In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and -B Enzymatic Activity

Article abstract of DOI:10.1021/acschemneuro.5b00223

The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine, and norepinephrine. Recently, changes in concentrations of MAO-B have been proposed to be an in vivo marker of neuroinflammation associated with Alzheimer's disease. Previous developments of in vivo radiotracers for imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based suicide inhibitors or high-affinity reversibly binding inhibitors. As an alternative approach, we have investigated 1-[¹¹C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases. MAO-mediated oxidation and spontaneous hydrolysis yield 1-[¹¹C]methyl-2,3-dihydro-4-pyridinone as a hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl, and 7-coumarinyl ethers were evaluated using microPET imaging in rat and primate brains. No isozyme selectivity for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain, the phenyl ether demonstrated MAO-A selectivity and the coumarinyl ether showed MAO-B selectivity. These are lead compounds for further development of 1-[¹¹C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with optimized brain pharmacokinetics and isozyme selectivity.

Full text of DOI:10.1021/acschemneuro.5b00223

Subscriber access provided by CMU Libraries - http://library.cmich.edu
Article
In Vivo Metabolic Trapping Radiotracers for Imaging
Monoamine Oxidase-A and –B Enzymatic Activity.
Allen F Brooks, Xia Shao, Carole A. Quesada, Phillip Sherman, Peter J. H. Scott, and Michael R. Kilbourn
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 22 Sep 2015
Downloaded from http://pubs.acs.org on September 25, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 11
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
In Vivo Metabolic Trapping Radiotracers for Imaging
Monoamine Oxidase-A and –B Enzymatic Activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Allen F. Brooks†, Xia Shao†, Carole A. Quesada†, Phillip Sherman†, Peter J.H. Scott*†,‡, and
Michael R. Kilbourn*†
†Division of Nuclear Medicine, Department of Radiology, University of Michigan Medical School, Ann Arbor,
Michigan 48109
‡The Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109
KEYWORDS: Alzheimer's disease, neuroimaging, positron emission tomography, carbon-11, monoamine oxidase
ABSTRACT: The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the
metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine and
norepinephrine. Recently, changes in concentrations of MAO-B have been proposed as an in vivo marker of
neuroinflammation associated with Alzheimer’s disease. Previous developments of in vivo radiotracers for
imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based
suicide inhibitors, or high-affinity reversibly-binding inhibitors. As an alternative approach, we have investigated
1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases.
MAO-mediated oxidation and spontaneous hydrolysis yields 1-[11C]methyl-2,3-dihydro-4-pyridinone as a
hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl and 7-
coumarinyl ethers were evaluated using microPET imaging in rat and primate brain. No isozyme selectivity
for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain the
phenyl ether demonstrated MAO-A selectivity, and the coumarinyl ether showed MAO-B selectivity.
These are lead compounds for further development of 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with
optimized brain pharmacokinetics and isozyme selectivity.
The enzymes monoamine oxidase-A (MAO-A) and –B (MAO-B) are responsible for the oxidation of a wide
variety of amines, including the amine neurotransmitters dopamine, norepinephrine, and serotonin. Inhibitors
of monoamine oxidases (MAOIs) are important in clinical medicine: inhibition of MAO-A is utilized in the
management of depression, and inhibition of MAO-B forms one of the therapeutic approaches to treating
Parkinson’s disease1. More recently, the potential for using changes of MAO-B as a marker of astrogliosis in
neurodegenerative diseases (such as Alzheimer's disease) has become of interest2. The importance of the
monoamine oxidases in numerous disease processes stimulated interest for in vivo non-invasive imaging of
changes of MAO in the human brain, leading to the development of numerous carbon-11 and fluorine-18 labeled
radiotracers useful for Positron Emission Tomography (PET) studies3. Prior approaches to isoform-selective MAO
radiotracer development have mostly concentrated on (a) irreversibly trapped suicide inhibitors (e.g.,
[11C]deprenyl and [11C]clorgyline) that form covalent bonds between the radiotracer and the flavin cofactor of the
enzymes, and (b) reversibly-binding high-affinity inhibitors (e.g., [11C]befloxatone, [11C]harmine). Several of these
have been successfully introduced into human PET studies, including evaluation in Alzheimer’s disease4, but
there is continued interest in the development of new MAO radiotracers labeled with carbon-11 and fluorine-18 in
the search for optimal in vivo radiotracers3b, 5
.
An alternative approach for measuring enzymatic activity in vivo is the use of metabolic trapping, where the
product of the catalytic activity of the enzyme is not covalently bound but still retained within the target tissue.
This is the mechanism behind such in vivo imaging radiotracers as 2-[18F]fluoro-2-deoxyglucose ([18F]FDG,
glucose metabolism), N-[11C]methyl-4-piperidinyl-propionate or acetate ([11C]PMP and [11C]MP4A,
acetylcholinesterase)6, and [18F]fluoroDOPA (dopamine synthesis) among other examples. The concept of non-
covalent metabolic trapping has been applied to MAO but the effort has been severely limited. The syntheses of
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 2 of 11
1
2
3
4
5
6
7
8
9
radiolabeled forms of N,N-dimethylphenylethylamine were reported, initial biodistribution studies reported in
rodents, and even a single human PET image presented7. Preliminary studies in both rat and primate were also
done with carbon-11 labeled MPTP8 and a radioiodinated tetrahydropyridine (N-methyl-4-(4’-hydroxy-3’-
[125I]iodophenyl)-1,2,3,6-tetrahydropyridine)9, but no additional reports were made for use of either compound.
