Synthesis and cerebral uptake of 1-(1-[11C]methyl-1H-pyrrol-2- yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone, a novel tracer for positron emission tomography studies of monoamine oxidase type A

Article abstract of DOI:10.1021/jm701378e

(R)-(-)- and (S)-(+)-1-(1-[¹¹C]methyl-1H-pyrrol-2-yl)-2-phenyl- 2-(1-pyrrolidinyl)ethanone 4 and 5 were synthesized, and their properties as tracers for positron emission tomography (PET) studies of monoamine oxidase type A (MAO-A) in the brain

Full text of DOI:10.1021/jm701378e

J. Med. Chem. 2008, 51, 1617–1622
1617
Synthesis and Cerebral Uptake of 1-(1-[11C]Methyl-1H-pyrrol-2-yl)-2-phenyl-2-
(1-pyrrolidinyl)ethanone, a Novel Tracer for Positron Emission Tomography Studies of
Monoamine Oxidase Type A
Svend Borup Jensen,*,† Roberto Di Santo,*,‡ Aage Kristian Olsen,† Kasper Pedersen,† Roberta Costi,‡ Roberto Cirilli,§ and
Paul Cumming†
PET Centre, Aarhus UniVersity Hospital, Nørrebrogade 44, 8000 Århus C, Denmark, Istituto PasteursFondazione Cenci Bolognetti,
Dipartimento di Studi Farmaceutici, UniVersità di Roma “La Sapienza”, P. le A. Moro 5, I-00185 Roma, Italy, and Dipartimento del Farmaco,
Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
ReceiVed August 13, 2007
(R)-(-)- and (S)-(+)-1-(1-[11C]methyl-1H-pyrrol-2-yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone 4 and 5 were
synthesized, and their properties as tracers for positron emission tomography (PET) studies of monoamine
oxidase type A (MAO-A) in the brain of living pigs were tested. Parametric maps of the distribution volume
(Vd) 4 in pig brain were qualitatively similar to those obtained with [11C]harmine, with prominent binding
in the ventral forebrain and mesencephalon. Its binding was highly vulnerable to MAO blockade, suggesting
a binding potential as high as 2 for MAO-A sites. The slow plasma metabolism of 4 and 5 may present
advantages over [11C]harmine for routine PET studies of MAO-A.
Chart 1. Pyrrolyletanoneamines 1-3 Endowed with Potent
Activities against MAOs and Newly Designed PET Tracers 4
and 5a
Introduction
Monoamine oxidase (MAO, EC 1.4.3.4) types A and B are
flavin-containing enzymes that contribute importantly to the
catabolism of dopamine and other biogenic primary monoamines
in the brain.1 The discovery of the antidepressant property of
the irreversible MAO-inhibitor N′-propan-2-ylpyridine-4-car-
bohydrazide (iproniazid) was an early breakthrough in psy-
chopharmacology.2 However, there have been relatively few
ligands suitable for studies of MAO by positron emission
tomography (PET). The propargylamine-compound [11C N-me-
thyl]N-[3-(2,4-dichlorophenoxy)propyl]-N-methyl-prop-2-yn-1-
amine ([11C]clorgyline) is a suicide MAO substrate with
considerable selectivity for MAO-A,3,4 and [11C N-methyl]N-
methyl-N-(1-methyl-2-phenyl-ethyl)-prop-2-yn-1-amine ([11C]de-
prenyl) is an irreversible inhibitor of MAO-B in living brain.5,6
However, the kinetic analysis of the cerebral binding of these
propargylamine ligands is hampered by the very high net
clearance from brain and rapid irreversible binding to MAOs,
such that net clearance from the brain approaches the limit
imposed by cerebral perfusion. Deuterium substitution of the
R-carbon of [11C]deprenyl results in a slower rate of association
with the MAO-B, which is more favorable for kinetic analysis.7,8
However, for reasons that remain uncertain, the deuterated
derivative of [11C]clorgyline yields cerebral-binding maps
endowed with relatively poorer contrast between regions of low
and high MAO-A activity than those produced by [11C]clor-
gyline itself.8,9 Several clorgyline analogues synthesized for PET
or SPECT (11C-fluoroclorgyline,10 18F-fluroclorgyline,11,12 and
a The position with the asterisk shows that the ligands have been labeled
with carbon-11.
clinical study of depression in humans).17 This tracer is very
rapidly metabolized in peripheral tissues (only 10% of the
plasma radioactivity remains as untransformed [11C]harmine at
10 min), which can be an encumbrance to the quantification of
its cerebral binding. Another MAO-A PET ligand, [11C-1](5R)-
5-(methoxymethyl)-3-[4-[(3R)-4,4,4-trifluoro-3-hydroxybutox-
y]phenyl]-2-oxazolidinone ([11C]befloxatone), is characterized
by relatively slower peripheral metabolism.18,19 However,
[11C]befloxatone is synthesized via [11C]phosgene, a dangerous
reagent available only at a few PET centers.
The lack of a ideal ligand and the emerging importance of
MAO-A in PET studies of depression17 and tobacco addiction20,21
justify the continued search for a ligand with optimal properties.
