11C-Labeling of aryl ketones as candidate histamine subtype-3 Receptor PET radioligands through Pd(0)-Mediated 11C-Carbonylative coupling

Article abstract of DOI:10.3390/molecules22050792

Pd(0)-mediated coupling between iodoarenes, [¹¹C]carbonmonoxide and aryltributylstannanes has been used to prepare simple model [¹¹C]aryl ketones. Here, we aimed to label four 2-Aminoethylbenzofuran chemotype based molecules ([¹

Full text of DOI:10.3390/molecules22050792

molecules
Article
11C-Labeling of Aryl Ketones as Candidate Histamine
Subtype-3 Receptor PET Radioligands through
Pd(0)-Mediated 11C-Carbonylative Coupling
Fabrice G. Siméon, William J. Culligan, Shuiyu Lu and Victor W. Pike *
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health,
Bethesda, MD 20892-1003, USA; simeonf@mail.nih.gov (F.G.S.); wjculligan@gmail.com (W.J.C.);
shuiyu.lu@mail.nih.gov (S.L.)
* Correspondence: pikev@mail.nih.gov; Tel.: +1-301-594-5986
Academic Editor: Diego Muñoz-Torrero
Received: 17 April 2017; Accepted: 7 May 2017; Published: 12 May 2017
Abstract: Pd(0)-mediated coupling between iodoarenes, [11C]carbon monoxide and aryltributylstannanes
has been used to prepare simple model [11C]aryl ketones. Here, we aimed to label four
2-aminoethylbenzofuran chemotype based molecules ([11C]
1–4) in the carbonyl position,
as prospective positron emission tomography (PET) radioligands for the histamine subtype 3
receptor (H3R) by adapting this methodology with use of aryltrimethylstannanes. Radiosynthesis
was successfully performed on a platform equipped with a mini-autoclave and a liquid handling
robotic arm, within a lead-shielded hot-cell. Candidate radioligands were readily formulated in
saline containing ethanol (10%, v/v) and ascorbic acid (0.5 mg/10 mL). Yields for preclinical use were
in the range of 5–9%, decay-corrected from cyclotron-produced [11C]CO2 and molar activities were
>115 GBq/µmol at end of synthesis. Radiochemical purities exceeded >97%.
Keywords: [11C]carbon monoxide; histamine 3 subtype; carbon-11; carbonylative coupling;
radioligand
1. Introduction
Histamine subtype-3 receptors (H3Rs) are located on presynaptic nerve terminals where they
regulate the synthesis and release of histamine and also modulate the release of other neurotransmitters,
notably dopamine, serotonin, norepinephrine and γ-aminobutyric acid [1,2]. H3R inverse agonists
have proven clinical efficacy for the treatment of neuropsychiatric and neurodegenerative disorders,
such as excessive diurnal sleepiness in patients with narcolepsy or Parkinson’s disease, Alzheimer’s
disease, schizophrenia, narcolepsy and attention deficit disorders [3,4]. H3R inverse agonists are also a
source of leads for developing positron emission tomography (PET) radioligands for H3R, which could
be powerful tools for (i) elucidating differences in H3R distribution and density between healthy
and disease states; and (ii) for determining the dose-dependence and duration of brain H3R receptor
occupancy by drug candidates.
Several 18F (t1/2 = 109.8 min) and 11C (t1/2 = 20.4 min) labeled H3R radioligands have emerged
based on imidazole and on non-imidazole chemotypes [5–9]. The non-imidazole chemotype has
provided radioligands with imaging performance in non-human primates and human subjects
superior to that of the imidazole chemotype. In our continuing efforts to develop more selective
PET radioligands for H3R based on a 2-aminoethylbenzofuran chemotype, we recently reported the
2-aminoethylbenzofuran derivative [18F]
with PET and discovered that the nitro-precursor (
H3R ligand [ ]. We hypothesized that could be labeled with carbon-11 and that similar labeling of
2
as a putative high-affinity radioligand to image H3R in vivo
) of [18F]
was also a selective and high-affinity
1
2
7
1
Molecules 2017, 22, 792; doi:10.3390/molecules22050792
www.mdpi.com/journal/molecules

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potent congeners of
2
might provide alternative radioligands with more choice of starting material,
labeling site, and metabolic profile. An added advantage of a carbon-11 label over a fluorine-18 label is
that it allows two PET scans in one day in the same subject.
[11C]Aryl ketones have been prepared by Pd(0)-mediated [11C]carbonylative coupling reactions
of diaryliodonium salts with aryltributylstannanes [10], and iodoarenes or aryl triflates with
boronic acids (Suzuki reactions) [11–13] or aryltrialkylstannanes (Stille reactions) [14–16] (Scheme 1).