Further development and validation (demonstration of isoform selectivity, pharmacokinetic analyses) of non-
covalent metabolic trapping radiotracers for MAO have not been pursued, and there have been no applications of
this concept to studies of MAO in human diseases.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1:
Monoamine oxidase-mediated oxidation of 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines,
followed by spontaneous hydrolysis, yields a phenol and 1-[11C]methyl-2,3-dihydro-4-pyridinone.
We have investigated here the metabolic trapping of 1-methyl-4-aryloxy-1,2,3,6-tetrahydropyridines, molecules
that undergo an initial single oxidation by monoamine oxidases (Fig. 1), forming dihydropyridinium species that
are then rapidly hydrolyzed to a phenol and 1-methyl-2,3-dihydro-4-pyridinone10. As that ketone is hydrophilic
(cLog P = -0.18), labeling with a carbon-11 at the N-methyl substituent was hypothesized to provide a metabolite
that would be trapped within brain tissues. Furthermore, as a second oxidation step of the dihydropyridinium
intermediate never occurs, the 4-aryloxy-1,2,3,6-tetrahydropyridines are non-toxic (in contrast to 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP)).
A series of 4-aryloxy substituted tetrahydropyridines were
synthesized by Castagnoli and coworkers11 and evaluated as substrates for the monoamine oxidases; those studies
demonstrated that a broad range of in vitro kinetic parameters (Km and Kcat) and MAO isoform selectivity are
possible by selection of the 4-aryloxy substituent.
RESULTS AND DISCUSSION
Chemistry. Using the prior studies of the in vitro kinetics of MAO-mediated hydrolysis as a guide, three target
compounds were selected to test the concept of using 1-methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as isoform-
selective MAO-catalyzed metabolic radiotracers (Fig. 2). The simplest molecule, 1-methyl-4-phenyloxy-1,2,3,6-
tetrahydropyridine (PHXY, 1a), was reported as a mixed MAO-A/MAO-B substrate with a slight (2-fold) higher
reactivity towards MAO-B12. The biphenyl-substituted derivative (BiPHEN, 1c) demonstrated 8-fold higher in
vitro reactivity towards MAO-A over MAO-B (Wang and Castagnoli 1995)11a. Finally, the 7-coumarinyl derivative
(COU, 1b) was reported by Long and coworkers13 to have a 22-fold selectivity for MAO-B.
O
O
O
O
O
N
N
H3C
H3C
N
H3C
MAO-B
selectivity
MAO-A
selectivity
BiPHEN (1c)
PHXY (1a)
cLogP = 2.97, tPSA = 12.47
COU (1b)
cLogP = 3.40, tPSA = 42.7
cLogP = 4.72, tPSA = 12.47
MAO A Km= 0.232 mM, Kcat= 1292 min-1 MAO A Km= 0.055 mM, Kcat= 130 min-1
MAO B Km= 0.058 mM, Kcat= 241 min-1
MAO B Km= 0.346 mM, Kcat= 220 min-1
MAO B Km= 0.058 mM
Vmax = 1.42 nmol mg min-1
Figure 2: Target carbon-11 radiotracers and reported in vitro enzyme kinetics.
ACS Paragon Plus Environment

Page 3 of 11
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
The syntheses of 1-methyl-4-aryloxy-1,2,3,6-tetrahydropyridines in isotopically unmodified forms were done
following modifications of the literature procedure11a, 13 and utilized the reaction of 4-chloro-1-methylpyridin-1-
ium triflate with the appropriate phenols (2a-c) to form intermediate 1-methyl-4-aryloxypyridines (3a-c),
followed by sodium borohydride reduction of the pyridine ring (Scheme 1). The products 1a-c were isolated in
good yields (12-47%) using flash silica gel chromatography. The syntheses of the carbon-11 forms of PHXY, COU
and BiPHEN were done using a one-pot procedure of N-[11C]methylation of the 4-aryloxypyridines 4a-c using no-
carrier-added [11C]methyl triflate, followed by rapid (5 min) sodium borohydride reduction in ethanolic solution
(Scheme 1). The required pyridine precursors (4a-c) were prepared by reaction of 4-chloropyridinium chloride
with the corresponding phenols 2a-c. The radiotracers ([11C]PHXY (1a), [11C]COU (1b) and [11C]BiPHEN (1c)) were
isolated and purified by HPLC, with overall synthesis times of 30 min. Although isolated radiochemical yields
were low (1-5%, not corrected for decay) they were not optimized, and specific activities averaged >55.5
TBq/mmol. Radiotracers were then formulated in isotonic saline for microPET imaging studies in rats and
monkey.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1: Syntheses of unlabeled and carbon-11 labeled tetrahydropyridine MAO substrates.a
aReagents and Conditions (i) NaOCH3, DMF, 4-chloro-1-methylpyridin-1-ium triflate; (ii) NaBH4, CH3OH; (iii)
KOtBu, DMF, 4-chloropyridin-1-ium; (iv) [11C]CH3OTf, EtOH, NaBH4
Biology. The in vivo brain distributions of all three radiotracers were evaluated using microPET imaging, with
the initial evaluations of pharmacokinetics and isozyme selectivity performed in the rat brain. However, due to
the well-known potential for species differences in the selectivity and rates of substrate oxidations by the
monoamine oxidases7a, 14, additional studies were performed in the rhesus monkey.
