Thioesters for the in vitro evaluation of agents to image brain cholinesterases

Article abstract of DOI:10.3109/14756366.2011.647008

Cholinesterases are associated with pathology characteristic of conditions such as Alzheimer's disease and are therefore, considered targets for neuroimaging. Ester derivatives of N-methylpiperidinol are promising potential imaging agents; however, method

Full text of DOI:10.3109/14756366.2011.647008

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 447–455
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.647008
ReseaRch aRtIcle
ioesters for the in vitro evaluation of agents to image brain
cholinesterases
Ian R. Macdonald1, Courtney T. Jollymore2, G. Andrew Reid1, Ian R. Pottie2,3, Earl Martin2, and
Sultan Darvesh1,2,4
1Department of Anatomy & Neurobiology and the Neuroscience Institute, Dalhousie University, Halifax, Nova Scotia,
Canada, B3H 4R2, 2Department of Chemistry and Physics, Mount Saint Vincent University, Halifax, Nova Scotia,
Canada, B3M 2J6, 3Department of Chemistry, Saint Mary’s University, Halifax, Nova Scotia, B3H 3C3, and 4Department
of Medicine (Neurology and Geriatric Medicine), Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4R2
abstract
Cholinesterases are associated with pathology characteristic of conditions such as Alzheimer’s disease and are
therefore, considered targets for neuroimaging. Ester derivatives of N-methylpiperidinol are promising potential
imaging agents; however, methodology is lacking for evaluating these compounds in vitro. Here, we report the
synthesis and evaluation of a series of N-methylpiperidinyl thioesters, possessing comparable properties to their
corresponding esters, which can be directly evaluated for cholinesterase kinetics and histochemical distribution in
human brain tissue. N-methylpiperidinyl esters and thioesters were synthesized and they demonstrated comparable
cholinesterase kinetics. Furthermore, thioesters were capable, using histochemical method, to visualize cholinesterase
activity in human brain tissue. N-methylpiperidinyl thioesters can be rapidly evaluated for cholinesterase kinetics
and visualization of enzyme distribution in brain tissue which may facilitate development of cholinesterase imaging
agents for application to conditions such as Alzheimer’s disease.
Keywords: Butyrylcholinesterase, acetylcholinesterase, brain histochemistry, neuroimaging, Alzheimer’s disease
Introduction
were synthesized as AChE-specific imaging agents. Two
such compounds, 4-(N-[11C]methyl)piperidinyl acetate
(4-[11C]AMP) and 4-(N-[11C]methyl) piperidinyl propio-
nate (4-[11C]PMP), were examined17–21 and provided some
e cholinergic system is affected in a number of dis-
orders, such as Alzheimer’s disease (AD)1–3, multiple
sclerosis4,5 and primary brain tumors6. In the brain cho-
linergic system, two enzymes, acetylcholinesterase
(AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC
3.1.1.8), catalyze the hydrolysis of acetylcholine, thereby
regulating the actions of this neurotransmitter7. Changes
in the activity and location of cholinesterases in neuro-
logical disorders render these enzymes suitable targets
for neuroimaging in the living brain, to facilitate early
diagnosis and treatment monitoring. In AD, cholinest-
erases, particularly BuChE, are associated with charac-
teristic pathological hallmarks of the disease, β-amyloid
reproduction of the known brain distributions of AChE18,21
.
Specific BuChE visualization was also attempted with
4-(N-[11C]methyl) piperidinyl n-butyrate (11C-MP4B)22
but was unsuccessful23. Alternate types of BuChE imaging
ligands, derived from N-methylpiperidinol and N-methyl
pyrrolidinol, were subsequently synthesized and tested in
a normal rat model system24. Using autoradiographic anal-
ysis these para-123iodobenzoate esters were found to have
entered the brain and their distribution recapitulated, for
the most part, the known distribution of BuChE therein.
Based on these observations, N-methylpiperidinyl com-
pounds remain promising agents for the imaging of cho-
linesterases in the living brain.
(Aβ) plaques and neurofibrillary tangles (NFTs)8–16
Inearlierattemptstorealizecholinesteraseimaging,sev-
eral 11C-radiolabelled alkyl esters of N-methylpiperidinol
.
Address for Correspondence: Dr. Sultan Darvesh, Room 1308, Camp Hill Veterans’ Memorial Building 1, 5955 Veterans’ Memorial Lane,
Halifax, Nova Scotia, Canada, B3H 2E1. Phone: 1-902-473-2490. Fax: 1-902-473-7133. E-mail: sultan.darvesh@dal.ca
(Received 31 October 2011; revised 29 November 2011; accepted 29 November 2011)
447

448 I. R. Macdonald et al.
abbreviations
AV, anteroventral nucleus
4-[11C]AMP,4-(N-[11C]methyl)piperidinyl acetate
4-[11C]PMP, 4-(N-[11C]methyl) piperidinyl propionate
11C-MP4B, 4-(N-[11C]methyl) piperidinyl n-butyrate
Aβ, β-amyloid; AChE, acetylcholinesterase
AD, Alzheimer’s disease
BuChE, butyrylcholinesterase
DAB, 3, 3′-diaminobenzidine tetrahydrochloride
Km, affinity constant
MMFF, Merk Molecular Force Field
NFT, neurofibrillary tangles.
