NMR d 1.14 (t, J 5 7 Hz, 3H), 1.25–1.73 (m, 6H), 2.05–2.12 (m, 2H), 2.12 (s, 3H), 2.37 (q, J 5 8 Hz, 2H), 2.87 [d (br), J 5 12 Hz, 1H], 4.14 (m, 2H). Mass spec (CI) 186 (M1 1 H), 172 (M1-CH2). HRMS calculated for C10H20NO2 186.1494, found 186.1485. RESULTS AND DISCUSSION Although AChE is a remarkably efficient enzyme for the hydrolysis of acetylcholine (turnover rate of 50,000 molecules/s) (6), it is promiscuous and will hydrolyze a wide variety of unrelated esters (10). The enzyme also binds, and can often react with, a number of other chemical structures (carbamates, ketones), several of which form the basis of important reversible and irreversible inhibitors (4, 20). Taken together, those data suggest that the enzyme binding site can accommodate a wide variety of structural types of molecules, with however widely varying affinities and efficiencies in subsequent hydrolysis. The prior studies of N-methylpiperidinyl esters had shown, in general, that the higher homologs of the esters had slower rates of reaction with the enzyme (7, 8, 12), culminating in no observable hydrolysis for the benzoate esters (2). As none of these previous studies had examined the effects of increased steric bulk in other positions of the piperidine ester structure, or the effects of rearrangement of the heteroatoms, we synthesized such esters. All of the synthesized compounds are structural analogs or derivatives of the parent radiotracer, N-[11C]methylpiperidin-4-yl propionate (4[11C]PMP).

Position of Heteroatoms: N-Methylpiperidinecarboxylic Acid Esters Examination of the structure of N-methylpiperidin-4-yl propionate (4-PMP) indicates an identical order of N, O, and C 5 O moieties as found in acetylcholine (Me2NCH2CH2OCOCH3). This relative order can be easily changed to N, C 5 O, and O (the “reverse” ester), resulting in the structures 1–3 (Fig. 1), which are more commonly referred to as N-methylnipecotic, N-methylisonipecotic, and N-methylpipecolinic acid ethyl esters, respectively. Nipecotic acid, isonipecotic acid, and pipecolinic acid ethyl esters are commercially available, and were easily converted to the N-methyl derivatives by simple alkylation with methyliodide. The carbon-11labeled form of each ester were readily prepared by alkylation of the secondary amines with [11C]methyltriflate in DMSO solution. For unexplained reasons, the apparent specific activity of [11C]3 was significantly lower than that obtained with any of the other [11C]ester prepared in this study.

N-Methyl-4-Hydroxypiperidine Esters: Variation of Acid Chain We have previously reported syntheses of the acetate (4-[11C]AMP) and propionate (4-[11C]PMP) esters of N-[11C]methylpiperidin-4-ol (13). To examine the effect of a larger ester chain, the isobutyric acid ester was first prepared in a single step by reaction of N-methyl-4-hydroxypiperidine with isobutyryl chloride, providing the ester in 57% yield. For labeling with carbon-11 the desmethyl ester (12) was required: this compound was prepared by by a standard sequence of (a) protection of the amine using benzylchloroformate, (b) esterification of the alcohol with isobutyryl chloride, and (c) removal of the amine protecting group by catalytic hydrogenation (Fig. 2). Attempts to shorten the synthesis of the

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FIG. 4. Syntheses of 3,N-dimethylpiperidin-4-yl propionate.

desmethyl ester (12) by treating 4-hydroxypiperidine with 1 eq of n-butyllithium in THF at 278°C to form the lithium alkoxide, followed by reaction with isobutyl chloride at low temperature, led to a mixture of the desired ester (12) and the isobutylamide produced by N-acylation. Alkylation of 4-piperidinyl isobutyrate (12) with methyl iodide provided a product identical with that prepared by the O-acylation of N-methylpiperidin-4-ol. The secondary amine (12) could also be easily alkylated with [11C]methyltriflate to provide the desired 4-N-[11C]methyl-4-hydroxypiperidinyl isobutyrate (4, 4-[11C]iBMP).

