Synthesis of 11C-labelled ureas by palladium(II)-mediated oxidative carbonylation

Article abstract of DOI:10.3390/molecules22101688

Positron emission tomography is an imaging technique with applications in clinical settings as well as in basic research for the study of biological processes. A PET tracer, a biologically active molecule where a positron-emitting radioisotope such as carbon-11 has been incorporated, is used for the studies. Development of robust methods for incorporation of the radioisotope is therefore of the utmost importance. The urea functional group is present in many biologically active compounds and is thus an attractive target for incorporation of carbon-11 in the form of [¹¹C]carbon monoxide. Starting with amines and [¹¹C]carbon monoxide, both symmetrical and unsymmetrical ¹¹C-labelled ureas were synthesised via a palladium(II)-mediated oxidative carbonylation and obtained in decay-corrected radiochemical yields up to 65%. The added advantage of using [¹¹C]carbon monoxide was shown by the molar activity obtained for an inhibitor of soluble epoxide hydrolase (247 GBq/μmol-319 GBq/μ mol). DFT calculations were found to support a reaction mechanism proceeding through an 11C-labelled isocyanate intermediate.

Full text of DOI:10.3390/molecules22101688

molecules
Article
Synthesis of 11C-Labelled Ureas by
Palladium(II)-Mediated Oxidative Carbonylation
Sara Roslin 1,
*
ID , Peter Brandt 1 ID , Patrik Nordeman 1, Mats Larhed 2 ID , Luke R. Odell 1
ID
and Jonas Eriksson 1
ID
1
Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, BMC, Uppsala University, Box 574,
SE-751 23 Uppsala, Sweden; peter.brandt@orgfarm.uu.se (P.B.); patrik.nordeman@akademiska.se (P.N.);
luke.odell@orgfarm.uu.se (L.R.O.); jonas.p.eriksson@akademiska.se (J.E.)
Science for Life Laboratory, Department of Medicinal Chemistry, BMC, Uppsala University, Box 574,
SE-751 23 Uppsala, Sweden; mats.larhed@orgfarm.uu.se
2
*
Correspondence: sara.roslin@orgfarm.uu.se; Tel.: +46-018-4714282
Received: 25 September 2017; Accepted: 4 October 2017; Published: 10 October 2017
Abstract: Positron emission tomography is an imaging technique with applications in clinical settings
as well as in basic research for the study of biological processes. A PET tracer, a biologically
active molecule where a positron-emitting radioisotope such as carbon-11 has been incorporated,
is used for the studies. Development of robust methods for incorporation of the radioisotope is
therefore of the utmost importance. The urea functional group is present in many biologically
active compounds and is thus an attractive target for incorporation of carbon-11 in the form
of [11C]carbon monoxide. Starting with amines and [11C]carbon monoxide, both symmetrical
and unsymmetrical 11C-labelled ureas were synthesised via a palladium(II)-mediated oxidative
carbonylation and obtained in decay-corrected radiochemical yields up to 65%. The added advantage
of using [11C]carbon monoxide was shown by the molar activity obtained for an inhibitor of soluble
epoxide hydrolase (247 GBq/µmol–319 GBq/µmol). DFT calculations were found to support a
reaction mechanism proceeding through an 11C-labelled isocyanate intermediate.
Keywords: carbon-11; 11C-labelling; urea; carbonylation; positron emission tomography;
carbon monoxide
1. Introduction
Positron emission tomography (PET) is a non-invasive imaging technique used to visualise and
study biological processes in vivo by use of a molecular probe, a PET tracer, where a positron-emitting
radioisotope has been incorporated. PET has found extensive use in clinical applications such as oncology,
neurology and cardiology [1–3]. PET also serves as a useful technique in drug development, where PET
offers the possibility to study the distribution, kinetics and target occupancy of potential drugs in vivo [4–6
].
A radioisotope commonly used in PET is carbon-11 (11C), with a half-life of 20.4 min. The natural
abundance of carbon in biologically active molecules makes carbon-11 an appealing isotope to
incorporate in PET tracers. The short half-life offers the possibility to perform several scans in
one patient during a day but it also puts time-restraints on the production of the 11C-labelled
PET tracer. Efficient incorporation of carbon-11 is therefore a key step for a successful production.
The labelling reaction should preferably be performed as the last step in the synthetic route and be a
fast, high-yielding and robust method.
Carbon-11 can be incorporated in the PET tracer via a carbonylation reaction using [11C]carbon
monoxide ([11C]CO). There are numerous carbonyl-containing biologically active molecules, thus the
11C-carbonylative labelling reaction has great potential in PET tracer development but the minute amounts
(typically 10–100 nmol), the short physical half-life and the low solubility of [11C]CO in organic solvents
Molecules 2017, 22, 1688; doi:10.3390/molecules22101688
www.mdpi.com/journal/molecules