Recently, we designed and synthesized a series of pyrrolyle-
thanonamines (general structure 1; Chart 1) that showed potent
activities as MAO inhibitors, as well as high selectivity against
[
125I]iodoclorgyline) showed low cerebral uptake.13
One of the best-characterized reversible PET ligands for
imaging MAO-A is [11C-MeO]7-MeO-1-Me-9H-pyrido[3,4-b]-
indole ([11C]harmine, a ꢀ-carboline alkaloid, which has been
employed in both preclinical studies on animals14–16 and a
* To whom correspondence should be addressed. Telephone: +45-8949-
3034. Fax: +45-8949-3020. E-mail: svend@pet.auh.dk (S.B.J.); Telephone/
Fax: +39-6-49913150. E-mail: roberto.disanto@uniroma1.it (R.D.S.).
a Abbreviations: Vd, distribution volume; t0, dead time of the column;
R, enantioselectivity; CSP, chiral stationary phase; Ca(T), arterial input
function; PR(t), total plasma radioactivity; PPR(t), plasma precipitate
radioactivity; ftracer, unmetabolized parent radioligand.
†
Aarhus University Hospital.
Università di Roma “La Sapienza”.
Istituto Superiore di Sanità.
‡
§
10.1021/jm701378e CCC: $40.75
2008 American Chemical Society
Published on Web 02/29/2008

1618 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Jensen et al.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) AlCl3, CH2Cl2, -20 °C for 20 min and
(b) pyrrolidine, K2CO3, acetone, reflux for 96 h.
This synthetic pathway was not immediately transferable to
the synthesis of (R)-(-)- and (S)-(+)-2-phenyl-1-(1H-pyrrol-
2-yl)-2-(1-pyrrolidinyl)ethanone (desmethyl-R- and S-ROMAO,
6 and 7). Derivatives 6 and 7 were therefore obtained as
described in Scheme 2. The 1H-pyrrole underwent a Friedel-
–Crafts reaction with 2-chloro-2-phenylacetyl chloride to give
2-chloro-2-phenyl-1-(1H-pyrrol-2-yl)ethanone 8 in 20% yield.
This compound was treated with pyrrolidine in refluxing acetone
using K2CO3 as a base, to obtain 6 and 7 in 20% yield. The
racemic mixture was separated by enantioselective high-
performance liquid chromatography (HPLC) at a semiprepara-
tive scale on a polysaccharide-based chiral stationary phase
(CSPa) (Chiralpack IA) to obtain the enantiomers 6 and 7
(80–90% yield) with enantiomeric excess (ee) > 99%, checked
by an analytical Chiralpack IA column (Figure 1).
a Reagents and conditions for the synthesis of isomer 2: (a)
CF3COOC2H5, tetramethyl-guanidine, MeOH, room temperature for 24 h,
93%, (b) Vilsmeier reactive, n-butyl acetate, -15 °C for 3 h, (c)
1-methylpyrrole, AlCl3, CH2Cl2, -5 °C for 2 h, 13%, (d) concentrated HCl,
MeOH, 40°C for 15 h, 80%, and (e) 1,4-dibromobutane, NaI, KHCO3,
reflux, 12 h, 67%. The same conditions were used to synthesize the isomer
3, using (S)-phenylglycine as the starting material.
MAO type A. This selectivity was demonstrated against the two
specific isoforms on MAO in a mitochondrial preparation from
bovine brain, according to the method of Basford.22 Within this
series of MAO inhibitors, we identified (R)-(-)-1-(1-methyl-
1H-pyrrol-2-yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone (R-RO-
MAO, 2) as a candidate for a PET study for MAO-A based on
its 200 000-fold selectivity for MAO-A over the B form,23
exceeding even the 52 000-fold selectivity found of the S
enantiomer (S-ROMAO, 3). Furthermore, the chemical structure
of these compounds is amenable for [11C]-labeling (Chart 1).
Our strategy to obtain the compounds (R)-(-)-1-(1-[11C]methyl-
1H-pyrrol-2-yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone (R-[11C]RO-
MAO, 4) and (S)-(+)-1-(1-[11C]methyl-1H-pyrrol-2-yl)-2-
phenyl-2-(1-pyrrolidinyl)ethanone (S-[11C]ROMAO, 5) (Chart
1) was based on the synthesis of (R)-(-)- and (S)-(+)-2-phenyl-
1-(1H-pyrrol-2-yl)-2-(1-pyrrolidinyl)ethanone (6 and 7), which
underwent a [11C]-methylation on position 1 of the pyrrole ring
to afford the required labeled derivatives 4 and 5.
We have obtained more than a decade of experience with
the pig as an animal for preclinical investigations of radioligands
targeted at neurotransmitter systems.24–26 The large volume of
the pig brain (75 mL) is sufficient for the discernment of the
anatomical structure by PET, and we have already described in
some detail the spatial pattern of binding of [11C]harmine to
MAO-A in the brain of living pig.16 In the present study, we
tested the plasmas metabolism and cerebral uptake of 4 and 5
in anesthetized pigs. Specificity of, the binding of R-[11C]RO-
MAO in pig brain was tested in a blocking study performed by
the pretreatment of the animal with pargyline at a dose known
to block MAO-A in pig brain, 1 h before the tracer administration.
Although the racemic mixture 6 and 7 was obtained in only
about 4% global yield, no efforts were made to improve this
yield, because the optimization of the synthesis was deemed
beyond the scope of the present study.