The reagents used in these methods are not overly sensitive to atmospheric oxygen and moisture.
These methods complement each other and widen the choice of synthetic strategies and starting
materials. Nevertheless, up to now, literature examples exploring methodology development have
been largely limited to the labeling of simple substrates with limited structural complexity and
functionality. Only one example of a PET radioligand labeled with carbon-11 in a ketone carbonyl
position has been reported in the open literature [17]. Here, we aimed to synthesize four candidate
H3R radioligands ([11C]
aryltrimethylstannyl derivatives (12–15) via Pd(0)-mediated [11C]CO coupling.
1–4) by reaction of a common iodoarene precursor (11) with one of four
Scheme 1. Methods for synthesis of [11C]aryl ketones.
2. Results and Discussion
2.1. Lead Compound Selection and Synthesis of Non-Radioactive Standards
We initially selected 1–4 (Table 1) as lead compounds based on their reported high affinities for
human H3R and some reported attractive pharmacokinetic properties (e.g., good brain penetration,
adequate half-life in vivo) [18], plus moderate computed lipophilicities (cLogD values), all of which
were considered promising for PET radioligand development [19,20].
Table 1. Ki values determined from in vitro competitive binding assays and calculated LogD for 1–4.
Affinity (Ki, nM)
cLogD
Ligand
Ar
c
c
H3 (lit) a
H2
H1
b
H3
1
2
3
4
4-NO2-C6H4
4-F-C6H4
3-F-C6H4
-
2.1
706
923
1368
630
1030
7055
>10,000
>10,000
2.69
2.90
2.79
2.75
0.27
0.10
0.09
4.2
4.9
2.9
3,5-di-F-C6H3
a
Assayed by displacement of [3H]
α
-methylhistamine from cell membranes isolated from C6 cells expressing
-methylhistamine from guinea pig cells
cloned human H3 receptors [18]; b Assays performed by displacing [3H]
expressing H3 receptors at the NIMH’s Psychoactive Drug Screening Program (PDSP); Assays performed by
displacing [3H]tiotidine for H2R, and [3H]pyrilamine for H1R from human recombinant receptors expressed in
HEK293T cells at PDSP.
α
c

Molecules 2017, 22, 792
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Compounds
1
–
3
were prepared as reported previously [18] to provide reference materials and
samples for screening in pharmacological binding assays. The preparation of
4 was described
previously as a 6-step synthesis, starting with 4-benzyloxybenzoic acid, in low overall yield (<6%).
Instead of using this procedure, we designed and implemented a simplified 4-step procedure starting
with 3,5-difluorobenzoyl chloride that afforded
3,5-difluorobenzoyl chloride was treated with anisole in the presence of scandium triflate to give
(59%). The was demethylated with boron tribromide to give (89%). The resultant phenol was
treated with iodine and potassium iodide in basic solution to give (67%). Coupling of with
4 in an improved 22% overall yield (Scheme 2). First,
5
5
6
7
7
(R)-1-(but-3-ynyl)-2-methylpyrrolidine gave the target compound 4 in 61% yield.
◦
Scheme 2. New synthetic route for compound
4
. Conditions: (
a
) Sc(OTf)3; (
b) BBr3,
−
78 C, DCM; (c) KI, I2
,
NH4OH; (d) (R)-1-(but-3-ynyl)-2-methylpyrrolidine in MeCN, Pd(PPh3)2Cl2, CuI, NEt3, KI, DMF.
In our evaluation, all four ligands exhibited high human H3 receptor affinity and high selectivity
(>200 fold) over other histamine receptor subtypes (Table 1), thereby affirming their suitabilities in
these regards for consideration as potential candidate PET radioligands.
2.2. Synthesis of Iodo-Precursor (11) and Stannyl Derivatives (12–15)
The common (R)-1-(2-(5-benzofuran-2-yl)ethyl)-2-methylpyrrolidine structural feature in ligands
1
–
4
led us to synthesize a single iodo-precursor 11 in four steps from commercially available
2-iodophenol (Scheme 3). The 2-Iodophenol was first converted into 2-iodo-4-nitrophenol ( ) by
reaction with nitric acid [21] in moderate but useful 29% yield. Sonogashira cross-coupling [22] of
with (R)-1-(but-3-ynyl)-2-methylpyrrolidine gave the nitro derivative in 80% yield. Compound
8
8
9
9
was reduced to the corresponding amine 10 in 82% yield with palladium on carbon in a solution of
potassium formate. Iodination in the presence of sodium nitrite and potassium iodide [23] afforded
iodoarene 11 in moderate yield (47%).