In vivo testing of the N-methylpiperidinyl cholinester-
ase imaging agents, 4-[11C]AMP, 4-[11C]PMP and 11C-MP4B,
has been extensive. However, methodology for determina-
tion of their specificity and direct measurement of their
actual affinity constants for AChE and BuChE has been dif-
ficult due to the lack of a suitable chromophore that would
allow the use of sensitive spectrophotmetric methods.
Tracking hydrolysis by radiolabelling reactants and prod-
ucts25 or by employing the chromophore of m-nitrophenol
to tag reactants in a spectrophotometric approach22 have
both proved cumbersome for screening of compounds as
potential imaging agents. us, a rapid in vitro evaluation
method would be beneficial for the continued develop-
ment of imaging agents derived from N-methylpiperidinol.
eEllmanspectrophotometricmethod26, orvariations
thereof27, remains the most rapid, reliable and sensitive
means to measure AChE or BuChE activity. e principle
on which this assay is based is that, thioesters, such as
acetylthiocholineandbutyrylthiocholine,shouldbekinet-
ically analogous to their corresponding esters with respect
to cholinesterase catalysis. A kinetic analysis28 comparing
benzoylcholine and benzoylthiocholine supports this
notion. In addition to their use in Ellman cholinesterase
assays, thioesters are employed for in vitro histochemical
detection of cholinesterase activity in tissues. One of the
commonly used histochemical techniques for detection
of AChE and BuChE is the Karnovsky-Roots method29.
In this procedure, hydrolysis of acetylthiocholine and
butyrylthiocholine generates a thiolate anion that leads to
a precipitate in the area of enzyme activity. is precipi-
tation facilitates detection of enzyme distribution under
the microscope. us, in theory, any thioester susceptible
to cholinesterase hydrolysis could be characterized using
Ellman enzyme kinetics and should enable histochemical
visualization of areas containing enzyme activity in brain
tissue using the Karnovsky-Roots method.
Compound characterization methods are also presented
therein.
Brain tissues
Brain tissues used in this study were provided by the
Maritime Brain Tissue Bank, (Halifax, Nova Scotia,
Canada), following approval from the Capital Health
Ethics Board, from the brain of a 90 year old female,
removed within 20 h of death. e brain was immersion
fixed in 10% formalin in 0.1 M phosphate buffer (pH 7.4)
for approximately 94 h and cut in 1–2 cm slabs. ese
slabs were cryoprotected by immersion in increasing
concentrations of sucrose, ranging from 10% to 40% in
0.1 M phosphate buffer (pH 7.4). e brain tissue was in
each concentration of sucrose solution for approximately
48 h and was stored in 40% buffered sucrose (pH 7.4) with
0.6% sodium azide until used.
Synthesis of compounds
Synthesis of N-methylpiperidin-4-yl 4-cyanobenzoate (1)
Under an argon atmosphere, N-methylpiperidin-4-ol
(0.92 g, 8.00 mmol) was dissolved in THF (15 mL). To
this solution was added 1.6 M butyl lithium in hexanes
(5.00 mL, 8.00 mmol) at −78°C followed by a solution
of 4-cyanobenzoyl chloride (1.21 g, 7.30 mmol) in THF
(20 mL) and the mixture was stirred at −78 °C for 16 h.
Water (20 mL) was added to the reaction mixture and
extracted with ethyl acetate (3 × 20 mL). e combined
organic layers were dried over Na2SO4. e solvent was
removed in vacuo to produce a yellow solid. e product
was purified by silica gel chromatography (10:90 MeOH/
CH2Cl2) to give a white solid (1.06 g, 59%). e solid was
recrystallized from hexanes to afford white crystals.
Analytical data: MP (hexanes): 119–120˚C. IR (Nujol): 2228,
1718, 1278, 1122, 1030 cm−1. 1H NMR (CDCl3): δ 1.92–2.03
(m, 2H), 2.10–2.18 (m, 2H), 2.44–2.56 (m, 2H), 2.42 (s, 3H),
2.72–2.87 (m, 2H), 5.08–5.19 (m, 1H), 7.77–7.79 (m, 2H),
8.15–8.17 (m, 2H). 13C NMR (CDCl3): δ 30.9 (2), 46.2 (3),
52.9 (2), 71.4 (1), 116.4 (0), 118.0 (0), 130.1 (1), 132.2 (1),
134.5 (0), 164.3 (0). EI-MS m/z: 244 (M+, 44%), 130 (22%),
114 (20%), 102 (29%, 98 (80%), 97 (100%), 96 (80%), 82
(32%), 70 (14%), 57 (10%), 55 (29%). HRMS (EI): M+ found
244.1216, calcd for C14H16N2O2+ = 244.1206.
Herein is described the synthesis, enzyme kinetics and
histochemical evaluation, as cholinesterase substrates,
of alkyl and aryl thioesters of N-methyl-4-piperidinethiol
as an indirect means to evaluate potential cholinesterase
imaging agents, such as the corresponding N-methyl-4-
piperidinol esters.
Materials and methods
Synthesis of N-methylpiperidin-4-yl 4-fluorobenzoate (2)
Under an argon atmosphere, N-methylpiperidin-4-ol
(0.94 g, 8.16 mmol) was dissolved in THF (15 mL). To
this solution was added 1.6 M butyl lithium in hexanes
Materials and compound characterization
Materials used for synthesis and enzymatic evaluation
of compounds are found in Supplementary Material.