The microPET studies in rat brain demonstrated similar pharmacokinetics for [11C]PHXY and [11C]COU, with
rapid initial uptakes peaking at 90-150 seconds and equivalent maximum brain concentrations (1.1 % injected
dose/g for both). For both [11C]PHXY and [11C]COU, there followed a rapid washout of radioactivity until constant
levels of trapped radioactivity were reached by 20-30 min, with trapping fractions (plateau/peak) of 55-60% for
[11C]PHXY and 10-15% for [11C]COU. [11C]BiPHEN showed different pharmacokinetics, with a significantly lower
and broader initial uptake (0.43 % injected dose/g) and a more gradual washout that did not quite reach a
constant level by the end of the 60 min imaging period.
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 4 of 11
1
2
3
4
5
6
7
8
9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1.2
1
1.4
control
control
control
+deprenyl
+clorgyline
+both
1.2
+deprenyl
+clorgyline
+clorgyline
A
C
B
+deprenyl
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time, min
Time, min
Time, min
Fig. 3. Representative curves (single scans) for in vivo trapping of [11C]PHXY (panel A), [11C]COU (panel B) and
[11C]BiPHEN (panel C) in rat brain cerebrum. Data is expressed as percent injected dose per gram for rat brain
cerebrum. Pharmacological interventions were done using pretreatment with MAO-A inhibitor (clorgyline) or
MAO-B inhibitor (deprenyl) or both together.
To test for isozyme selectivity, studies of trapping efficiencies were performed after pretreatments with deprenyl
(selective MAO-B inhibitor, 10 mg/kg i.p. 90 min prior to scan) or clorgyline (selective MAO-A inhibitor, 10
mg/kg i.p. 90 min prior to scan) or both irreversible inhibitors. The in vivo trapping of [11C]PHXY and [11C]BiPHEN
were more sensitive to MAO-A inhibition, and [11C]COU more sensitive to MAO-B inhibition, but none of the
three radiotracers examined here exhibited specificity for either isozyme in the rat brain. The pharmacological
blocking studies support little non-specific binding of [11C]PHXY and [11C]COU, as residual trapped radioactivity
levels are very low after enzyme inhibition. The lower brain uptake and slower kinetics of [11C]BiPHEN suggests
the radiotracer might exhibit higher non-specific distribution, consistent with its higher lipophilicity.
The rat studies were encouraging and supported the hypothesis that MAO-mediated oxidation of 1-methyl-4-
aryloxy-1,2,3,6-tetrahydropyridines could be imaged in the mammalian brain. Metabolite studies of rat brain
extracts at 10 minutes after injection of [11C]PHXY confirmed the formation of a single polar radioactive
metabolite, consistent with formation of 1-[11C]methyl-2,3-dihydro-4-pyridinone. The failure to achieve isozyme
selectivity (or specificity) for any of the radiotracers in the rat brain was however not discouraging. The prior
studies11a, 12 of 1-methyl-4-aryloxy-1,2,3,6-tetrahydropyridines had utilized MAO isolated from bovine or human
tissues (or both), and numerous studies have demonstrated significant species variability in the behavior of both
inhibitors and substrates towards the two isozymes of MAO7a, 14. This variability supported the notion that our
new radiotracers needed to be evaluated in a second species.
The imaging studies in the rhesus monkey brain showed pharmacokinetics of all three radiotracers that were
different than seen in the rat brain, with more encouraging isozyme selectivity for [11C]PHXY and [11C]COU.
[11C]PHXY, the lowest molecular weight and least lipophilic compound, exhibited the highest uptake and
trapping, with clear heterogeneity between the major regions of the brain (striatum > thalamus > cerebellum >
cortex) (Fig. 4A). The coumarin derivative [11C]COU showed similar pharmacokinetics, with slightly lower
trapping of radioactivity, and little or no distinction between levels in the striatum and thalamus (Fig 4D).
Finally, the kinetics of [11C]BiPHEN were significantly different, with much lower brain uptake and trapping, and
less heterogeneity in the regional brain distribution (Fig. 4G). No attempts have been made to correlate the
observed regional radiotracer trapping concentrations with in vitro values for protein concentrations or enzyme
activities of MAO-A or MAO-B, as there is very limited data available for rhesus monkey brain15. The rank order
of striatum > cerebellar cortex > cortex for [11C]COU is however consistent with the in vitro studies of Riachi and
Harik15a using either binding of [3H]pargyline or oxidation of substrates (MPTP, benzylamine).