Scheme 3. Synthesis of iodo-precursor 11. Conditions: (a) HNO3; (b) (R)-1-(but-3-ynyl)-2-methylpyrrolidine,
Pd(PPh3)2Cl2, CuI, DMF; (c) Pd/C, HCOOK; (d) NaNO2, KI.

Molecules 2017, 22, 792
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Trimethyl(4-nitrophenyl)stannane (12) was obtained in 90% yield by coupling p-iodonitrobenzene
with hexamethylditin in the presence of Pd(MeCN2)Cl2, as previously reported [24]. The three other
stannyl derivatives (13–15) were prepared with literature methods [25,26] by reaction of the appropriate
Grignard reagent with trimethyltin bromide.
2.3. Radiosynthesis
Initial labeling experiments with 11 and 12 using Pd(0) and xantphos in DMF or THF did
not produce the target aryl ketone [11C] , but yielded the corresponding [11C]acid by-product.
The 11C-carbonylation reaction succeeded when DMSO was used as solvent and P(o-tol)3 as palladium
1
ligand (Scheme 4). Subsequently, [11C]
1–4 were prepared according to a standard procedure using
Pd(0) catalyzed 11C-carbonylation with iodoarene 11 and an appropriately substituted arylstannyl
reagent (12–15) in DMSO.
Scheme 4. Radiosyntheses of [11C] with Pd(0)-mediated 11C-carbonylative coupling of an iodoarene
1–4
with aryltrimethylstannanes.
When labeling was performed in the presence of air and moisture, we observed that more [11C]acid
by-product was generated and lower yields of target [11C]aryl ketones were obtained. Therefore,
all reagents were handled in a dry glove box under nitrogen gas. The Pd(0) reagent was instantly
generated when Pd2(dba)3 and P(o-tol)3 were dissolved in anhydrous DMSO (80
µL). The palladium
mixture was then added to the iodo-precursor in a capped polypropylene vial (250
µL) and kept in
the glove box until 5–10 min before the beginning of radiosynthesis. Three min before the start of
radiosynthesis, the tin reagent was added to the reaction mixture outside the glove box while the vial
cap remained on and the final mixture in DMSO was uploaded to the reagent loop of the Synthia
radiosynthesis platform. Cyclotron-produced [11C◦]CO2 was converted into [11C]CO in 75% yield
through one pass over molybdenum wires at 875 C with helium carrier gas flow at 10 mL/min.
The cryogenically concentrated [11C]CO plus the reaction mixture in DMSO were compressed into the
autoclave and sealed. The optimized temperature for 11C-carbonylation was 130 ◦C, and the reaction
time 4 min.
The radioactive product was purified with semi-preparative scale reverse phase HPLC.
After removal of solvent, the residue was reconstituted in a saline solution (10 mL) containing 10% (v/v)
EtOH and ascorbic acid (0.5 mg), and filtered through a 0.2 µm sterile filter before being released for
an imaging experiment. The addition of ascorbic acid helped to reduce radiolysis or decomposition of
the radioligands.
Key radiosynthesis parameters are summarized in Table 2. No significant reactivity difference
was observed between the four stannyl compounds. Overall decay-corrected yields of radiotracers
for clinical use were in the range of 5–9% for each new radioligand whether substituted in meta or
para position and whether bearing an electron-withdrawing or an electron-donating group. For the
HPLC purification, we observed that a small change in pH of the aqueous mobile phase caused a shift
in the product peak retention time. Therefore, it was good practice to use freshly prepared aqueous
NH4OH solution. Incidentally, the lower molar activity of [11C]
2 coincided with the replacement of a

Molecules 2017, 22, 792
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new molecular sieves 13X column. It is possible that due to less rigorous heating activation in order to
prolong its life, residual CO2 had been retained inside the molecular sieve pores and then released
along with [11C]CO2. However, the molar activity obtained at the end of synthesis was still adequate
for brain receptor imaging purposes. Molar activity data for the other three labeled compounds were
consistently high.
Table 2. Summary of key radiosynthesis parameters.
Molar activity at EOS b
Radioligand
Yields a (%)
Prep-HPLC Method
tR (min)
(GBq/µmol)
11C]1
11C]2
11C]3
11C]4
a
4.9 (n = 10)
6.8 (n = 5)
6.1 (n = 4)
8.6 (n = 3)
293 ± 84
115 ± 45
420 ± 60
398 ± 141
A
A
B
13.1
14.4
14.3
15.1
[
[
[
[
B
Based on the formulated dose from cyclotron produced [11C]CO2; b EOS = end of synthesis.