Journal of Enzyme Inhibition and Medicinal Chemistry

Thioesters for the evaluation of imaging agents 449
(5.00 mL, 8.00 mmol) at −78 °C followed by a 4-fluo-
robenzoyl chloride (0.88 g, 7.30 mmol) and the mixture
was stirred at −78 °C for 16 h. Water (20 mL) was added
to the reaction mixture and extracted with ethyl acetate
(3 × 20 mL). e combined organic layers were dried over
Na2SO4. e solvent was removed in vacuo to produce a
yellow solid. e product was purified by silica gel chro-
matography (10:90 MeOH/CH2Cl2) to give a pale yellow
solid (0.72 g, 42%). e solid was recrystallized from
hexanes to afford white crystals. Analytical data: MP
(hexanes): 48–49˚C. IR (Nujol): 1726, 1274, 1114, 852 cm−1.
1H NMR (CDCl3): δ 1.83–1.88 (m, 2H), 2.00-2.05 (m,
2H), 2.32 (s, 3H), 2.33–2.40 (m, 2H), 2.63–2.77 (m, 2H),
4.96–5.10 (m, 1H), 7.08–7.13 (m, 2H), 8.04–8.08 (m, 2H).
13C-NMR (CDCl3): δ 30.9 (2), 46.2 (3), 52.9 (2), 70.3 (1),
115.4 (JCF = 22 Hz, 1) 126.9 (0), 132.0 (JCF = 10 Hz, 1), 164.7
(JCF = 254 Hz, 0), 166.7 (0). EI-MS m/z: 237 (MH+, 30%),
123 (30%), 114 (15%), 98 (53%), 97 (100%), 96 (82%), 95
(31%), 82 (35%), 75 (12%), 70 (14%), 55 (28%). HRMS (EI):
M+ found 237.1173, calcd for C13H16FNO2+ = 237.1160.
J= 7.0, 1H), 1.64–1.71 (m, 2H), 1.99–2.04 (m, 4H), 2.26 (s,
3H), 2.67–2.80 (m, 3H). 13C NMR (CDCl3): δ 35.5 (1), 36.9
(2), 46.1 (3), 55.2 (2). EI-MS m/z: 131 (M+ 32%), 98 (100%),
96 (12%), 70 (18%), 55 (27%). HRMS (ESI): MH+ found
132.0841, calcd for C6H14NS+ = 132.0841.
Synthesis of (N-methylpiperidin-4-yl)
4-cyanobenzenecarbothioate (4)
Under an argon atmosphere, triethylamine (0.53 mL, 3.80
mmol) was added to dichloromethane (20 mL), followed
by N-methyl-4-piperidinethiol (0.45 mL, 3.80 mmol) and
4-cyanobenzoyl chloride (0.629 g, 3.80 mmol). e reac-
tion mixture was stirred for 24 h at room temperature. e
reaction was extracted with saturated NaHCO3 (3 × 20 mL)
and the organic layer dried over Na2SO4. e solvent was
removed in vacuo to yield a light yellow solid which was
recrystallized from hexanes to afford pale yellow crystals
(0.24 g, 24%). Analytical data: MP (hexanes): 152–153°C. IR
1
(Nujol): 2229, 1655, 1208, 860 cm-1. H NMR (CDCl3): δ
1.82–1.89 (m, 2H), 2.10–2.15 (m, 2H), 2.25–2.33 (m, 2H),
2.35 (s, 3H), 2.74-2.85 (m, 2H), 3.72-3.82 (m, 1H), 7.74-
7.76 (m, 2H), 8.02-8.04 (m, 2H). 13C NMR (CDCl3): δ 32.1
(2), 40.4 (1), 46.3 (3), 55.1 (2), 116.5 (0), 117.8 (0), 127.6
(1), 132.4 (1), 140.3 (0), 190.2 (0). EI-MS m/z: 260 (M+,
4%), 130 (54%), 102 (20%), 98 (100%), 97 (81%), 96 (30%),
70 (15%), 55 (22%), 44 (10%), 42 (18%). HRMS (ESI): MH+
found 261.1056, calcd for C14H17N2OS+ = 261.1056.
Synthesis of N-methylpiperidin-4-yl 4-iodobenzoate (3)
Synthesized according to a previously described proce-
dure24 and the analytical data was consistent and purity
greater than 98%. Analytical data: MP: 128–130°C (Lit
MP = 130°C26). IR (Nujol): 1711, 1585, 1283, 1268, 1118,
and 754 cm−1. 1H NMR (CDCl3): δ 1.84–1.91 (m, 2H),
2.01–2.08 (m, 2H), 2.31–2.39 (m, 5H), 2.67–2.73 (m, 2H),
5.02–5.08 (m, 1H), 7.75 (d, J= 8.5 Hz, 2H), and 7.81 (d,
J= 8.5 Hz, 2H). 13C NMR (CDCl3): δ 31 (2), 46 (3), 53 (2),
70 (1), 100 (1), 130, 131, 138, and 166. EI-MS m/z: 345(M+,
16%), 231 (12%), 203 (9%), 114 (11%), 97 (100%), 82 (22%),
70 (7%), 55 (14%). HRMS (EI): M+ found 345.0233, calcd
for C13H16NO2I = 345.0226.