Stereochemical Characterization of the Enantiomers 6
and 7. The absolute configuration of 6 and 7 was assigned by
a comparison of their circular dichroism (CD) spectra with those
of the structurally related compounds in the ROMAO series
(Figure 2). As previously reported,23,27 the dextrorotatory form
3 displays negative CD bands at around 254 and 319 nm and
two positive CD bands at around 229 and 290 nm. The CD
profile of the (+)-7 enantiomer in ethanol is strikingly similar
to that of reference compound (S)-(+)-ROMAO 3, while the
(-)-6 enantiomer showed opposite Cotton effects. Therefore,
we could empirically assign the S configuration to the dextroro-
tatory enantiomer 7 and the R configuration to the levorotatory
enantiomer 6.
Radiochemistry. The carbon-11 labeling of compound 6 to
obtain S-[11C]ROMAO (4) took 42 min ((5 min) (Scheme 3).
The same conditions were applied to transform its enantiomer
compound 7 into R-[11C]ROMAO (5). Yields were 1.2 GBq
((0.5 GBq) from 6 to 9 GBq [11C]CH3I. When the 20 min
physical half-life of carbon-11 was taken into account, the
calculated yield of the labeling reaction was 60–70%. The
products were 99% ((1%) radiochemically pure; the residual
content of precursor was below 50 µg/production; and the
contents of S- or R-ROMAO 4 or 5 were below 10 µg/
production. The specific activity was higher than 50 GBq/µmol.
Metabolic Stability of Carbon-11-Labeled S- and
R-ROMAO (4 and 5). Four pigs were used for both PET scan
imaging studies and studies of the plasma metabolism of the
carbon-11-labeled S- and R-ROMAO (4 and 5). Each pig was
Results and Discussion
Chemistry. The syntheses of (R)-(-)- and (S)-(+)-1-(1-
methyl-1H-pyrrol-2-yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone (R-
and S-ROMAO, 2 and 3; Scheme 1) have been described
previously.23

NoVel Tracer for PET Studies of MAO-A
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1619
Scheme 3a
a Reagents and conditions: (a) 11CH3I, NaH, DMF, room temperature
for 1 min. The same conditions were used to obtain 5 using 7 as the starting
material.
Figure 1. (Traces a and b) Analytical separation of desmethyl-ROMAO
with UV and polarimetric detection. (Traces c and d) Purity control of
the first and second fractions collected at a semipreparative scale. k1,
retention factor of the first eluted enantiomer, defined as (t1 - t0)/t0,
where t0 is the dead time of the column. R, enantioselectivity factor
defined as k2/k1. Rs, resolution factor defined as 2(t2 - t1)/(w1 + w2),
where t1 and t2 are retention times and w1 and w2 are band widths at
the baseline in time units.
Figure 3. Normalized uptake of the R- and S-[11C]ROMAO 4 and 5
in pig 3. The values are the average concentrations of the interval
between 10 and 60 min, as a percentage of the injected dose per liter
(% ID/L). Extracerebral voxels have been removed, and the images
are blurred (4 mm fwhm). Maps are shown in coronal, sagittal, and
horizontal planes.
declined to 98% at 2 min, 68% at 10 min, 41% at 30 min, and
24% untransformed at 50 min. Reverse-phase radio-HPLC
analysis of the plasma showed that the radio-metabolite(s) eluted
early in the radiochromatogram, indicating that radio-metabo-
lite(s) are considerably more polar than the parent compound.
This predicts that the metabolites do not enter the brain,28 which
is a precondition for kinetic modeling. Consequently, no attempt
was made to identify the polar plasma metabolites.
Figure 2. CD profiles of reference compound (S)-(+)-ROMAO (3)
and the enantiomers of desmethyl-ROMAO (6 and 7) in ethanol.
premedicatedandanaesthetisedasdescribedindetailpreviously.24–26
Catheters had been placed in an femoral artery for blood
sampling and in a femoral vein for radiotracer injection. Arterial
blood samples were collected at intervals during PET scans for
pigs 2, 3, and 4 only for HPLC plasma metabolite measure-
ments. Carbon-11 analogues 4 and 5 were relatively stable
toward metabolism in the plasma of living pigs, compared to
[11C]harmine.16 Metabolite HPLC analysis of the two stereoi-
somers shows no notable differences between the two isomers.
At 30 s after intravenous injection, 99.7% of the extracted
plasma radioactivity was the untransformed parent. This fraction
PET Examination of R- and S-[11C]ROMAO 4 and 5. The
anaesthetised pig was placed in the PET scanner before injection
of the radiotracer. On the basis of its higher selectivity for MAO-
A, we expected that R-[11C]ROMAO 4 might have superior
binding properties in the living brain when compared to its
enantiomer 5. Contrary to this expectation, maps of the
normalized uptake of the two enantiomers in pig 3 show that

1620 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Jensen et al.
to generate the arterial input concentration as a function of the
circulation time (arterial input function, Ca(T)):
Ca(T) ) PR(t)[PR(t) - PPR(t)/PR(t)]ftracer
(1)
where PR(t) is the total plasma radioactivity, PPR(t) is the
plasma precipitate radioactivity, and ftracer is the unmetabolized
parent radioligand, measured by radio-HPLC. This equation
corrects for both protein binding and metabolism of the free
tracer.
The first term of eq 1 referred to the measured plasma
radioactivity (PR); the second term [PR(t) - PPR(t)/PR(t)] takes
into account the amount unavailable for brain uptake because
of protein binding; and the third term (ftracer) considers the
removal of the metabolites from the input function.