3. Materials and Methods
Reference compounds 1–3 were prepared as reported previously [18] using the appropriately
substituted benzophenones obtained from Combi-Blocks (San Diego, CA, USA). All other chemicals
and solvents were purchased from Sigma-Aldrich (Milwaukee, WI, USA) or Alfa Aesar (Ward Hill,
MA, USA) and used without further purification unless otherwise indicated.
The 1H- (400 MHz), 19F- (376.5 MHz) and 13C-NMR (100 MHz) spectra of all compounds were
acquired on an Advance (Bruker) spectrometer using the chemical shifts of residual deuterated solvent
as internal standard; chemical shifts (
δ) for the proton and carbon resonance are reported in parts
per million (ppm) downfield from TMS (
δ
= 0). Thin layer chromatography (TLC) was performed
with silica gel layers (type 60 F254; EM Science) and compounds visualized under ultraviolet (UV)
light. Mass spectra were acquired with a PolarisQ GC-MS instrument (Thermo Finnigan, San Jose,
CA, USA) equipped with a capillary RTX-5ms column (30 m
carrier gas, He). LC-MS was performed on a LCQ Deca instrument (Thermo Fisher Scientific Corp.;
Waltham, MA, USA) equipped with a reversed-phase HPLC column (Luna C18, 3
m, 50 mm × 2 mm
Phenomenex, Torrance, CA, USA), eluted at 200 L/min with a mixture of A (H2O:MeOH:AcOH,
×
0.25 mm; flow rate, 1 mL/min;
µ
;
µ
90:10:0.5 by vol.) and B (MeOH:AcOH, 100:0.5 v/v), initially composed of 20% B and linearly reaching
80% B in 3 min. Melting points were measured with a Digital SMP20 (Stuart) melting point apparatus
and are uncorrected. Yields are reported for spectroscopically (1H-NMR) or chromatographically pure
materials. HRMS data were acquired at the Mass Spectrometry Laboratory, University of Illinois at
Urbana-Champaign (Urbana, IL, USA) under electron ionization conditions using a double-focusing
high-resolution mass spectrometer (Micromass, Waters, Milford, MA, USA). All HRMS are provided
for ESI+ (M+ + 1) except for compound 8, obtained in EI+ (M).
Radioactive products were separated with HPLC on an apparatus comprising a solvent module
(System Gold 126; Beckman Coulter, Fullerton, CA, USA) coupled with an absorbance detector
operating at 254 nm (Model 166; Beckman Coulter) and a sodium iodide radioactivity detector
(Bioscan, Washington, DC, USA). The HPLC apparatus for radioligand analysis comprised a solvent
module (System Gold 126; Beckman Coulter) coupled with an absorbance detector operating at
254 nm (Model 168; Beckman Coulter) and a radioactivity detector (PMT, Flow-count; Bioscan).
Radioactivity was measured with a calibrated dose ionization chamber (Atomlab 300; Biodex Medical
Systems Inc., Shirley, NY, USA). All radioactivity measurements were corrected for background and
were decay-corrected.
(3,5-Difluorophenyl)(4-methoxyphenyl)methanone (5). Scandium triflate (0.12 g, 0.25 mmol) was added to
a solution of anisole (0.27 g, 0.271 mL, 2.5 mmol) and 3,5-difluorobenzoyl chloride (0.44 g, 2.5 mmol) in
nitroethane (5 mL). The resultant dark-purple solution was stirred at 60 ◦C for 5 days at which point

Molecules 2017, 22, 792
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TLC showed no remaining starting material. The solution was quenched with saturated NaHCO3
(10 mL) and extracted thrice with DCM. The solvent from the combined organic layers was dried over
MgSO4, evaporated under vacuum, and the residue was purified on silica gel (hexanes:EtOAc, 90:10)
◦
to give
5
(0.36 g, 59%) as white crystals. m.p.: 90–92 C. 1H-NMR (CDCl3)
δ
7.81 (m, 2H), 7.26 (m, 2H)
,
7.00 (m, 3H), 3.90 (s, 3H). 13C-NMR (CDCl3)
δ
192.64, 163.80, 162.64 (dd, J1 = 251.0 Hz, J2 = 11.8 Hz),
141.35 (t, J1 = 251.0 Hz, J2 = 11.8 Hz), 132.55, 128.98, 113.87, 112.56 (dd, J1 = 25.2 Hz, J2 = 11.2 Hz),
107.13 (t, J = 25.3 Hz), 55.58. 19F-NMR (CDCl3) δ −108.41. HRMS ESI: calc’d 249.0722 for C14H11O2F2;
found 249.0735.