ACS Paragon Plus Environment

Page 5 of 11
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
0.05
0.04
0.03
0.02
0.01
0
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
striatum
thalamus
cortex
control striatum
+lazabemide
control cortex
+lazabemide
+moclobemide
A
B
C
cerebellum
+moclobemide
0
10
20
30
Time, min
40
50
60
0
10
20
30
Time, min
40
50
60
0
10
20
30
Time, min
40
50
60
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
control cortex
+lazabemide
+moclobemide
control cortex
control striatum
+lazabemide
control striatum
control thalamus
control cerebellum
F
D
E
+moclobemide
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time, min
Time, min
Time, min
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.06
0.05
0.04
0.03
0.02
0.01
0
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
striatum
thalamus
cortex
control striatum
+moclobemide
+lazabemide
control cortex
+moclobemide
+lazabemide
G
I
H
cerebellum
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
10
20
30
Time, min
40
50
60
Time, min
Time, min
Fig. 4. Representative curves (single scans) for in vivo trapping of [11C]PHXY (panels A-C), [11C]COU (panel D-F)
and [11C]BiPHEN (panels G-I) in striatum, thalamus, cerebral cortex and cerebellum of rhesus monkey brain.
Pharmacological interventions were done using pretreatment with MAO-A inhibitor (moclobemide) or MAO-B
inhibitor (lazabemide).
Pharmacological studies for isozyme selectivity in the monkey were done using the reversible MAO inhibitors
moclobemide (MAO-A, 1.0 mg/kg i.v.) and lazabemide (MAO-B, 0.5 mg/kg i.v.), to avoid the long-lasting
irreversible inactivation of the enzymes known to occur with deprenyl and clorgyline16. The doses were
administered as a 10 min infusion prior to radiotracer injection. The doses of inhibitors were limited to amounts
supported by prior safe administration to monkeys, based on literature reports; without independent evidence
that the doses were sufficient for complete inhibition of enzyme activities, or that the radiotracers themselves do
not effectively displace these reversibly-bound inhibitors. These studies with moclobemide and lazabemide are
considered here as pharmacological intervention studies but not necessarily full blocking studies (in the sense
the enzymes can be fully and irreversibly inhibited, as was achieved with deprenyl and clogyline in the rat brain).
Radiotracer trapping of the simplest substrate [11C]PHXY was significantly reduced in all brain regions by
inhibition of MAO-A by moclobemide, with data for striatum and cortex shown in Figs 4B-C. In contrast,
administration of the MAO-B inhibitor lazabemide produced no change in the cortex and a small and perhaps
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 6 of 11
1
2
3
4
5
6
7
8
9
insignificant increase in the striatum. These preliminary results suggest that, contrary to the in vitro assays which
used a combination of enzymes from human (MAO-A) and bovine (MAO-B) tissue sources and contrary to the
rat imaging studies, [11C]PHXY exhibits MAO-A selectivity in the monkey brain.
As was hoped based on the reported in vitro selectivity of [11C]COU, where human tissues provided the sources
for both MAO-A and MAO-B, the in vivo blocking studies in the monkey brain showed a significant reduction in
trapping in striatum and cortex after inhibition of MAO-B (Fig. 4E-F), with lesser effects effects for inhibition of
MAO-A. Again, the monkey imaging studies are more encouraging than those of the rat where significant effect
of both MAO-A and MAO-B inhibition were observed for [11C]COU. These studies emphasize the importance of
species selection when evaluating MAO substrates or inhibitors.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, the results for [11C]BiPHEN, a compound which was more selective for MAO-A in vitro (using human
and bovine sources of MAO), were less clear. Inhibition of MAO-B had mixed effects on [11C]BiPHEN
pharmacokinetics, and the inhibition with the MAO-A inhibitor moclobemide produced an unexpected increase
in trapping of radioactivity in all brain regions (Figs. 4 H-I). One possible explanation is that moclobemide
inhibition of peripheral MAO-A activity increased the available [11C]BiPHEN in the blood, resulting in increased
radiotracer delivery to the brain. We had observed such changes in brain pharmacokinetics in previous studies of
radiolabeled trapping agents for the enzyme acetylcholinesterase, where inhibition of blood enzymes
significantly increased radiotracer delivery to the brain. Studies of [11C]BiPHEN employing metabolite-corrected
blood input data would allow us to verify this hypothesis, however, the lower initial brain uptake, poor regional
heterogeneity, mixed pharmacological selectivity and potential for significant non-specific distribution due to its
high lipophilicity (clog P = 4.72) make [11C]BiPHEN at this point a less interesting candidate MAO-A radiotracer
than [11C]PHXY.
SUMMARY
These preliminary studies have demonstrated the feasibility of using 1-methyl-4-aryloxy-1,2,3,6-
tetrahydropyridines as substrates for in vivo PET imaging studies of the enzymatic activity of brain monoamine
oxidases. Two of the initial radiotracers examined here, [11C]PHXY and [11C]COU, already show encouraging
isozyme selectivity in the monkey brain, and are of immediate interest for further evaluation. The general
synthetic strategy shown in Scheme I can be used to prepare a wide variety of substituted tetrahydropyridines in
the continued search for radiotracers having the optimal combinations of lipophilicity, affinity for the enzyme
(Km) and rate of enzymatic hydrolysis (Kcat). Additional modifications of the rate of MAO-mediated oxidation of
1-methyl-4-aryloxy-1,2,3,6-tetrahydropyridines should also be possible by selective incorporation of deuterium at
the 6-position of the tetrahydropyridine ring, as demonstrated to be effective for MPTP17.