In our previous study using [11C]harmine to visualize the
MAO enzyme, we found that the Vd in the pituitary gland was
partly resistant to pargyline blockage, suggesting some phar-
macological heterogeneity in that structure.16 However, [11C]RO-
MAO (4 nor 5) did not discernibly label the pig pituitary; indeed,
with [11C]ROMAO (4 or 5), the only region with distinctly
greater binding was the ventral forebrain (Figure 4), although
there was some evidence of greater uptake in the diencephalon.
The blocking study revealed a global fall in Vd throughout out
the brain, which was most conspicuous in the ventral forebrain,
consistent with the global pattern of distribution of MAO-A.
Conclusions
On the basis of these preliminary results, both R- and
S-[11C]ROMAO 4 and 5 appear to be very promising PET
tracers for the MAO-A enzyme in brain. There are several
positive attributes to these ligands, which may present advan-
tages over [11C]harmine and other existing ligands. In particular,
the carbon-11-labeling reaction is fairly simple and robust.
Yields of more than 1 GBq were routinely obtained and with
high specific activity of the final product. The metabolism of
R- and S-[11C]ROMAO (4 and 5) is relatively slow in plasma
of living pigs, in contrast to [11C]harmine, which is difficult to
detect in plasma at times after 10 min. Parametric maps of
[11C]ROMAOs 4 and 5 binding are qualitatively very compa-
rable to those of [11C]harmine.16 There was extensive displace-
ment of binding after the blockade of the MAO-A enzyme with
pargyline at a dose know to block entirely [11C]harmine binding
in pig brain.
Figure 4. Parametric images of R-[11C]ROMAO (4) distribution
volume in pig 4 before (A) and after (B) pargyline. Extracerebral voxels
have been removed, and the images are blurred (4 mm). Maps are shown
in coronal, sagittal, and horizontal planes.
net uptake of the S form 5 was 2-fold greater than for the R
form 4 (Figure 3). The specific activities of the two injections
were both very high (>100 GBq/µmol), and the dose injected
was very similar, at 480 MBq ((20 MBq). Compounds 4 and
5 have very similar metabolic patterns; therefore, differences
in plasma metabolism cannot explain the higher brain uptake
of the S form compared to the R form.
Comparison of the parametric maps of the equilibrium
distribution volume (Vd,a mL g-1) relative to the metabolite-
corrected arterial input for 4 and 5 (R- and S-[11C]ROMAO)
with previously obtained results for [11C]harmine16 showed that
all three tracers had preferential radioactivity accumulation in
the ventral forebrain/ventral striatum. In the parametric maps
of 4 and 5 (R- and S-[11C]ROMAO) Vd had only this single
“hotspot” (Figure 3), in contrast to [11C]harmine, which had a
somewhat more complex pattern of cerebral binding.
To verify the pharmacological nature of its binding in brain,
a blocking PET study was carried out with 4 in pig 4. A baseline
recording was followed by a second scan, initiated at 1 h after
pargyline treatment (5 mg/kg, i.v.). At intervals after tracer
injection, we measured the radioactivity in (i) whole arterial
blood, (ii) plasma, and (iii) acetonitrile precipitate of plasma.
The extract after acetonitrile precipitation plasma was examined
by HPLC to measure the metabolism at selected times. The Vd
maps were calculated as relative to the untransformed tracer in
the plasma extracts. The fraction precipitated with acetonitrile
was reduced to 25–30% after treatment with pargyline, sug-
gesting that bound R-[11C]ROMAO (4) can be trapped in a
plasma protein compartment by a MAO-dependent process.
Taking into account the above, we used the following equation
Experimental Section
Chemistry. General. Melting points were determined with a
Buchi 530 capillary apparatus and are uncorrected. Infrared (IR)
1
spectra were recorded on a Spectrum-one spectrophotometer. H
NMR spectra were recorded on a Bruker AC 400 spectrometer,
using tetramethylsilane (Me4Si) as an internal standard. All
compounds were routinely checked by thin-layer chromatography
(TLC) and 1H nuclear magnetic resonance (NMR). TLC was
performed using aluminum-baked silica gel plates (Fluka F254) and
aluminum-baked aluminum oxide plates (Fluka F254). The concen-
tration of solutions after reactions and extractions was obtained
using a rotatory evaporator operating at a reduced pressure of
approximately 20 Torr. Organic solutions were dried over anhydrous
sodium sulfate. Solvents and reagents were obtained from com-
mercial suppliers (Sigma-Aldrich) and were used without further
purification.
Syntheses. 2-Chloro-2-phenyl-1-(1H-pyrrol-2-yl)ethanone
(8). AlCl3 (9.9 g, 74.2 mmol) was added in small portions during
30 min into a well-stirred solution of pyrrole (10 g, 149.0 mmol)
and 2-chloro-2-phenylacetyl chloride (14.1 g, 74.5 mmol) in CH2Cl2
(120 mL) cooled at -20 °C. The mixture was stirred at -20 °C
for 20 min, and then the mixture was added to a slush consisting

NoVel Tracer for PET Studies of MAO-A
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1621
of crushed ice (250 g) and concentrated HCl (17 mL). Extraction
with CHCl3 (3 × 200 mL) provided an organic solution that was
washed with brine (3 × 250 mL), saturated NaHCO3 solution (1
× 250 mL), then with brine again (3 × 250 mL), before it was
dried. Removal of the solvent by evaporation yielded a residue,
which was chromatographed on an alumina column (chloroform
as the eluent) to afford pure 8 (racemic mixture) (3.27 g, 20%).