(3,5-Difluorophenyl)(4-hydroxyphenyl)methanone (
DCM (5 mL) was placed in a round-bottom flask under argon and cooled to
boron tribromide (2.9 mL, 2.9 mmol, 0.1 M in DCM) was added dropwise at
6
). A solution of compound
5
(0.240 g, 0.97 mmol) in
78 ◦C. A solution of
78 ◦C and the reaction
−
−
mixture was allowed to warm to room temperature (RT) and stirred until no starting material was
observed with TLC. The reaction mixture was cooled to 0 ◦C in an ice-bath and quenched by slow
addition of water (1 mL). After stirring at 0 ◦C for 5 min, more water was added and the layers were
separated. The aqueous layer was extracted twice with DCM (10 mL) and the combined organic layers
were dried over MgSO4 and evaporated to dryness. Chromatography on silica gel (pentane:Et2O,
60:40) gave
6
(0.202 g, 89%) as a beige powder. m.p.: 150–151 ◦C. 1H-NMR (MeOD)
194.30, 164.28, 164.14 (dd, J1 = 249.6 Hz, J2 = 12.2 Hz),
δ 7.73 (m, 2H),
7.26 (m, 3H), 6.90 (m, 2H). 13C-NMR (MeOD)
δ
143.31 (t, J = 7.8 Hz), 134.00, 128.82, 116.44, 113.36 (dd, J1 = 26.4 Hz, J2 = 11.7 Hz), 107.84 (t, J = 25.9 Hz).
19F-NMR (CDCl3) δ −110.46. HRMS calc’d 235.0571 for C13H9O2F2; found 235.0568.
(3,5-Difluorophenyl)(4-hydroxy-iodophenyl)methanone (7). 6 (0.92 g, 3.93 mmol) was dissolved in 28–30%
aq. ammonium hydroxide (60 mL) for 20 min at RT. An aqueous solution (15 mL) of potassium
iodide (3.26 g, 19.7 mmol) and iodine (1.00 g, 3.93 mmol) was added and the mixture was stirred
for 10 min. A solution of 6 M HCl was then added until neutral pH was reached and then the
resultant solution was extracted thrice with AcOEt (40 mL). The organic layers were combined,
washed with water (40 mL), brine (40 mL), dried over MgSO4 and then evaporated to dryness.
Chromat◦ography on silica gel eluting with DCM gave
J2 = 2.1 Hz, 2H), 7.56 (m, 1H),
7.35 (d, J = 5.56 Hz, 2H), 7.01 (d, J = 8.5 Hz, 1H). 13C-NMR (DMSO-d6)
7 (0.956 g, 67%) as an off-white solid. m.p.:
208–210 C. H-NMR (DMSO-d6) δ 11.50 (br s, 1H), 8.11 (d, J = 2.1 Hz, 1H), 7.66 (dd, J1 = 8.5 Hz,
1
δ
190.56, 162.01 (dd, J1 = 248.7 Hz, J2 = 12.4 Hz), 161.56, 141.02, 140.97, 132.57, 128.69, 114.48, 112.23
(dd, J1 = 26.4 Hz, J2 = 11.4 Hz), 107.27 (t, J = 25.8 Hz), 85.04. 19F-NMR (DMSO-d6) δ −108.37. HRMS ESI:
calc’d. 360.9537 for C13H8O2F2I; found 360.9540.
(R)-(3,5-Difluorophenyl)(2-(2-(2-methylpyrrolidin-1-yl)ethyl)benzofuran-5-yl)methanone (4). (R)-1-(But-3-ynyl)-
2-methylpyrrolidine (0.1 M in acetonitrile) was first prepared by reaction of (R)-2-methylpyrrolidine
(0.60 g, 7.0 mmol), potassium carbonate (2.03 g, 14.7 mmol) and 3-butynyl 4-toluenesulfonate (1.57 g,
◦
◦
7.0 mmol) in MeCN (60 mL). The mixture was stirred at 23 C for 1 h and then heated to 50 C for 24 h.
The mixture was allowed to cool to RT. The precipitated salts were filtered off and the filter cake was
washed with small amounts of MeCN. The filtrate was diluted to a total volume of 70 mL and used as
a solution of 0.1 M reagent.