These studies also demonstrate some of the difficulties in developing new radiotracers for in vivo imaging
studies of the monoamine oxidases. The potential for significant species differences is well recognized for the
isozymes of MAO, and were reflected in the relatively little selectivity of [11C]PHXY and [11C]COU in the rat brain.
A correlation of regional in vivo brain trapping of our new radiotracers with in vitro values for enzyme activities is
essentially impossible given the extreme paucity of available data on the regional distributions of enzymatic
activities in the monkey brain. Finally, pharmacological studies in the monkey have to be planned carefully, as
the use of irreversible inhibitors (e.g., deprenyl and clogyline) that unequivocally block enzyme action poses a
challenge given the now recognized long-lasting effects of such inhibitors on the concentrations of active enzyme
molecules16.
EXPERIMENTAL SECTION
Chemistry. General Considerations. All solvents and reagents were commercially available and used without
further purification unless otherwise stated. 7-Methylumbeliferone and 3-phenylphenol were obtained from
Sigma-Aldrich; 4-Phenoxypyridine was purchased from TCI America and used directly as a precursor for
radiolabeling. NMR spectra were recorded with a Varian 400 MHz instrument at room temperature with
tetramethylsilane (TMS) as an internal standard. Mass spectra were performed on a Micromass LCT Time-of-
Flight mass spectrometer or an Agilent Q-TOF HPLC-MS employing the electrospray ionization (ESI) method.
High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT system
equipped with a Bioscan B-FC-1000 radiation detector.
General Procedure for preparation of 1-methyl-4-aryloxy-1,2,3,4-tetrahydropyridines (1a-c): Phenol starting
material (0.72 mmol) was added to sodium methoxide (0.87 mmol) dissolved in DMF (3 mL) and stirred for 10
minutes. 4-Chloro-1-methylpyridin-1-ium triflate (0.73 mmol) was added and the reaction was stirred for 18 h.
ACS Paragon Plus Environment

Page 7 of 11
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
The solvent was removed in vacuo and the resulting intermediate was suspended in methanol (3 mL). The
reaction mixture was cooled to 0 °C in a water ice bath, and NaBH4 (2.9 mmol) was added slowly. After 1 h the
solvent was removed in vacuo. Water and ethyl acetate were added to the residue and the mixture was transfer to
a separatory funnel. The product was extracted with ethyl acetate (3X), dried over sodium sulfate, filtered, and
concentrated in vacuo. The product was purified by flash silica gel chromatography (dichloromethane, methanol
gradient).
1-Methyl-4-phenoxy-1,2,3,6-tetrahydropyridine (PHXY, 1a). Starting from 4-phenoxypyridine, the reduction step
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
reaction yielded 0.056 g (41 % yield) of 1a as a white solid. H NMR (400 MHz; CH3OD-d4)/δ (ppm): 7.32 (2H, t,
J= 7.8, 2H), 7.08 (1H, t, J=7.4), 7.00 (2H, d, J= 7.8), 4.77 (1H, t, J= 3.2), 2.98 (2H, d J=3.1), 2.70 (2H, t, J=5.9), 2.38
(2H, m), 2.37 (3H, s); HRMS: calculated for [M+H]+(M = C12H15NO), 190.1226, found 190.1230.
4-Methyl-7-((1-methyl-1,2,3,6-tetrahydropyridin-4-yl)oxy)-2H-chromen-2-one (COU, 1b). Starting with 4-
methylumbeliferone, the reaction sequence yielded 0.091 g (47 % yield) of 1b as an off-white solid. 1H NMR (400
MHz; CH3OD-d4)/δ (ppm): 7.74 (1H, d, J=8.7), 7.06 (1H, dd, J=2.4, 8.7), 7.00(1H, d, J=2.4), 6.23(1H, d, J=1.1), 5.21
(1H, t, J=3.4); 3.17 (2H, d, J=3.4) 2.84 (2H, t, J=5.9), 2.48 (3H, s), 2.46 (3H, s), 2.42 (2H, m); HRMS: calculated for
[M+H]+(M = C16H17NO3), 272.1281, found 272.184.
4-([1,1'-Biphenyl]-3-yloxy)-1-methyl-1,2,3,6-tetrahydropyridine (BiPHEN, 1c). Starting from 3-hydroxybiphenyl, the
1
reaction sequence yielded 0.0743 g (39 % yield) of 1c as a pale yellow oil. H NMR (400 MHz; CH3OD-d4)/δ
(ppm): 7.57 (2H, d, J=7.2), 7.42-7.30 (5H, m), 7.27 (1H, t, J=1.9), 6.98 (1H, dq J=7.8, 1.2), 4.86 (1H, t, J=3.5), 2.96 (2H,
dd, J=5.8, 2.5), 2.67 (2H, t, J=5.9) 2.39 (2H, m), 2.34 (3H, s); HRMS: calculated for [M+H]+(M = C18H19NO),
266.1539, found 266.1538.