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pose particular challenges [
7
–
9
]. To achieve sufficiently fast reaction kinetics, high reagent concentrations
are needed, something that can be accomplished by confining the [11C]CO in low volume reaction
vessels [8
]. However, conventional bubbling of [11C]CO into the reaction mixture in this setting typically
results in low recovery due to the low solubility of [11C]CO and efficient purging of the gas phase
as the carrier gas is vented. This problem has been approached in different ways, for example using
high-pressure autoclave reactors [10– –18], microfluidic reactors [19–21]
12], [11C]CO-trapping agents [13
and an ambient pressure system [22]. The ambient pressure system, developed by Eriksson et al., uses
xenon as a carrier gas instead of helium or nitrogen. Most of the carrier gas is absorbed by the reaction
solvent, thus precluding the need for high-pressure autoclave reactors or [11C]CO-trapping reagents.
The urea moiety is a structural motif with a long history in medicinal chemistry and is found in
various drugs and biologically active compounds [23]. By offering possibilities for hydrogen bonding,
modulation of physicochemical properties and unique binding modes, therapeutic agents acting as
inhibitors at targets such as protein kinases, Hepatitis C NS3 protease and methionyl–tRNA synthetase
all contain the urea functional group (Figure 1) [23–26]. With the incorporation of the urea motif in
aspiring drugs, there is a rationale for developing a simple and reliable method for incorporation of
carbon-11 into the urea functional group.
Figure 1. Biologically active ureas.
[11C]Urea and 11C-labelled urea-derivatives have traditionally been synthesised from
[11C]phosgene ([11C]COCl2) [27
– –
31] but also from [11C]cyanide [32 34], [11C]carbon dioxide
([11C]CO2) [35 41] and [11C]CO [14
–
,42–49]. The different methods come with their own unique
limitations. Production of [11C]COCl2 is rather complicated, and the method is burdened by low
product molar activities (Am) and is less suited for the labelling of unsymmetrical ureas. [11C]HCN
also suffers from low Am and the production requires a series of chemical transformations, hence
the long reaction times. Methods utilising [11C]CO2 and [11C]CO offer improved Am and fewer or
no subsequent chemical transformations. Cyclotron-produced [11C]CO2 can be utilised directly but
fixation agents and drying agents are needed in addition to the amine/amines to be incorporated in the
11C-labelled urea. Great care must be taken to make sure that all agents used are freed of atmospheric
CO2 to avoid isotopic dilution and reduced Am. The Am reached with [11C]CO2-fixation methods
have been in the range of 25–148 GBq/µmol [36–38,41,50]. High Am is of particular importance when
imaging a less abundant target, especially in the central nervous system [51,52].
Different approaches have been employed for the synthesis of 11C-labelled ureas from [11C]CO
and amines. The first method published was a selenium-mediated carbonylation for the synthesis of a
number of symmetrical and unsymmetrical 11C-labelled ureas, where secondary amines were difficult