A round-bottom flask under nitrogen was charged with
7 (0.47 g, 1.3 mmol), Pd(PPh3)2Cl2 (0.04 g,
0.06 mmol, 3.5%) and CuI (0.012 g, 0.07 mmol, 5%) in DMF (3 mL). A mixture of triethylamine (0.26 g,
2.6 mmol) in DMF (2 mL) and (R)-1-(but-3-ynyl)-2-methylpyrrolidine (17 mL, 1.7 mmol from the
stock solution) was then introduced into the flask. The mixture was stirred at RT for 30 min and then
heated to 60 ◦C for 12 h. The reaction mixture was cooled, diluted with water and extracted three
times with AcOEt. The combined organic layers were washed thrice with water, dried over MgSO4,
and filtered. Solvent was removed under vacuum, and the crude residue was purified with silica gel
1
(DCM:MeOH:NH4OH, 97:2.9:0.1) to give
4
(0.289 g, 61%) as a heavy oil. H-NMR (DMSO-d6)
δ
7.82
(d, J = 1.4 Hz, 1H), 7.58 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 7.41 (d, J = 8.6 Hz, 1H), 7.14 (m, 3H), 6.56
d, J = 0.6 Hz, 1H), 3.17 (m, 2H), 2.95 (m, 2H), 2.48 3.17 (m, 2H), 2.26 (qd, J = 8.9 Hz, 1H), 1.91 (m, 1H)
(
,

Molecules 2017, 22, 792
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1.70 (m, 2H), 1.36 (m, 1H), 1.07 (d, J = 6.2 Hz, 3H). 13C-NMR (DMSO-d6)
δ 195.03, 165.36 (dd, J1 = 249.8 Hz,
J2 = 12.1 Hz), 160.43, 158.81, 142.85 (t, J = 7.9 Hz), 132.67, 130.42, 127.13, 124.76, 113.71 (dd, J1 = 26.6 Hz
,
J2 = 11.5 Hz), 112.01, 108.27 (t, J = 25.3 Hz), 104.59, 62.11, 54.69, 52.75, 33.34, 28.19, 22.40, 18.35. 19F-NMR
(DMSO-d6) δ −108.37. HRMS ESI: calc’d. 370.1613 for C22H22F2NO2; found 370.1610.
2-Iodo-4-nitrophenol (8). A 70% nitric acid solution (0.70 mL, 10.9 mmol) was slowly added to a
solution of 2-iodophenol (2.0 g, 9.1 mmol) in DCM (20 mL). The reaction was stirred for 6 h at RT,
then diluted with water and extracted thrice with DCM. The solvent from the combined organic layers
was evaporated off, and the compound was purified on silica gel (hexanes:EtOAc, 95:5 to 80:20) to
1
give
8
(0.705 g, 29%) as a yellow solid. m.p.: 85–86 ◦C. (lit. m.p.: 84–86 ◦C [27]). H-NMR (DMSO-d6)
δ
11.96 (br s, 1H), 8.51 (d, J = 2.76 Hz, 1H), 8.15 (dd, J1 = 8.98 Hz, J2 = 2.78 Hz, 1H), 7.02 (d, J = 9.00 Hz,
1H). 13C-NMR (DMSO-d6)
161.86, 138.88, 133.24, 124.50, 113.03, 83.13. HRMS EI+ calc’d 264.92362 for
C6H4O3NI; found 264.92486.
δ
(R)-2-Methyl-1-(2-(5-nitrobenzofuran-2-yl)ethyl)pyrrolidine (9). A round-bottom flask was charged with
8
(0.66 g, 2.5 mmol), Pd(PPh3)2Cl2 (0.06 g, 0.09 mmol, 3.5%) and CuI (0.02 g, 0.13 mmol, 5%) in
DMF (5 mL). A mixture of triethylamine (0.51 g, 5.0 mmol) in DMF (2 mL) and (R)-1-(but-3-ynyl)-
2-methylpyrrolidine (35 mL, 3.5 mmol) was then introduced into the flask. The mixture was stirred at
RT for 30 min and then heated to 60 ◦C for 18 h. The reaction mixture was cooled, diluted with water
and extracted thrice with DCM. The combined organic layers were washed thrice with 0.5 M NaOH
and thrice with water, dried over MgSO4, and filtered. Solvent was removed under vacuum, and the
crude residue was purified with silica gel (DCM:MeOH:NH4OH, 97:2.7:0.3) to give
9 (0.547 g, 80%) as
1
a heavy brown oil. H-NMR (CDCl3)
δ 8.40 (d, J = 2.36 Hz, 1H), 8.14 (dd, J1 = 8.98 Hz, J2 = 2.38 Hz,
1H), 7.47 (d, J = 8.88 Hz, 1H), 6.60 (d, J = 0.92 Hz, 1H), 3.25 (m, 2H), 3.05 (t, J = 7.72 Hz, 2H), 2.53 (m, 1H)
2.43 (m, 1H), 2.24 (q, J = 8.79 Hz, 1H), 1.95 (m, 1H), 1.77 (m, 2H), 1.47 (m, 1H), 1.15 (d, J = 6.08 Hz,
3H). 13C-NMR (CDCl3)
161.42, 157.49, 143.97, 129.32, 119.28, 116.73, 110.95, 103.34, 60.12, 53.79, 51.52,
,
δ
32.67, 28.07, 21.70, 18.88. HRMS ESI: calc’d 275.1396 for C15H19O3N2; found 275.1394.