General Procedure for preparation of 4-aryloxypyridines (4b,c): Phenol starting material (1.6 mmol) was added to
potassium tert-butoxide (2.9 mmol) dissolved in DMF (7 mL). The reaction mixture was heated to 140 °C, 4-
chloropyridin-1-ium (1.33 mmol) was added, and the reaction was stirred for 18 h. The reaction was cooled to
room temperature and quenched with aqueous saturated NH4Cl. The product was extracted with ethyl acetate
(3X), dried over sodium sulfate, filtered, and concentrated in vacuo. The product was purified by flash silica gel
chromatography (hexanes, ethyl acetate gradient).
4-Methyl-7-(pyridin-4-yloxy)-2H-chromen-2-one (4b). Starting with 4-methylumbeliferone the reaction yielded
0.040 g (12 % yield) of 4b as an off-white solid. 1H NMR (400 MHz; CH3OD-d4)/δ (ppm): 8.48 (2H, d, J=6.1), 7.88
(1H, d, J=9.3), 7.170-7.155 (2H, m), 7.08 (2H, d, J=6.1), 6.33 (1H, s), 2.50 (3H, s); HRMS: calculated for [M+H]+(M =
C15H11NO3), 254.0812, found 254.0815.
4-([1,1'-Biphenyl]-3-yloxy)pyridine (4c): Starting with 3-hydroxybiphenyl the reaction yielded 0.1831 g (56 % yield)
1
of 4c as a yellow solid. H NMR (400 MHz; CH3OD-d4)/δ (ppm): 8.41 (2H, d, J=4.9, 1.5), 7.62 (2H, d, J=7.5), 7.55
(2H, m), 7.44 (2H, t, J=7.5), 7.40-7.34 (2H, m), 7.13 (1H, dt, J=7.1, 2.2), 7.00 (2H, dd, J=4.9, 1.5); HRMS: calculated
for [M+H]+(M = C17H13NO), 248.1070, found 248.1068.
Radiochemistry. General Considerations. Reagents and solvents were commercially available and used without
further purification, unless otherwise noted: sodium chloride (0.9% USP) and sterile water for Injection (USP)
were purchased from Hospira; Dehydrated Alcohol for Injection (USP) was obtained from Akorn Inc. Shimalite-
Nickle was purchased from Shimadzu; iodine was obtained from EMD; phosphorus pentoxide was acquired from
Fluka; molecular sieves were purchased from Alltech; and HPLC columns were acquired from Phenomenex.
Other synthesis components were obtained as follows: sterile filters were acquired from Millipore; C18-light Sep-
Paks and Porapak Q were purchased from Waters Corporation; 10 cc sterile vials were obtained from
HollisterStier. Sep-Paks were flushed with 10 mL of ethanol followed by 10 mL of sterile water prior to use.
General Procedure for Radiochemical Syntheses. Production of carbon-11 labeled radiotracers was carried out using
a TracerLab FXC-Pro automated radiochemistry synthesis module (General Electric, GE). [11C]Carbon dioxide was
produced using a GE PETTrace cyclotron (40 µA beam for 20 min) and converted by standard procedures into
carbon-11 labeled methyl triflate ([11C]CH3OTf). The [11C]CH3OTf in helium carrier gas was bubbled into a vial
containing a solution of precursor (1 mg) dissolved in ethanol (0.2 mL). At completion of transfer of radioactivity
into the reaction vial, the ethanol solution was then transferred to a second conical vial containing sodium
borohydride (2 mg) in ethanol (0.3 mL). The resulting mixture was stirred for 5 min at room temperature and
then the reaction was quenched by addition of HPLC buffer. The crude product was loaded onto a semi-
preparative HPLC loop. The product was purified by reverse phase chromatography (Prodigy ODS prep, 250 x 10
mm, 10µ, 4 mL/min), collected and diluted into H2O (40 mL) and reformulated using a C-18 extraction disk into a
final 5 mL total volume of 10 % ethanol in saline. The doses produced were assessed via standard quality control
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 8 of 11
1
2
3
4
5
6
7
8
9
techniques and were appropriate for rodent and non-human primate studies. Average specific activity was 69116
GBq/mmol (range of 23236-178081 GBq/mmol). Overall synthesis times were 30 min from end-of-bombardment.
[11C]1-Methyl-4-phenoxy-1,2,3,6-tetrahydropyridine ([11C]PHXY, [11C]1a). In non-optimized yields, 270 ± 173 mBq
of the phenyl ether (0.8 % yield from [11C]CH3OTf, not decay corrected; n=8) were collected. The product was
purified by semi-preparative reverse phase chromatography (250 x 10, 10µ, ODS prep column eluted with 40 %
CH3CN, 60 % H2O, 10 mM NH4OAc).