Molecules 2017, 22, 1688
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to employ [42]. Rhodium(I) has mainly been used in the synthesis of unsymmetrical 11C-labelled ureas
and necessitates the use of an azide as precursor, which, according to Doi et al., converts to a nitrene
intermediate and subsequently to the 11C-labelled isocyanate when reacting with [11C]CO. [43
–48].
In contrast to the case with Rh(I), palladium(II)-mediated 11C-urea syntheses can use amines as sole
precursors. Kealey et al. reported the use of a Cu(I)scorpionate complex for the trapping of [11C]CO
for a Pd(II)-mediated formation of symmetrical and unsymmetrical 11C-labelled ureas [49]. Primary,
aliphatic amines performed well as substrates, whereas anilines were found to be more challenging.
Since no 11C-labelled ureas were isolated nor any Am determined, the practical utility of the method
was difficult to assess.
To address some of the issues with the related methods and to improve the access to 11C-labelled
ureas, we here report on Pd(II)-mediated oxidative 11C-carbonylation of amines for the synthesis
of symmetrical and unsymmetrical 11C-labelled ureas. The developed protocol utilised [11C]CO,
with xenon as a carrier gas, a palladium source and amines for the isolation of 14 11C-labelled ureas.
Additionally, to demonstrate the advantage of using [11C]CO to reduce isotopic dilution, the Am was
determined for an inhibitor of soluble epoxide hydrolase (sEH, Figure 1).
2. Results and Discussion
The minute amounts of [11C]CO available for reaction and the requisite for a finished synthesis
within 2–3 half-lives of carbon-11 set the framework for a transition-metal-mediated 11C-carbonylation.
We have previously used the xenon system for ambient pressure carbonylations and demonstrated
its feasibility in synthesising amides [53,54] and sulfonyl carbamates [55]. The report by Kealey et al.
as well as our own observations that 11C-labelled ureas can form as byproducts in the synthesis of
11C-labelled amides, especially when a Pd(II) source is used as a pre-catalyst for the aminocarbonylation,
sparked our interest in exploring the Pd(II)-mediated formation of 11C-labelled ureas [14].
The investigation into the synthesis of symmetrical 11C-labelled ureas began with using
benzylamine (
of the [11C]CO was converted to non-volatile 11C-labelled compounds and the selectivity for
[11C]-N,N0-dibenzylurea
was >99%, giving a radiochemical yield (RCY) of 55% calculated from
1) as a model amine and Pd(Xantphos)Cl2 as a Pd source [56,57]. Initially, 56%
2
the conversion and product selectivity (Table 1, entry 1) [58].
Table 1. Optimisation of reaction conditions for synthesis of symmetrical 11C-labelled urea.
◦
Product Selectivity b (%)
Entry
T ( C) Time (min)
Conversion a (%)
RCY c (%)
1
2
3
120
120
150
150
120
5
10
5
10
10
56 ± 2.2
83 ± 3.9
66 ± 4.5
90 ± 2.5
66 ± 1.0
>99
>99
96 ± 2.6
97 ± 2.1
>99
55 ± 2.1 (3)
82 ± 3.9 (3)
63 ± 4.3 (3)
87 ± 3.4 (3)
65 ± 1.0 (2)
4
5 d
Conditions:
1
(30
µmol), Pd(Xantphos)Cl2 (4
µmol), THF (400
µ
L). a Percentage of [11C]CO converted to non-volatile
b
products. Decay-corrected. Percentage of product formed, assessed by analytical HPLC of crude reaction mixture,
after volatiles were purged. Radiochemical yield, calculated from the conversion and product selectivity. Number
of experiments in brackets. 10 µmol of 1.
c
d
With these encouraging results, the reaction time and temperature were altered in order to
improve the conversion. Extending the reaction time to 10 min (entry 2) increased the conversion
◦
to 83% and returned a RCY of 82%. Raising the reaction temperature to 150 C (entry 3) whilst
keeping the reaction time at 5 min did not improve the reaction to the same extent as prolonging the