(R)-2-(2-(2-Methylpyrrolidin-1-yl)ethyl)benzofuran-5-amine (10). Pd/C (10%, 0.30 g) and potassium
formate (1.24 g, 15.1 mmol) [28] were added to a solution of 9 (0.473 g, 1.72 mmol) in MeOH (10 mL),
and the mixture refluxed at 70 ◦C for 2 h. The reaction mixture was allowed to cool to RT, and then
filtered over Celite. Concentrated HCl (12 M) was added dropwise to the filtrate until no more
effervescence was observed. The mixture was filtered again. The filtrate was concentrated under
vacuum, and the resultant residue was partitioned between EtOAc and aq. KHCO3 (1.0 M). The organic
layer was evaporated under vacuum to give a crude product. The crude compound was purified by
partitioning between EtOAc and HCl (aq. 1 M), and the pH of the aqueous layer was then adjusted to
10 with NaOH (aq. 2 M). The resulting liquid was extracted thrice with EtOAc. The combined organic
layers were dried over MgSO4 and filtered. Removal of solvent under vacuum gave 10 (0.344 g, 82%)
1
as a brown semi-solid. H-NMR (CD3CN)
δ
7.12 (d, J = 8.56 Hz, 1H), 6.70 (dd, J1 = 2.38 Hz, J2 = 0.54 Hz
,
1H), 6.54 (dd, J1 = 8.62 Hz, J2 = 2.34 Hz, 1H), 6.32 (d, J = 0.96 Hz, 1H), 3.93 (br s, NH2), 3.15 (m, 2H),
2.87 (m, 2H), 2.35 (m, 2H), 2.14 (m, 1H), 1.90 (m, 1H), 1.67 (m, 2H), 1.32 (m, 1H), 1.05 (d, J = 6.04, 3H).
13C-NMR (CD3CN)
δ 158.13, 148.05, 143.26, 129.47, 111.41, 110.14, 104.16, 101.64, 59.35, 52.99, 51.27,
32.26, 27.45, 21.13, 18.06. HRMS ESI: calc’d 245.1654 for C15H21N2O; found 245.1650.
(R)-1-(2-(5-Iodobenzofuran-2-yl)ethyl)-2-methylpyrrolidine (11). p-Toluenesulfonic acid (0.70 g, 3.7 mmol)
was added to a solution of 10 (0.30 g, 1.2 mmol) in MeCN (6 mL). The resulting suspension was cooled
to 5 ◦C. A solution of sodium nitrite (0.17 g, 2.5 mmol) and potassium iodide (0.51 g, 3.1 mmol) in
water was added dropwise, releasing N2 gas. The reaction mixture was stirred for 10 min, and then
gradually warmed to RT over 50 min. The mixture was diluted with water and pH adjusted to 10 by
NaHCO3 (aq. 1.0 M). Liquid was extracted thrice with EtOAc. The combined organic layers were dried
over MgSO4, and solvent was evaporated under reduced pressure. Crude product was purified on
1
silica gel (DCM:MeOH:NH4OH, 95:4.5:0.5) to give 11 (0.204 g, 47%) as a brown semi-solid. H-NMR

Molecules 2017, 22, 792
8 of 10
(CD3CN)
J2 = 0.42 Hz, 1H), 6.51 (d, J = 0.96 Hz, 1H), 3.19 (m, 2H), 2.95 (m, 2H), 2.44 (m, 2H), 2.20 (m, 1H),
1.91 (m, 1H) 159.21, 153.56,
1.68 (m, 2H), 1.34 (m, 1H), 1.07 (d, J = 7.40 Hz, 3H). 13C-NMR (CD3CN)
δ 7.87 (d, J = 1.80 Hz, 1H), 7.51 (dd, J1 = 8.56 Hz, J2 = 1.84 Hz, 1H), 7.24 (dd, J1 = 8.58 Hz,
,
δ
131.56, 131.42, 128.83, 112.45, 101.46, 85.19, 59.60, 52.93, 50.90, 32.14, 27.06, 21.10, 17.83. HRMS calc’d
356.0511 for C15H19NOI; found 356.0504.
Trimethyl(4-nitrophenyl)stannane (12). 1-Iodo-4-nitrobenzene (0.249 g, 1.00 mmol) and Sn2Me6 (0.426 g,
1.30 mmol) were added to DMF (5 mL). Pd(CH3CN)2Cl2 (0.006 g, 0.03 mmol) was added, and the
reaction was stirred at RT for 10 min. The reaction mixture was diluted with water and extracted
thrice with diethyl ether. The combined organic layers were washed thrice with water, dried over
MgSO4, and filtered. The solvent from the filtrate was evaporated under reduced pressure, and the
crude residue was purified on a silica gel plug column (hexane: diethyl ether, 90:10) to give
90%) as a light yellow solid. m.p.: 49–51 ◦C (lit. m.p.: 47–48 ◦C [29]).