Synthesis of (N-methylpiperidin-4-yl)
4-fluorobenzenecarbothioate (5)
Under an argon atmosphere, triethylamine (0.53 mL, 3.80
mmol) was added to dichloromethane (20 mL), followed
by N-methyl-4-piperidinethiol (0.45 mL, 3.80 mmol) and
4-fluorobenzoyl chloride (0.18 mL, 3.80 mmol). e reac-
tion mixture was stirred for 4 d at reflux temperature. e
reaction was extracted with saturated NaHCO3 (3 × 20 mL)
and the organic layer dried over Na2SO4. e solvent
was removed in vacuo to yield a light yellow solid which
was purified by silica gel chromatography (7% MeOH in
CH2Cl2) and recrystallized from hexanes to afford pale
Synthesis of N-methyl-4-piperidinethiol
N-Methyl-4-piperidone (52.92 g, 469.7 mmol) was dis-
solved in isopropanol (200 mL). Hydrogen sulfide was
bubbled through the solution using a glass frit at 0°C
resulting in the precipitation of white solid, which was
collected by suction filtration after 1 hour. e filtrate
was again treated with hydrogen sulfide for an additional
30 min and all further white solid was collected by suc-
tion filtration for a total of 80.47 g. Sodium borohydride
(16.62g, 439.3 mmol) was suspended in isopropanol
(150 mL) followed by the slow addition of the white solid
at 0˚C. e reaction mixture was returned to room tem-
perature and stirred under an argon atmosphere for 16 h.
Excess solvent was removed under reduced pressure to
produce a white paste which was partitioned between
150 mL of both water and ether. e aqueous layer was
extracted with ether (4 × 50 mL) and the combined
organic layers washed with brine (2× 30 mL) and dried
over anhydrous sodium sulfate. e solvent was removed
in vacuo to yield a colourless liquid which was distilled
(BP: 168–172°C, lit. BP = 62°C at 0.8mm30); to yield the
product as a clear colourless oil (21.22 g, 34%). IR (Neat):
yellow crystals (0.11 g, 28%). Analytical data: MP
:
(hexanes)
66–68°C. IR (Nujol): 1655, 1225, 1202, 1155, 916, 843
cm-1. 1H NMR (CDCl3): δ 1.88–1.95 (m, 2H), 2.11–2.20 (m,
2H), 2.41 (s, 3H), 2.81–2.94 (m, 2H), 3.69–3.81 (m, 1H),
7.10–7.15 (m, 2H), 7.95–7.99 (m, 2H). 13C NMR (CDCl3):
δ 32.5 (2), 40.2 (1), 46.6 (3), 55.5 (2), 116.2 (JCF = 22 Hz, 1),
130.2 (JCF = 9 Hz, 1), 134.0 (0), 166.4 (JCF = 255 Hz, 0), 190.4
(0). EI-MS m/z: 253 (M+, 3%), 130 (39%), 123 (24%), 98
(82%), 97 (100%), 96 (36%), 95 (24%), 82 (10%), 75 (10%),
70 (17%), 55 (21%), 42 (17%). HRMS (ESI): MH+ found
254.1009, calcd for C13H17FNOS+ = 254.1009.
Synthesis of (N-methylpiperidin-4-yl)
4-iodobenzenecarbothioate (6)
Under an argon atmosphere, triethylamine (0.53 mL, 3.80
mmol) was added to dichloromethane (20 mL), followed
by N-methyl-4-piperidinethiol (0.45 mL, 3.80 mmol) and
4-iodobenzoyl chloride (1.01 g, 3.80 mmol). e reaction
1
3338, 2944, 1278, 1132 cm-1. H NMR (CDCl3): δ 1.55 (d,
© 2013 Informa UK, Ltd.

450 I. R. Macdonald et al.
mixture was stirred for 24 h at reflux temperature. e
reaction was extracted with saturated NaHCO3 (3 × 20 mL)
and the organic layer dried over Na2SO4. e solvent was
removed in vacuo to yield a light yellow solid which was
recrystallized from hexanes to afford pale yellow crystals
(0.41 g, 30%). Analytical data: MP (hexanes): 136–137°C. IR
Synthesis (N-methylpiperidin-4-yl) butanethioate (9)
N-Methyl-4-piperidinethiol (0.5 mL, 4.2 mmol) was
dissolved in dry CH2Cl2 (5 mL) under an argon atmo-
sphere. To this was added butyryl chloride (1.3 mL,
13 mmol) and the reaction refluxed for 1.5 h. The
solvent was removed in vacuo and the resulting oil
dissolved in saturated NaHCO3 (20 mL). This solu-
tion was extracted with CH2Cl2 (4 × 20 mL), dried
over Na2SO4 and the solvent removed in vacuo to
produce a clear colourless viscous liquid. This oil
was purified by silica gel column chromatography
(1:19 MeOH/ CH2Cl2) to produce a clear colourless
oil (0.56 g, 67%). Analytical data: IR (Neat): 2938,
1
(Nujol): 1650, 1208, 824 cm-1. H NMR (CDCl3): δ 1.80–
1.87 (m, 2H), 2.09–2.14 (m, 2H), 2.25–2.32 (m, 2H), 2.34 (s,
3H), 2.74–2.83 (m, 2H), 3.67–3.78 (m, 1H), 7.64–7.67 (m,
2H), 7.79–7.82 (m, 2H). 13C NMR (CDCl3): δ 31.9 (2), 39.6
(1), 46.0 (3), 54.9 (2), 100.8 (0), 128.2 (1), 136.2 (0), 137.6
(1), 190.6 (0). EI-MS m/z: 361 (M+, 1%), 230 (12%), 130
(30%), 98 (60%), 97 (100%), 96 (22%), 76 (14%), 70 (10%),
55 (12%), 42 (10%). HRMS (ESI): MH+ found 362.0070,
calcd for C13H17INOS+ = 362.0070.