For analytical data, see the Supporting Information.
tomograph. Pig 4 was scanned with R-[11C]ROMAO (4) before
and after blocking with pargyline. Blood samples were collected
during scans for pigs 2, 3, and 4 for metabolite measurements.
Radiochemistry. Acetonitrile (HPLC), dichloromethane, iodine,
sodium phosphate monohydrate, and bufotenine (1 mg/mL) in
acetonitrile were purchased from Bie and Berntsen, Denmark.
Pargyline HCl, were obtained from Sigma-Aldrich, Denmark. The
synthetic route to obtain (R)-(-)- and (S)-(+)-1-(1-[11C]methyl-
1H-pyrrol-2-yl)-2-phenyl-2-(pyrrolidin-1-yl)ethanoneR-andS-[11C]RO-
MAO 4 and 5 is shown in Scheme 3. [11C]Carbon dioxide was
prepared by a General Electric Medical Systems PET trace 200
cyclotron by a 14N(p,R)11C nuclear reaction. [11C]carbon dioxide
was reduced to [11C]methane, which was then converted to
[11C]methyl iodide using the General Electric methyl iodide box.
The [11C]methyl iodide (6–9 GBq after 30 min of bombardment at
40 µA) was transferred in a stream of nitrogen and bubbled though
the reaction mixture in a capped vial via a needle and vent needle.
The premade reaction mixture was a suspension containing the
precursor (6 or 7, ca. 1.5 mg) and NaH (0.3 mg) in DMF (300
µL); it was shaken before the [11C]methyl iodide gas was bubbled
through it. The reaction mixture was left for 1 min at room
temperature before it was quenched with HPLC eluent (0.3 mL)
and transferred into the HPLC loop.
The reaction mixture was purified using a semipreparative HPLC
(PerkinElmer model 200) equipped with a 5 mL injection loop.
Product elution was monitored with online γ detection of in-house
design, in conjunction with UV-visible detection (Applied Bio-
systems model 759A, λ ) 300 nm). The mobile phase, consisting
of 30–40% ammonium formate (0.1 M, pH adjusted to 8.5 with
NaOH) and 60–70% acetonitrile, was delivered at a rate of 10 mL/
min to a SpherClone ODS (2) column. In both cases, the products
eluted as broad peaks: the first one has Rf of 10–16 min (40%
acetonitrile), and the second one has Rf of 25–32 min (30%
acetonitrile). The fraction corresponding to the labeled product (ca.
25 mL) was collected, concentrated to ca. 5 mL under reduced
pressure at 90 °C, and formulated with isotonic saline solution (5
mL).
2-Phenyl-1-(1H-pyrrol-2-yl)-2-(1-pyrrolidinyl)ethanone (6
and 7) (Desmethyl-ROMAO). A solution of 8 (2.6 g, 11.6 mmol)
in acetone (52 mL) was added dropwise into a well-stirred
suspension of pyrrolidine (1.6 g, 23.1 mmol) and K2CO3 (3.0 g,
21.6 mmol) in acetone (52 mL). The mixture was refluxed with
stirring for 96 h and then treated with water. After extraction with
CHCl3 (100 mL), the organic solution was washed with brine (5 ×
50 mL) and dried. Evaporation of the solvent gave a crude product,
which was chromatographed on a silica column (5:1 ethyl acetate/
chloroform as the eluent) to afford 6 and 7 (racemic mixture) (590
mg, 20%). For analytical data, see the Supporting Information.
Enantioseparation. HPLC-grade solvents were supplied by
Carlo Erba (Milan, Italy). Diethylamine (DEA) was obtained from
Fluka Chemie (Buchs, Switzerland). HPLC enantioseparations were
carried out by using a Perkin-Elmer (Norwalk, CT) 200 liquid
chromatographic pump equipped with a Rheodine (Cotati, CA)
injector with a 20 µL (analytical) or 1 mL (semipreparative) sample
loop, a Perkin-Elmer column oven, and a Perkin-Elmer 290 UV
detector. The sign of the optical rotations of enantiomers of
compounds tested was measured online at a wavelength of 365
nm by a Perkin-Elmer polarimeter model 241 equipped with Hg/
Na lamps and a 40 µL flow-cell. The signal was acquired and
processed by Clarity (Data Apex, Prague, Czech Republic) software.
The chiral analytes were dissolved in ethanol, and their CD spectra
were measured in a quartz cell (0.1 cm path length) using a Jasco
J-710 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan),
maintained thermostatically at 25 °C. The mean spectra from three
instrumental scans were calculated as ellipticity values (millide-
grees).
Semipreparative enantioseparations of 6 and 7 (desmethyl-
ROMAO) were accomplished by HPLC on a polysaccharide-based
chiral stationary phase (CSP) column type Chiralpak IA 250 ×
4.6 mm I.D., with a mixture of 60:40:1:0.1 n-hexane/ethyl acetate/
ethanol/DEA (v/v/v/v) as the eluent. The column temperature was
set at 25 °C, with a flow rate of 4 mL/min and detection wavelength
at 280 nm. After each semipreparative chromatographic run (25
mg), the fractions corresponding to a single enantiomer were pooled
and evaporated. The collected fractions of each enantiomer were
analyzed by analytical Chiralpak IA chiral columns, with a flow
rate at 1.0 mL/min, at 25 °C with UV detection at 280 nm, to
determine their enantiomeric excess (ee > 99%). The yields ranged
from 80 to 90% (Figure 1).