9 (0.258 g,
Radiosynthesis and purification. Radiosynthesis was performed with a modified Synthia [11C]CO
module [30]. In a typical procedure, iodoarene 11 (1.4 mg, 3.9 mol), 4-fluoroaryltrimethylstannane
13, 1.4 mg, 5.3 mol), tris(dibenzylidene-acetone)dipalladium(II) (0.4 mg, 0.45 mol), and P(o-tol)3
(0.9 mg, 2.9 mol) were mixed with DMSO (80 L) and loaded into the autoclave of the apparatus.
µ
(
µ
µ
µ
µ
Each compound and reagent was measured and mixed under nitrogen gas protection in a glove
box. [11C]CO, generated by a single pass of cyclotron-produced [11C]CO2 over heated (875 ◦C) Mo
wires, was cryogenically concentrated and then directed into the autoclave containing reagent mixture.
◦
The autoclave was sealed and heated at 130 C for 4 min. The reaction mixture was then flushed
out with THF (0.7 mL) into a collection vial (5-mL), diluted with water (3 mL) and injected onto a
Luna C18 column (250
×
10 mm) eluted using semi-prep HPLC method A or B for purification of
the radioligand (See Figures S1–S4 for examples of HPLC chromatograms). Upon removal of solvent,
the final dose-for-injection was formulated in saline-10% v/v EtOH containing ascorbic acid (0.5 mg)
for intravenous injection. Radioligand purities and molar activities were measured with analytical
reverse phase HPLC, and radioligand identities were confirmed by LC-MS of associated carrier and
co-elution with the non-radioactive standards.
Semi-prep HPLC method A. The reaction mixture was eluted on Luna C18 column (5
µ
m, 250
×
10 mm).
The mobile phase consisted of aqueous NH4OH (A, 1 mM, pH = 8.5) and MeCN (B) with B initially
at 40% (v/v) for 2 min and increased to 88% in 2 min at a flow rate of 6 mL/min. The eluate was
monitored for radioactivity and UV absorbance (254 nm).
Semi-prep HPLC method B. The reaction mixture was eluted on Luna C18 column (5
µ
m, 250
×
10 mm).
The mobile phase consisted of aqueous NH4OH (A, 5 mM, pH = 9.0) and MeCN (B) with B initially
at 40% (v/v) for 2 min and increased to 82% in 2 min at a flow rate of 6 mL/min. The eluate was
monitored for radioactivity and UV absorbance (254 nm).
Analytical HPLC method for QC and SA determination. The column was a Luna C18 column (10 µ,
250 × 4.0 mm). For [11C]
1
, the mobile phase was an isocratic mixture of MeCN (85%) and aq. NH4OH
(1 mM, pH = 8.5, 15%) eluted at 2 mL/min. For [11C]
2–4
, the mobile phase consisted aq. NH4OH
(A, 1 mM, pH = 8.5) and MeCN (B) with B initially at 45% (v/v) for 1 min and increased to 91% in 4 min
at a flow rate of 2.75 mL/min. The eluate was monitored for radioactivity and UV absorbance (254 nm).
4. Conclusions
Pd(0)-mediated 11C-carbonylative coupling of iodoarenes with aryltrimethylstannanes proved
to be an effective method to synthesize 11C-labeled aryl ketones [11C]
1–4 as prospective high-affinity
and selective H3R radioligands with adequate yield, chemical purity, radiochemical purity,
and molar activity.

Molecules 2017, 22, 792
9 of 10
Supplementary Materials: The supplementary materials are available online. Examples of radio-HPLC
chromatograms (A) Semi-prep, (B) QC for [11C]1–4.
Acknowledgments: This study was supported by the Intramural Research Program of the National Institutes of
Health (NIMH, ZIA MH002793). We thank the NIH Clinical PET Department (Chief P. Herscovitch) for carbon-11
production. Receptor binding assays were performed by the National Institute of Mental Health’s Psychoactive
Drug Screening Program, Contract # HHSN-271-2008-00025-C (NIMH PDSP). The NIMH PDSP is directed by
Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at
NIMH, Bethesda MD, USA.
Author Contributions: V.W.P., F.G.S. and S.L. conceived and designed the experiments; F.G.S., S.L. and W.J.C.
performed the experiments; F.G.S. and S.L. analyzed the data; S.L., F.G.S. and V.W.P. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).