1
2782, 1686, 1447, 1129, 994 cm-1. H NMR (CDCl3): δ
0.95 (t, J = 7.3, 3H), 1.62-1.71 (m, 4H), 1.89–2.01 (m,
2H), 2.08–2.22 (m, 2H), 2.25 (s, 3H), 2.49 (t, J = 7.3,
2H), 2.59–2.81 (m, 2H), 3.38–3.53 (m, 1H). 13C NMR
(CDCl3): δ 13.3 (3), 19.0 (2), 32.1 (2), 39.1 (1), 45.9
(2), 46.20 (3), 55.1 (2), 198.9 (0). EI-MS m/z: 201 (M+,
16%), 130 (65%), 98 (100%), 97 (28%), 96 (26%), 70
(17%), 55 (20%). HRMS (ESI): MH+ found 202.1260,
calcd for C10H20NOS+ = 202.1260.
Synthesis of (N-methylpiperidin-4-yl) ethanethioate (7)
N-Methyl-4-piperidinethiol (0.5 mL, 4.2 mmol) was
dissolved in dry CH2Cl2 (5 mL) under an argon atmo-
sphere. To this was added acetyl chloride (0.90 mL, 13
mmol) and the reaction refluxed for 1.5 h. e solvent
was removed in vacuo and the resulting oil dissolved in
saturated NaHCO3 (20 mL). is solution was extracted
with CH2Cl2 (4 × 20 mL), dried over Na2SO4 and the sol-
vent removed in vacuo to produce a clear colourless oil.
is oil was purified by silica gel column chromatogra-
phy (1:19 MeOH/CH2Cl2) to produce a clear colourless
viscous liquid (0.69 g, 96%). Analytical Data: IR (Neat):
Enzyme kinetics
Measurement of aryl ester hydrolysis
Cholinesterase specificity and maximum absorbance
change during hydrolysis for each benzoyl ester was
determined spectrophotometrically, making use of
differences in the aryl chromophores of substrates and
products determined through repetitive absorbance
scans (e.g. Figure 1). Briefly, 15 μL of AChE (2.5 U) or
BuChE (5.4 U) dissolved in 0.1% gelatin(aq), containing
0.01% sodium azide, and 1.44 mL of 0.1 M phosphate
buffer (pH 7.4) were placed in a quartz cuvette of 1-cm
path length. e reaction was commenced with the
1
2941, 1686, 1448, 1219, 915 cm-1. H NMR (CDCl3): δ
1.58–1.65 (m, 2H), 1.88–1.92 (m, 2H), 2.08–2.18 (m, 2H),
2.21 (s, 3H), 2.24 (s, 3H), 2.59–2.70 (m, 2H), 3.33–3.48
(m, 1H). 13C NMR (CDCl3): δ 30.6 (3), 31.8 (2), 39.3 (1),
46.0 (3), 54.9 (2), 195.2 (0). EI-MS m/z: 173(M+, 17%),
130 (52%), 98 (100%), 97 (30%), 96 (33%), 70 (26%), 55
(45%). HRMS (ESI): MH+ found 174.0947, calcd for
C8H16NOS+ = 174.0947.
addition of 50 μL of 5 mM ester in 50% acetonitrile(aq)
.
e absorbance was scanned from 200–300 nm every
2 min for a total of 30 min using a Ultrospec 2100 pro
UV/Visible Spectrophotometer (Biochrom) with Swift
II software (Amersham). e wavelength correspond-
ing to the maximum absorbance change during hydro-
lysis (235 nm for compounds 1-3) was used for the
subsequent determination of BuChE affinity constants
(Km values, Table 1) using Lineweaver-Burk double
reciprocal plots.
Determination of affinity constants (Km) and maxi-
mum velocity values (Vmax) was accomplished by measur-
ing change in absorbance per min (ΔA/min) at 235nm,
using a fixed amount of enzyme AChE (8.3 U) or BuChE
(5.9 U) and varying amounts of compounds (1.67 × 10−4−
1.67 × 10−5 M), using a Spectronic 1001 (Milton Roy)
UV-visible spectrophotometer. e plot of 1/v against
1/s gave Km as the negative reciprocal of the intercept on
the 1/s-axis and gave Vmax as the reciprocal of the inter-
cept of the 1/v-axis. As defined previously31, 0.1 U is the
amount of cholinesterase that gives a ΔA/min of 1.0 in
the presence of 1.6 × 10−4 M substrate (acetylthiocholine
for AChE, butyrylthiocholine for BuChE).
Synthesis of (N-methylpiperidin-4-yl) propanethioate (8)
N-Methyl-4-piperidinethiol (0.5 mL, 4.2 mmol) was
dissolved in dry CH2Cl2 (5 mL) under an argon atmo-
sphere. To this was added propionyl chloride (0.94 mL,
11 mmol) and the reaction refluxed for 2 h. e solvent
was removed in vacuo and the resulting oil dissolved in
saturated NaHCO3 (20 mL). is solution was extracted
with CH2Cl2 (4 × 20 mL), dried over Na2SO4 and the sol-
vent removed in vacuo to produce a clear colourless
viscous liquid. is oil was purified by silica gel column
chromatography (1:19 MeOH/CH2Cl2) to produce a
clear colourless viscous liquid (0.45 g, 57%). Analytical
data: IR (Neat): 2939, 2782, 1689, 1463, 1129, 937 cm-1.