Animals, Anesthesia, Surgery, and PET Scanning. The study
procedure was approved by the Danish Experimental Animal
Inspectorate. A total of 4 Danish Landrace/Yorkshire pigs (females;
38.25–40.5 kg of body weight; approximately 3 months old) were
used. For experimental details, see the Supporting Information.
In three cases, the pigs had been used for other PET-scanning
protocols before this study. They had previously been treated with
6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-
methyl-3-pyridinecarboxamide hydrochloride (GSK189254) (pig 1),
7-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy]-3,4-dihydro-1H-
quinolin-2-one (aripiprazole) (pig 2), and 3,5-dichloro-N-[(1-
ethylpyrrolidin-2-yl)methyl]-2-hydroxy-6-methoxy-benzamide (raclo-
pride), 3-(2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl)-2,4(1H,3H)-
quinazolinedione tartrate salt (kentaserin), 2-(5-methoxy-1H-indol-
The purity of the final product was determined by HPLC, using
a BondClone C18 column (5 µm, 250 × 4.6 mm) with an eluent
consisting of 50% aqueous 0.1 M ammonium formate (pH 8.5 with
NaOH) and 50% acetonitrile, with serial UV (250 nm) and γ
detection. Reference standards of ROMAO (50 µg/mL) (2 or 3)
and desmethyl-ROMAO (6 or 7) (50 µg/mL) were used for
quantification. The product solution was spiked with ROMAO (2
or 3) in a second run to verify product identity on the basis of the
co-elution position of the UV peak and the radioactivity peaks.
Pharmacokinetic Analysis. No arterial input was obtained for
pigs 1–3; therefore, the analysis of tracer uptake in these three pigs
were limited to the comparison of the region of interest (ROI)
analysis of the summation images. For pig 4, continuous arterial
inputs of untransformed R-[11C]ROMAO (4) and the sum of its
metabolites were calculated. The area under the curve for plasma
R-[11C]ROMAO (4) as a percentage of the total injected dose was
calculated for the interval of the PET recording (90 min). Plasma
metabolite analysis in pigs 3 and 4 was made from arterial blood
samples (1 mL) drawn at 0.5, 2, 5, 10, 20, 30, 40, 50, 70, and 90
min after injection. The blood samples were centrifuged (1300g)
for 1 min, and then 0.5 mL of the plasma was denatured by mixing
with 0.5 mL of acetronitrile. The mixture was shaken and then
centrifuged (1300 g) for 5 min. The supernatants were analyzed
using a BondClone C18 column (5 µm, 250 × 4.6 mm) with an
eluent consisting of 50% aqueous 0.1 M ammonium formate (pH
8.5 with NaOH) and 50% acetonitrile, with serial UV (280 nm)
and γ detection at a flow rate of 2.5 mL/min. The radioactivity of
the precipitate was measured in a well counter cross-calibrated to
the tomograph.
3-yl)-N,N-dimethylethanamine
(MeO-DMT),
2-ethenyl-4-
azabicyclo[2.2.2]oct-5-yl)- (6-methoxyquinolin-4-yl)-methanol
(quinidine) (pig 3) at pharmalogically active doses, in the hours
prior to scanning with [11C]ROMAO (4 or 5). The fourth pig was
dedicated to the [11C]ROMAO (4 or 5) study. After acquisition of
a brief attenuation scan, 90 min long dynamic emission recordings
were performed in the aperture of the Siemens ECAT Exact
Voxel-wise parametric maps of the distribution volume (Vd, mL/
g) of R-[11C]ROMAO (4) were calculated in the native PET space
relative to the metabolite-corrected arterial input by the method of
Turkheimer et al.29 for pig 4. In this method, Akaike-weighted
averages of the estimates of Vd for one- and two-tissue compartment

1622 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Jensen et al.
(14) Bergström, M.; Westerberg, G.; Långström, B. 11C-Harmine as a tracer
for monoamine oxidase A (MAO-A): In vitro and in vivo studies.
Nucl. Med. Biol. 1997, 24, 287–293.
(15) Bergström, M.; Westerberg, G.; Nemeth, G.; Traut, M.; Gross, G.;
Greger, G.; Müller-Peltzer, H.; Safer, A.; Eckernäs, S.-Å.; Grahnr,
A.; Långström, B. MAO-A inhibition in brain after dosing with
esuprome, moclobemide, and placebo in healthy volunteers: In vivo
studies with positron emission tomography. Eur. J. Clin. Pharmacol.
1997, 52, 121–128.
(16) Jensen, S. B.; Olsen, A. K.; Pedersen, K.; Cumming, P. Effect of
monoamine oxidase inhibition on amphetamine-evoked changes in
dopamine receptor availability in the living pig: A dual tracer PET
study with [11C]harmine and [11C]raclopride. Synapse 2006, 59, 427–
434.
(17) Meyer, J. H.; Ginovart, N.; Boovariwala, A.; Sagrati, S.; Hussey, D.;
Garcia, A.; Young, T.; Prashak-Rieder, N.; Wilson, A. A.; Houle, S.