Piperidine Ring Substitutions Alkyl group substitution of the piperidine ring of 4-PMP might also slow down the reaction with AChE, either by inhibiting the initial binding of the substrate to the enzyme, or interfering with the approach of the functional groups involved in ester hydrolysis. To examine this, we have synthesized derivatives of 4-PMP with methyl groups added at the 3 or 4 positions of the piperidine ring. The syntheses of 4,N-dimethylpiperidin-4-yl acetate (5) and 4,Ndimethylpiperidin-4-yl propionate (6) are shown in Figure 3. The amine group of piperidin-4-one (1) was first protected by reaction with benzyl chloroformate. Reaction of the N-protected piperidinone 13 with methyllithium in tetrahydrofuran at 278°C produced N-(benzylcarbamoyl)-4-methylpiperidin-4-ol (14) in 67% yield; this yield could be improved to 87% by first treating the methyllithium with dried CeCl3 before adding to a solution of the N-(benzylcarbamoyl)piperidinyl-4-one. The tertiary alcohol (14) was then esterified with either acetyl choride or propionyl chloride to provide the corresponding esters, N-(benzylcarbamoyl)-4-methylpiperidin-4-yl acetate 15a and N-(benzylcarbamoyl)-4-methylpiperidin-4-yl propionate 15b. Removal of the benzylcarbamate groups was again done using catalytic hydrogenation, providing the

secondary amines (16a, 16b) as free bases. Alkylation with methyliodide provided the two N-methyl-4-methylpiperidin-4-yl esters 5 and 6; the corresponding carbon-11-labeled esters were then prepared in good yields by N-alkylation with [11C]methyltriflate. The synthesis of 3,N-dimethylpiperidin-4-yl propionate (7) is shown in Figure 4. The protected amine (13), N-(benzylcarbamoyl)piperidin-4-one, was treated with lithium diisopropylamine at 278°C for 30 min, followed by the slow addition of methyliodide. The intermediate a-methylketone was then reduced with borohydride to give N-(benzylcarbamoyl)-3-methylpiperidin-4-ol (17). Addition of the methyl group and reduction of the ketone creates two chiral centers, and thus four potential isomers: no separation of these isomers was performed at this step. The steps of esterification (propionyl chloride) and removal of the N-protecting group (catalytic hydrogenation) were done as described previously, and the secondary amine was then alkylated with methyliodide to give the final product, 3,N-dimethylpiperidin-4-yl propionate (7), which again was isolated as the mixture of all four possible isomers. Alkylation of the desmethyl precursor 20 with [11C]methyltriflate gave the corresponding carbon-11 labeled ester [11C]7 in good yield, with the product again isolated as the mixture of isomers.

Other Piperidine Esters We recognized that a structure such as (N-methylpiperidin-2yl)methyl propionate (8) includes the backbone structure of acetylcholine; that is, the distance between N and O atoms is two methylene groups, and the molecule contains the tertiary amine and ester groups present and in the same order as in substrates such as 4-PMP. To the best of our knowledge, piperidines with an ester group at the 2-position on the ring as in 8 have not been previously examined as AChE substrates. Employing the same general synthetic route of N-protection, O-acylation, N-deprotection, and

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FIG. 5. Synthesis of (N-methylpiperidin-2-yl)methyl propionate.

N-methylation, (N-methylpiperidin-2-yl)methyl propionate 8 was synthesized from commercially available 2-piperidinemethanol (22) (Fig. 5). The starting material utilized was racemic, and no attempt was made to separate the isomers at either the secondary amine 23 or tertiary amine 8 stage. Similarly, no attempt was made to separate the isomers of (N-[11C]methylpiperidin-2-yl)methyl propionate ([11C]8), which was prepared in 22% yield by N-alkylation of the secondary amine 23 using [11C]methyltriflate. CONCLUSIONS We have prepared a series of simple esters, all of which are structurally related to 4-N-methylpiperidinyl propionate (4-PMP), but which incorporate changes in the order of the heteroatoms (piperidinecarboxylic acid esters), additional steric bulk (methyl substituents on acid chain or piperidine ring), or alkyl chain configuration (piperidinol vs. piperidinylmethanol). All of the esters (compounds 1– 8) were prepared in carbon-11-labeled form by a standard method of reaction of the secondary amine with no-carrier-added [11C]methyl triflate at room temperature, with purification by reverseD-phase HPLC. Yields were acceptable (10 – 50%, corrected for decay) and no attempts were made to optimize reaction conditions. Specific activities of the products were uniformly high with the exception of compound [11C]2, but even 150 Ci/mmol is more than sufficient for in vivo use of this radiotracer, as we have previously shown that the in vivo AChE-mediated hydrolysis of 4-[11C]PMP was not sensitive to specific activity (13). The in vivo pharmacokinetics of these radiolabeled esters in mouse brain will be reported elsewhere.

Financial support from the National Institutes of Health [grants NS24896 and T-32-CA09015 (to T.B.N.)] and from the Department of Energy (grant DOE-DE-FG021-87ERG0561) are gratefully acknowledged. We also thank the staff of the Cyclotron Facility at the University of Michigan for technical assistance.

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