1H NMR (CDCl3): δ 1.19 (t, J = 7.5, 3H), 1.65-1.72 (m, 2H),
1.95–1.98 (m, 2H), 2.13–2.25 (m, 2H), 2.28 (s, 3H), 2.55
(q, J = 7.5, 2H), 2.63–2.83 (m, 2H), 3.41–3.54 (m, 1H).
13C NMR (CDCl3): δ 9.6 (3), 32.1 (2), 37.5 (2), 39.1 (1),
46.2 (3), 55.1 (2), 199.8 (0). EI-MS m/z: 187 (M+, 17%),
130 (61%), 98 (100%), 97 (27%), 96 (28%), 70 (17%), 55
(20%). HRMS (ESI): MH+ found 188.1104, calcd for
C9H18NOS+ = 188.1104
Journal of Enzyme Inhibition and Medicinal Chemistry

Thioesters for the evaluation of imaging agents 451
Figure 1. Repetitive absorbance scans for N-methylpiperidin-4-yl 4-cyanobenzoate in the presence of butyrylcholinesterase (BuChE) or
acetylcholinesterase (AChE). Note change in absorbance over time when the compound was incubated with BuChE (left) reflecting hydrolysis
of the compound by this enzyme. No change in absorbance with AChE (right) indicates no hydrolysis by this enzyme. e absorbance was
measured every 2 min for 30 min.
Table 1. Affinity constants (Km) and maximum velocity values (Vmax) for N-methylpiperidinyl alkyl thioesters, N-methylpiperidinyl aryl
esters and corresponding thioesters with butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE). None of the aryl compounds
(1–6) were hydrolyzed by AChE under the conditions used (X).
AChE
BuChE
Km (µM)
X
Vmax (µM min−1)
X
Km (µM)
438 75
Vmax (µM min−1)
45.0 6.6
Compound
1
Structure
2
3
4
5
6
X
X
X
X
X
X
X
X
X
X
284 28
123 42
167 17
173 13
24.7 2.0
21.0 5.1
30.7 2.4
27.6 1.7
9.7 0.3
11
1
7
8
9
751 26
1108 106
X
18.3 0.2
21.4 2.2
X
684 54
16.7 1.3
20.7 0.1
7.0 0.3
302
2
129 12
© 2013 Informa UK, Ltd.

452 I. R. Macdonald et al.
Measurement of thioester hydrolysis
water, sections were placed in a 3, 3′-diaminobenzidine
tetrahydrochloride (DAB) solution consisting of 1.39 mM
in 0.1M phosphate buffer (pH 7.4). After 5 min, a solution
of 0.15% hydrogen peroxide in distilled water was added
at a ratio of 10:1 (DAB solution: hydrogen peroxide solu-
tion) and the reaction was carried out for approximately
2 min. Sections were then washed in 0.01 M acetate buf-
fer, pH 3.3, mounted on slides, coverslipped and exam-
ined with brightfield microscopy.
Sections were photographed with an AxioCam HRc
camera on a Zeiss Axioplan II microscope. e photo-
graphic plates were assembled using Adobe Photoshop
7.0. e images were colour balanced, contrast enhanced
and brightness adjusted to match the background from
different images.
e kinetics of both alkyl and aryl thioester hydrolysis by
AChE or BuChE was determined using a modification27 of
the method described by Ellman et al.26. Briefly, 1.40 mL
of buffered 5, 5′-dithio-bis(2-nitrobenzoic acid) solution
(pH 7.4) and 0.05 mL of AChE (8.3 units) or BuChE (5.9
units), in 0.1% aqueous gelatin, were mixed in a quartz
cuvette of 1-cm path-length and zeroed at 412 nm. e
reaction was initiated by the addition of thioester in a
50% aqueous acetonitrile solution to give a final sub-
strate concentration from 1.7 × 10−4 to 3.3 × 10−6 M. e
reactions were performed at 23˚C. e rate of change of
absorbance (ΔA/min), reflecting the rate of hydrolysis
of the thioester, was recorded every 5 s for 1min, using
a Milton-Roy 1201 UV–vis spectrophotometer. e molar
extinction coefficient for the Ellman product, 5-thio-2-
nitrobenzoic acid, used to convert the change in absor-
bance at λ= 412 nm to moles of product was 14,150M−1
cm−1. ese experiments were performed at least in trip-
licate and the values averaged.
Results and discussion
Organic synthesis
To synthesize N-methyl-4-piperidinethiol, N-methyl-4-
piperidone was mixed with hydrogen sulfide and the result-
ing reaction produced a dithiol. is intermediate was then
reduced with sodium borohydride (Scheme 1), in a reac-
tion adapted from an earlier procedure34. ioesters were
subsequently generated by esterification of this thiol with
an acid chloride (Scheme 1). Benzoate esters were also
synthesized, as previously described24, (Scheme 1, Table
1, 1–3) in order to directly compare cholinesterase kinetic
parameters for aryl thioesters and esters. Two classes of
thioesters were synthesized, those from aryl acid chlo-
rides (Table 1, 4–6), analogous to the benzoate esters, and
those from alkyl acid chlorides (Table 1, 7–9), analogous
Structural analysis
Computational chemistry studies, to determine the
most stable geometries of compounds, were carried
out at the molecular mechanics level of theory using
the Merck Molecular Force Field (MMFF), employing
Spartan’0632.