Elevated monoamine oxidase A levels in the brainsAn explanation
for the monoamine imbalance of major depression. Arch. Gen.
Psychiatry 2006, 63, 1209–1216.
(18) Bottlaender, M.; Dolle, F.; Guenther, I.; Roumenov, D.; Fuseau, C.;
Barmoulle, Y.; Curet, O.; Jegham, J.; Pinquier, J. L.; Valette, G. P.
Mapping the cerebral monoamine oxidase type A: Positron emission
tomography with the reversible selective inhibitor [11C]befloxatone.
J. Pharmacol. Exp. Ther. 2003, 305, 467–473.
(19) Dolle, F.; Valette, H.; Barmoulle, Y.; Guenther, I.; Fuseau, C.; Coulon,
C.; Lartizien, C.; Jegham, S.; George, P.; Curet, O.; Pinquier, J. L.;
Bottlaender, M. Synthesis and in vivo imaging properties of [11C]be-
floxatone: A novel highly potent positron emission tomography ligand
for mono-amine oxidase-A. Bioorg. Med. Chem. Lett. 2003, 13, 1771–
1775.
(20) Fowler, J. S.; Volkow, N. D.; Wang, G. J.; Pappas, N.; Logan, J.;
Shea, C.; Alexoff, D.; mecGregor, R. R.; Schlyer, D. J.; Zezulkova,
I.; Wolf, A. P. Brain monoamine oxidase A inhibition in cigarette
smokers. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14065–14069.
(21) Fowler, J. S.; Volkow, N. D.; Wang, G. J.; Pappas, N.; Logan, J.;
MacGregor, R.; Alexoff, D.; Wolf, A. P.; Warner, D.; Cilento, R.;
Zezulkova, I. Neuropharmacological actions of cigarette smoke: Brain
monoamine oxidase B (MAO B) inhibition. J. Addict. Dis. 1998, 17,
23–34.
models were obtained in each voxel using linearized equations.30
The sum of all emission frames from each PET session was
calculated and manually registered to the MR-based common
stereotaxic space for the minipig brain,29 using a rigid-body
transformation with 9 degrees of freedom in “Register”.31 Visual
inspection of the individual dynamic and summed emission
recordings did not reveal discernible head movement in the course
of each PET session. Therefore, individual parametric maps of Vd
were resampled into the common stereotaxic space using the
transformation matrix and averaged by condition.
Acknowledgment. This project was supported by the Italian
MUR and Denmark’s National Science Foundation.
Supporting Information Available: Analytical data on com-
pounds 6-8 and experimental data on animal handling. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Schoepp, D. D.; Azzaro, A. J. Specificity of endogenous substrates
for types A and B monoamine oxidase in rat striatum. J. Neurochem.
1981, 36, 2025–2031.
(2) Youdim, M. B. Monoamine oxidase. Its inhibition. Mod. Probl.
Pharmacopsychiatry 1975, 10, 65–88.
(3) MacGregor, R. R.; Halldin, C.; Fowler, J. S.; Wolf, A. P.; Arnett,
C. D.; Langstrøm, B.; Alexoff, D. Selective, irreversible in vivo binding
of [11C]clorgyline and [11C]-L-deprenyl in mice: potential for measure-
ment of functional monoamine oxidase activity in brain using positron
emission tomography. Biochem. Pharmacol. 1985, 34, 3207–3210.
(4) Fowler, J. S.; MacGregor, R. R.; Wolf, A. P.; Arnett, C. D.; Dewey,
S. L.; Schlyer, D.; Christman, D.; Logan, J.; Smith, M.; Sachs, H.;
Aquilonius, S. M.; Bjurling, P.; Halldin, C.; Hartvig, P.; Leenders,
K. L.; Lundqvist, H.; Oreland, L.; Stalnacke, C. G.; Langström, B.
Mapping human brain monoamine oxidase A and B with 11C-labeled
suicide inactivators and PET. Science 1987, 235, 481–485.
(5) Arnett, C. D.; Fowler, J. S.; MacGregor, R. R.; Schlyer, D. J.; Wolf,
A. P.; Långström, B.; Halldin, C. Turnover of brain monoamine
oxidase measured in vivo by positron emission tomography using
L-[11C]deprenyl. J. Neurochem. 1987, 49, 522–527.
(6) Lammertsma, A. A.; Bench, C. J.; Price, G. W.; Cremer, J. E.; Luthra,
S. K.; Turton, D.; Wood, N. D.; Frakowiak, R. S. J. Measurement of
cerebral monoamine oxidase B activity using L-[11C]deprenyl and
dynamic positron emission tomography. J. Cereb. Blood Flow Metab.
1991, 11, 545–556.
(7) Fowler, J. S.; Wolf, R. R.; MacGregor, R. R.; Dewey, S. L.; Logan,
J.; Schlyer, D. J.; Langstrom, B. Mechanistic positron emission
tomography studies: Demonstration of a deuterium isotope effect in
the monoamine oxidase catalyzed binding of [11C]L-deprenyl in living
baboon brain. J. Neurochem. 1988, 51, 1524–1534.