[11C]4-Methyl-7-(pyridin-4-yloxy)-2H-chromen-2-one ([11C]COU, [11C]1b). In non-optimized yields, 577 ± 141 MBq
of the coumarin ether (1.7 % yield from [11C]CH3OTf, not decay corrected; n=10) were collected. The product was
purified by semi-preparative reverse phase chromatography (250 x 10, 10µ, ODS prep column eluted with 30 %
CH3CN, 70 % H2O, 10 mM NH4OAc).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[11C]4-([1,1'-Biphenyl]-3-yloxy)-1-methyl-1,2,3,6-tetrahydropyridine ([11C]BiPHEN, [11C]1c). In non-optimized yields,
551 ± 126 MBq of the biphenyl ether (1.7 % yield from [11C]CH3OTf, non-decay corrected; n=7). The product was
purified by semi-preparative reverse phase chromatography (250 x 10, 10µ, ODS prep column eluted with 50 %
CH3CN, 50% H2O, 10 mM NH4OAc, pH 4.5).
ASSOCIATED CONTENT
Spectra for all novel compounds synthesized, chromatographic data for radiotracer purifications, and procedures
for microPET imaging. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Michael R. Kilbourn. E-mail: mkilbour@umich.edu. Tel.: +1 (734) 763-9246.
*Peter J. H. Scott. E-mail: pjhscott@umich.edu. Tel.: +1(734)615-1756.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final
version of the manuscript.
Funding
Financial support for this work from the National Institutes of Health (National Institute of Neurological
Diseases and Stroke, Award Number 1R21 NS075553 (MRK), and National Institute of Biomedical Imaging and
Bioengineering, Award Number T32-EB005172 (PJHS, MRK) is gratefully acknowledged. The content of this
article is solely the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health. Additional funding for this research from the Alzheimer's Association (NIRP-14-
305669 (PJHS)) is also acknowledged.
REFERENCES
1.
modulation of CNS monoamine neurotransmitter release. Pharmacology & Therapeutics 2014, 143 (2), 133-152.
2. Jacobs, A. H.; Tavitian, B., Noninvasive molecular imaging of neuroinflammation. J Cereb Blood Flow
Metab 2012, 32 (7), 1393-1415.
Finberg, J. P. M., Update on the pharmacology of selective inhibitors of MAO-A and MAO-B: Focus on
3.
(a) Kersemans, K.; Van Laeken, N.; De Vos, F., Radiochemistry devoted to the production of monoamine
oxidase (MAO-A and MAO-B) ligands for brain imaging with positron emission tomography. Journal of Labelled
Compounds and Radiopharmaceuticals 2013, 56 (3-4), 78-88; (b) Fowler, J. S.; Logan, J.; Shumay, E.; Alia-Klein, N.;
Wang, G.-J.; Volkow, N. D., Monoamine oxidase: radiotracer chemistry and human studies. Journal of Labelled
Compounds and Radiopharmaceuticals 2015, 58 (3), 51-64.
4.
(a) Carter, S. F.; Schöll, M.; Almkvist, O.; Wall, A.; Engler, H.; Långström, B.; Nordberg, A., Evidence for
Astrocytosis in Prodromal Alzheimer Disease Provided by 11C-Deuterium-L-Deprenyl: A Multitracer PET
Paradigm Combining 11C-Pittsburgh Compound B and 18F-FDG. Journal of Nuclear Medicine 2012, 53 (1), 37-46;
ACS Paragon Plus Environment

Page 9 of 11
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
(b) Choo, I.; Carter, S.; Schöll, M.; Nordberg, A., Astrocytosis measured by 11C-deprenyl PET correlates with
decrease in gray matter density in the parahippocampus of prodromal Alzheimer’s patients. Eur J Nucl Med Mol
Imaging 2014, 41 (11), 2120-2126.
5.
Tong, J.; Meyer, J. H.; Furukawa, Y.; Boileau, I.; Chang, L.-J.; Wilson, A. A.; Houle, S.; Kish, S. J.,
Distribution of monoamine oxidase proteins in human brain: implications for brain imaging studies. J Cereb
Blood Flow Metab 2013, 33 (6), 863-871.
6.
Journal of Labelled Compounds and Radiopharmaceuticals 2013, 56 (3-4), 172-179.
7. (a) Inoue, H.; Castagnoli, K.; Van der Schyf, C.; Mabic, S.; Igarashi, K.; Castagnoli, N., Species-Dependent
Kikuchi, T.; Okamura, T.; Zhang, M.-R.; Irie, T., PET probes for imaging brain acetylcholinesterase.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Differences in Monoamine Oxidase A and B-Catalyzed Oxidation of Various C4 Substituted 1-Methyl-4-phenyl-
1,2,3,6-tetrahydropyridinyl Derivatives. Journal of Pharmacology and Experimental Therapeutics 1999, 291 (2), 856-
864; (b) Shinotoh, H.; Inoue, O.; Suzuki, K.; Yamasaki, T.; Iyo, M.; Hashimoto, K.; Tominaga, T.; Itoh, T.; Tateno,
Y.; Ikehira, H., Kinetics of [11C]N,N-Dimethylphenylethylamine in Mice and Humans: Potential for Measurement
of Brain MAO-B Activity. Journal of Nuclear Medicine 1987, 28 (6), 1006-1011; (c) Halldin, C.; Bjurling, P.;
Stalnacke, C. G.; Jossan, S. S.; Oreland, L.; Langstrom, B., 11C-labelling of dimethylphenethylamine in two
different positions and biodistribution studies. International journal of radiation applications and
instrumentation. Part A, Applied radiation and isotopes 1989, 40 (7), 557-60.