Cholinesterase histochemistry with thioester
substrates
Brain tissue containing the thalamus was cut on a Leica
SM2000R microtome with a Physitemp freezing stage in
50 μm thick coronal sections. Sections were stored in
40% sucrose in 0.1 M phosphate buffer (pH 7.4) at −20˚C
until used. A modified33 Karnovsky-Roots method was
employed for AChE and BuChE staining. Substrates used
to provide staining were synthesized thioesters and the
standards, butyrylthiocholine and acetylthiocholine in
the absence of cholinesterase inhibitors. Briefly, tissue
sections were rinsed in 0.1 M maleate buffer (pH 7.4) for
30 min and then placed in 0.1 M maleate buffer, pH 7.4,
containing 0.15% hydrogen peroxide for 30 min to quench
endogenous peroxidase activity. Following a second rinse
in 0.1 M maleate buffer, pH 7.4, for 30 min, sections were
incubated for 1 h (acetylthiocholine, [N-methylpiperidin-
4-yl] ethanethioate (7)) or 18 h (butyrylthiocholine,
[N-methylpiperidin-4-yl] butanethioate (9) and
[N-methylpiperidin-4-yl] 4-cyanobenzenecarbothioate
(4)) in a medium containing 0.5 mM sodium citrate,
0.47 mM cupric sulfate, 0.05 mM potassium ferricyanide
and thioester substrate in 0.1 M maleate buffer (pH 7.4).
Substrate concentrations were 4 mM for acetylthio-
choline, butyrylthiocholine, (N-methylpiperidin-4-yl)
ethanethioate, (N-methylpiperidin-4-yl) butanethioate
and 0.5 mM for the less soluble (N-methylpiperidin-4-yl)
4-cyanobenzenecarbothioate. Sections were then rinsed
for 30 min in distilled water and placed in 0.1% cobalt (II)
chlorideinwaterfor10 min.Afterafurtherrinseindistilled
to previously developed alkyl ester imaging agents17,22
.
Kinetic analysis
ioester analogues of aryl esters such as benzoylcho-
line have been found28 to be kinetically comparable to
their ester counterparts for cholinesterase hydrolysis.
erefore, thioesters can provide a rapid, indirect enzyme
kinetic screening method for analysis of potential ester
imaging agents. All aryl and alkyl thioesters, producing
a thiol product, upon hydrolysis, are amenable to kinetic
analysis as cholinesterase substrates via an Ellman assay.
Scheme 1. Reaction scheme for the synthesis of (a) alkyl and aryl
thioesters and (b) aryl esters.
Journal of Enzyme Inhibition and Medicinal Chemistry

Thioesters for the evaluation of imaging agents 453
Furthermore, since all aryl esters showed distinct UV
spectral differences between reactant and product (Figure
1), they could be analyzed for comparison with thioester
kinetic parameters by an alternate method employing
changes in absorbance. Esters and thioesters of substi-
tuted benzoic acids gave comparable affinity constants
and maximum velocity values (Table 1), in agreement
with the earlier observation comparing benzoylcholine
and benzoylthiocholine analogues28. In general, the
thioester derivatives had a slightly greater affinity towards
BuChE than did the esters. In addition, none of the aryl
thioesters, as observed earlier for aryl esters24, were sus-
ceptible to hydrolysis by AChE (Figure 1).
Although it was not possible to directly obtain affinity
constants for the esters of N-methylpiperidinol derived
from acetic acid, propionic acid and butyric acid, it is rea-
sonable to expect, from comparable studies with aryl deriv-
atives (Table 1,28), these values to be similar to those for the
comparable thioesters (Table 1). is is supported by the
observation that for AChE, like the analogous thiocholine
esters, the acetyl ester of N-methyl-4-piperidinethiol (7,
Table 1) had the greatest affinity, followed by the propionyl
ester(8),whilethebutyrylderivative(9)wasnothydrolyzed
by AChE under these conditions. In addition, BuChE affin-
ity increased with longer substrate side chains, consistent
with the known preference of this enzyme for longer acyl
chains27,35. e observation that the thioester analogue of
MP4B is BuChE-specific is also in keeping with the earlier
report25 that the ester is only hydrolyzed by BuChE. Of
note is that the affinities of the acetyl (7) and propionyl
(8) thioesters for BuChE are greater than for AChE. Even
though the corresponding radiolabelled esters, 4-[11C]
AMP and 4-[11C]PMP, have been developed as AChE imag-
ing agents, these kinetic results suggest that BuChE is also
capable of efficiently hydrolyzing these compounds and
thus, both AChE and BuChE may be detected in molecular
imaging studies with these agents, rendering them to be
non-specific.
cerebral blood flow, penetration of the blood–brain
barrier, interaction with the target molecules and fate
of the metabolites. us, determining the in vitro dis-
tribution of the imaging agent in brain tissues would be
valuable before advancing to in vivo radioimaging stud-
ies. e cholinesterases are commonly stained in brain
tissues using thioesters such as butyrylthiocholine or
acetylthiocholine29,36. In order to explore cholinesterase
specificity and to determine their potential to detect the
distribution of cholinesterases in the brain, several of the
thioesters prepared in this study were tested as histo-
chemical substrates and compared with choline thioes-
ters for staining human thalamus brain tissue (Figure
2). is region of the brain was chosen because it has
been precisely mapped for cholinesterase activity and
certain nuclei, such as the anteroventral (AV) nucleus
(Figure 2A), exhibit distinct patterns of AChE and BuChE
activity36. In the AV nucleus of the thalamus, AChE activ-
ity is found only in the neuropil while BuChE activity is
observed only in neurons. As can be seen in Figure 2,
acetylthiocholine (Figure 2B) and N-methylpiperidinyl
acetylthioester (7; Table 1; Figure 2C) show comparable
neuropil staining typical of AChE activity in this nucleus.