(8) Fowler, J. S.; Wang, G. J.; Logan, J.; Xie, S.; Volkow, N. D.;
MacGregor, R. R.; Schlyer, D. J.; Pappas, N.; Alexoff, D. L.; Patlak,
C.; Wolf, A. P. Selective reduction of radiotracer trapping by deuterium
substitution: Comparison of carbon-11-L-deprenyl and carbon-11-
deprenyl-D2 for MAO B mapping. J. Nucl. Med. 1995, 36, 1255–
1262.
(9) Logan, J.; Fowler, J. S.; Ding, Y. S.; Franceschi, D.; Wang, G. J.;
Volkow, N. D.; Felder, C.; Alexoff, D. Strategy for the formation of
parametric images under conditions of low injected radioactivity
applied to PET studies with the irreversible monoamine oxidase A
tracers [11C]clorgyline and deuterium-substituted [11C]clorgyline.
J. Cereb. Blood Flow Metab. 2002, 22, 1376–1376.
(10) Ohmomo, Y.; Hirata, M.; Murakami, K.; Magata, Y.; Tanaka, C.;
Ypkoyama,A.Synthesisandcharacterizationof11C-labeledfluoroclorgylinesA
monoamine oxidase-A specific inhibitor fpr positron emission tomog-
raphy. Chem. Pharm. Bull. 1993, 41, 1994–1997.
(11) Mukherjee, J.; Yang, Z.-Y. Development of N-[3-(2′,4′-dichlorophe-
noxy)-2-18F-fluoropropyl]-N-methylpropargylamine (18F-fluoroclor-
gyline) as a potential PET radiotracer for monoamine oxidase-A. Nucl.
Med. Biol. 1999, 26, 619–625.
(22) Basford, R. E. Preparation and properties of brain mitochondria.
Methods Enzymol. 1967, 10, 96–101.
(23) Di Santo, R.; Costi, R.; Roux, A.; Artico, M.; Befani, O.; Meninno,
T.; Agostinelli, E.; Palmegiani, P.; Turini, P.; Cirilli, R.; Ferretti, R.;
Gallinella, B.; La Torre, F. Design, synthesis, and biological activities
of pyrrolylethanoneamine derivatives, a novel class of monoamine
oxidases inhibitors. J. Med. Chem. 2005, 48, 4220–4223.
(24) Lind, N. M; Moustgaard, A.; Jelsing, J.; Vajta, G.; Cumming, P.;
Hansen, A. K. The use of pigs in neuroscience: Modeling brain
disorders. Neurosci. BiobehaV. ReV. 2007, 31 (5), 728–751.
(25) Pedersen, K.; Simonsen, M.; Ostergaard, S. D.; Munk, O. L.; Rosa-
Neto, P.; Olsen, A. K.; Jensen, S. B.; Moller, A.; Cumming, P.
Mapping the amphetamine-evoked changes in [11C]raclopride binding
in living rat using small animal PET: Modulation by MAO inhibition.
Neuroimage 2007, 35 (1), 38–46.
(26) Jensen, S. B.; Smith, D. F.; Bender, D.; Jakobsen, S.; Peters, D.;
Nielsen, E. O.; Olsen, G. M.; Scheel-Kruger, J.; Wilson, A.; Cumming,
P. [11C]-NS 4194 versus [11C]-DASB for PET imaging of serotonin
transporters in living porcine brain. Synapse 2003, 49, 170–177.
(27) La Torre, F.; Cirilli, R.; Costi, R.; Di Santo, R.; Ferretti, R.; Gallinella,
B.; Salerno, A.; Zanitti, L. Enantiomers of new synthetic pyrrolylphe-
nylethanoneamine mono-amino oxidase inhibitor compounds: Analyti-
cal and semipreparative HPLC separations, and chiroptical charac-
teristics. Chromatographia 2004, 60, 171–178.
(28) Cumming, P.; Yokoi, F.; Chen, A.; Deep, P.; Dagher, A.; Reutens,
D.; Kapczinski, F.; Wong, D. F.; Gjedde, A. Pharmacokinetics of
radiotracers in human plasma during positron emission tomography.
Synapse 1999, 34, 124–134.
(29) Turkheimer, F. E.; Hinz, R.; Cunningham, V. J. On the undecidability
among kinetic models: From model selection to model averaging.
J. Cereb. Blood Flow Metab. 2003, 23, 490–498.
(30) Zhou, Y.; Brasic, J. R.; Ye, W.; Dogan, A. S.; Hilton, J.; Singer, H. S.;
Wong, D. F. Quantification of cerebral serotonin binding in normal
controls and subjects with Tourette’s syndrome using [11C ]MDL
100,907 and (+)[11C]McN 5652 dynamic PET with parametric
imaging approach. Neuroimage 2004, 22, T98–T99.
(31) MacDonald, D. Program for Registration of Images (Register);
McConnell Brain Imaging Center: Montreal, Canada, 1986.
(12) Mukherjee, J.; Yang, Z.-Y. Monoamine oxidase A inhibition by
fluoxetine: an in vitro and in vivo study. Synapse 1999, 31, 285–289.
(13) Hamid, R.; Chalon, S.; Ombetta, J.-E.; Frangin, Y.; Garreau, L.;
Dognon, A.-M.; Lena, I.; Bodard, S.; Vilar, M.-P.; Besnard, J.-C.;
Guilloteau, D. Synthesis and characterization of [125I]N-(2-aminoet-
hyl)-4-iodobenzamide as a selective monoamine oxidase B inhibitor.
Nucl. Med. Biol. 1995, 22, 617–623.
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