8.
Livni, E.; Spellman, J. P.; Correia, J. A.; Alpert, N. M.; Brownell, G. L.; Strauss, H. W.; Elmaleh, D. R.,
[11C]MPTP: A Potential Tracer for Parkinson's Disease Research in Laboratory Animals. Journal of Nuclear
Medicine 1986, 27 (10), 1600-1603.
9.
(a) Efange, S. M.; Mash, D.; Hefti, F.; Kung, H. F.; Billings, J., Selective visualization of rodent locus
ceruleus by a radiolabeled N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine analog. Journal of neurochemistry 1989,
53 (2), 459-64; (b) Efange, S. M.; Kung, H. F.; Mash, D. C.; Jabir, M.; Billings, J.; Pablo, J.; Dutta, A.; Freshler, A.,
Pargyline-sensitive selective accumulation of a radiolabeled MPTP analog in the primate cerebral cortex and
basal ganglia. Synapse (New York, N.Y.) 1990, 5 (3), 207-12.
10.
Zhao, Z.; Dalvie, D.; Naiman, N.; Castagnoli, K.; Castagnoli, N., Design, synthesis, and biological
evaluation of novel 4-substituted 1-methyl-1,2,3,6-tetrahydropyridine analogs of MPTP. Journal of Medicinal
Chemistry 1992, 35 (23), 4473-4478.
11.
(a) Wang, Y.-X.; Castagnoli, N., Studies on the Monoamine Oxidase (MAO)-Catalyzed Oxidation of
Phenyl-Substituted 1-Methyl-4-phenoxy-1,2,3,6-tetrahydropyridine Derivatives: Factors Contributing to MAO-A
and MAO-B Selectivity. Journal of Medicinal Chemistry 1995, 38 (11), 1904-1910; (b) Yu, J.; Castagnoli, N., Jr.,
Synthesis and MAO-B substrate properties of 1-methyl-4-heteroaryl-1,2,3,6-tetrahydropyridines. Bioorganic &
medicinal chemistry 1999, 7 (2), 231-9.
12.
of MPTP as monoamine oxidase A and B substrates. J Med Chem 1994, 37 (7), 944-9.
13. Long, S.; Chen, L.; Xiang, Y.; Song, M.; Zheng, Y.; Zhu, Q., An activity-based fluorogenic probe for
Kalgutkar, A. S.; Castagnoli, K.; Hall, A.; Castagnoli, N., Jr., Novel 4-(aryloxy)tetrahydropyridine analogs
sensitive and selective monoamine oxidase-B detection. Chemical communications (Cambridge, England) 2012, 48
(57), 7164-6.
14.
(a) Fowler, J. S.; Ding, Y.-S.; Logan, J.; MacGregor, R. R.; Shea, C.; Garza, V.; Gimi, R.; Volkow, N. D.;
Wang, G.-J.; Schlyer, D.; Ferrieri, R.; Gatley, S. J.; Alexoff, D.; Carter, P.; King, P.; Pappas, N.; Arnett, C. D., Species
differences in [11C]clorgyline binding in brain. Nuclear Medicine and Biology 2001, 28 (7), 779-785; (b) Novaroli,
L.; Daina, A.; Favre, E.; Bravo, J.; Carotti, A.; Leonetti, F.; Catto, M.; Carrupt, P. A.; Reist, M., Impact of species-
dependent differences on screening, design, and development of MAO B inhibitors. J Med Chem 2006, 49 (21),
6264-72.
15.
(a) Riachi, N. J.; Harik, S. I., Monoamine oxidases of the brains and livers of macaque and cercopithecus
monkeys. Experimental neurology 1992, 115 (2), 212-7; (b) Campbell, I. C.; Marangos, P. J.; Parma, A.; Garrick, N.
A.; Murphy, D. L., Localization of monoamine oxidases A and B in primate brains relative to neuron-specific and
non-neuronal enolases. Neurochemical research 1982, 7 (6), 657-66.
16.
Fowler, J. S.; Volkow, N. D.; Logan, J.; Wang, G. J.; MacGregor, R. R.; Schyler, D.; Wolf, A. P.; Pappas, N.;
Alexoff, D.; Shea, C.; et al., Slow recovery of human brain MAO B after L-deprenyl (Selegeline) withdrawal.
Synapse (New York, N.Y.) 1994, 18 (2), 86-93.
17.
Ottoboni, S.; Caldera, P.; Trevor, A.; Castagnoli, N., Jr., Deuterium isotope effect measurements on the
interactions of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidase B. The
Journal of biological chemistry 1989, 264 (23), 13684-8.
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 10 of 11
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 11 of 11
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
101x43mm (300 x 300 DPI)
ACS Paragon Plus Environment