at is, both the choline and N-methylpiperidinethiol
acetyl thioester substrates provided histochemical stain-
ing consistent with the previously described distribution
of AChE in this region of the brain36. is observation
is also in agreement with the in vitro enzyme kinetic
results indicating that AChE is capable of efficiently
hydrolyzing the N-methylpiperidinyl acetylthioester
(Table 1). N-methylpiperidinyl acetylthioester (7) is
hydrolyzed by both AChE and BuChE (Table 1), similar
to acetylthiocholine37, using Ellman method. However,
the modified Karnovsky-Roots method for histochemi-
cal staining requires shorter (1 h) incubation for staining
AChE and longer (18 h) for BuChE. Based on this dif-
ference in the incubation time, staining in Figure 2C is
predominantly for AChE. Similarly, to evaluate specific
histochemical visualization of BuChE activity in the
human thalamus, comparative staining with butyrylthio-
choline, N-methylpiperidinyl butyrylthioester (9; Table
1) and cyanobenzoate thioester (4; Table 1) was carried
out (Figure 2D, E, F). BuChE staining using butyrylthio-
choline as substrate (Figure 2D) was comparable to that
obtained with the butyryl (Figure 2E) and 4-cyanoben-
zoyl (Figure 2F) derivatives of N-methylpiperidinethiol.
Furthermore, the BuChE staining profiles (Figure 2D, E,
F) were distinct from those for AChE observed with the
acetyl thioesters (Figure 2B and C) in the same region
of the brain. High (>1 mM) thioester concentrations are
typically required in the staining medium to achieve ade-
quate visualization of cholinesterase activity. Because of
this, solubility limitations prohibited use of the iodo- and
fluorobenzoate thioesters in similar histochemical anal-
ysis to that provided by the cyanobenzoate derivative
(Figure 2F). However, successful histochemical recapitu-
lation of enzyme activity with an aryl thioester provides
a critical proof-of-principle that analogous radiolabelled
Structural analysis
e preferred geometries of the synthetic thioesters and
their ester counterparts were computed at the molecular
mechanics level in order to further explore any subtle dif-
ferences observed in the cholinesterase kinetic affinities.
In general, the preferred conformations for each oxygen
and sulfur analogous pair were comparable (Figure S1).
However, slight differences in molecular shape, because
of the presence of the bulkier and electronically distinct
sulfur atom, most likely contributed to observed differ-
ences in relative affinity constants for BuChE (Table 1)
in each analogous pair. e structural similarity further
suggests that thioesters are acceptable kinetic surrogates
for corresponding ester imaging agents.
Histochemical evaluation of N-methyl piperidinyl
thioesters
Distribution of an imaging agent within the living brain
is dependent upon a number of factors. ese include
© 2013 Informa UK, Ltd.

454 I. R. Macdonald et al.
Figure 2. Histochemical staining of human brain tissue at the level of the thalamus in the coronal plane. (A) Parcellation of the thalamus
in the region used to compare histochemical staining by various cholinesterase substrates; (B) Acetylthiocholine. (C) (N-methylpiperidin-
4-yl) ethanethioate. Note that both of these substrates produced a similar pattern of staining recapitulating the known distribution of
acetylcholinesterase in this region. (D) Butyrylthiocholine. (E) (N-methylpiperidin-4-yl) butanethioate. (F) (N-methylpiperidin-4-yl)
4-cyanobenzenecarbothioate. Note that these substrates produced a similar pattern of distribution that reflected the known distribution of
butyrylcholinesterase in this region. AV: anteroventral nucleus; CL: central lateral nucleus; MD: mediodorsal nucleus; R: reticular nucleus;
V: ventral nuclei. Scale bar = 200 µm.
compounds are potential imaging agents to be highly
acknowledgements
sensitive detectors of cholinesterase activity in brain
e authors thank Eric Joy for technical assistance.
tissue.
Declaration of interest
conclusion
Portions of this work were supported by grants from
Canadian Institutes of Health Research, Canada
is study indicates that N-methylpiperidinyl thioesters
are effective surrogate substrates for the evaluation
Foundation for Innovation, Natural Sciences and
and development of corresponding ester compounds
Engineering Research Council of Canada, Vascular
as AChE and BuChE imaging agents. ese thioester
Health and Dementia Initiative (through Canadian
analogues have comparable affinities towards AChE
Institutes of Health Research, Heart & Stroke Foundation
and BuChE to their ester counterparts. ese similari-
of Canada, the Alzheimer Society of Canada and Pfizer
ties permit screening of corresponding ester substrates
Canada Inc.), Capital District Health Authority Research
as potential radioligands for specific detection of AChE
Fund, Nova Scotia Health Research Foundation, Brain
or BuChE activity. ese thioester compounds can also
Tumour Foundation of Canada, Multiple Sclerosis Society
be employed for histochemical detection of enzyme
of Canada, National Institutes of Health, Dalhousie
activity in brain tissue, which could then be used for
Medical Research Foundation, Faculty of Medicine of
comparison with the in vivo images generated by the
analogous radiolabelled molecules. Development of
compounds, screened in this manner, could facilitate
discovery of suitable molecular imaging agents for
Dalhousie University and the Committee on Research
and Publications of Mount Saint Vincent University.
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