Molecules 2017, 22, 1688
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◦
reaction time (66% conversion and 63% RCY). When heating the reaction at 150 C for 10 min (entry 4),
the conversion and RCY were further improved to 90% and 87%, respectively. The gain in RCY was
minor for entry 4 as compared to entry 2 and we therefore decided to continue with the conditions as
in entry 2 to avoid unwanted side reactions caused by the high temperature. In a final experiment,
the amount of
1 was lowered to 10 µ
mol to test whether the conversion of [11C]CO and the selectivity
could be retained (entry 5). The conversion dropped somewhat but 2 was still obtained in 65% RCY.
Further experiments were conducted using 30 µmol of amine.
Next, the scope for symmetrical 11C-urea formation was investigated (Table 2). Symmetrical ureas
are not as abundant in medicinal chemistry as unsymmetrical ureas. There are, however, examples of
bioactive, symmetrical ureas such as [(7-amino(2-naphthyl)sulfonyl]phenylamines derivatives that
have been shown to activate insulin receptor tyrosine kinases or sulfonylated naphthyl urea derivatives
inhibiting protein arginine methyl transferases [59
HPLC purification and the radiochemical yields are based on the amount of [11C]CO transferred to the
reaction vial [58]. Primary, aliphatic amines ( and ) were found to be very good substrates and the
,60]. The products were isolated by semi-preparative
2,
3
4
products were isolated in good radiochemical yields and in >99% radiochemical purity (RCP). The gain
in conversion that was seen when increasing the reaction time from 5 min to 10 min manifested itself
as a gain in RCY for 2 (41% compared with 65%).
Table 2. Scope for symmetrical 11C-labelled ureas.
Compound
2
11C-Labelled Urea
Conversion a (%)
RCP c (%)
RCY b (%)
65 ± 1
81 ± 5
>99
41 d
3
4
67 ± 4
71 ± 2
40 ± 6
48 ± 4
>99
>99
5
15 ± 1
4 ± 1
>99
Conditions: Amine (30
µ
mol), Pd(Xantphos)Cl2 (4
µ
mol), THF (400
µ
L). All experiments were performed in
duplicate. Percentage of [11C]CO converted to non-volatile products. Decay-corrected. Radiochemical yield.
a
b
Based on the 11C-labelled product obtained after semi-preparative HPLC and amount of [11C]CO collected in the
reaction vial. Decay-corrected. Radiochemical purity. Determined by analytical HPLC of the isolated 11C-labelled
c
d
product. 5 min reaction time, single experiment.
Urea
2 was isolated with 0.44 GBq 34 min after end of nuclide production (EOB) following a
5-min reaction, whereas a 10 min reaction time returned 2 with 0.83–0.88 GBq 33–36 min after EOB.
Another example of a symmetrical urea in medicinal chemistry is N,N0-dicyclohexylurea, which has
been identified as a potent inhibitor of sEH (Ki 30 nM), a discovery that sparked interest in the urea as
a scaffold for designing sEH inhibitors [61]. Notably, [11C]N,N0-dicyclohexylurea (
4
) was isolated in
a high RCY (48%). Aniline was found to be a sluggish substrate and
5 was only isolated in 4% RCY.

Molecules 2017, 22, 1688
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This is in line with previous studies using either a Pd(II)-source and high concentrations of aniline
(90 36 49 50].
mol) or [11C]CO2-fixation methods using varying amounts of aromatic amines [35
Rh(I)-mediated synthesis of aromatic 11C-labelled ureas has returned higher RCYs, albeit starting from
µ
, , ,
an aromatic azide rather than an aromatic amine [44,45]. When piperidine 6 was used as substrate,
the tetra-substituted urea was not detected.
For optimisation of the synthesis of unsymmetrical 11C-labelled ureas,
7
was chosen as the model
compound (Table 3). Starting from the same conditions as in the synthesis of symmetrical 11C-labelled
ureas, was obtained in 42% RCY based on a conversion of 53% and a product selectivity of 79%
(entry 1) [58]. The ratio of symmetrical ( ) versus unsymmetrical ( ) formation was 12:88. Initial
optimisation was performed by changing the Pd-source (entries 2–5) [14 49]. Although the Pd-species
7
2
7
,
in entries 2–5 gave higher conversions than Pd(Xantphos)Cl2, the product selectivity was lower and
consequently the RCYs as well (7–32%). Using DMF as solvent was unfavorable for the product selectivity
(entry 6). An investigation of the temperature influence (entries 7 and 8), showed that a lowering of
the temperature to 80 ◦C, which has been used in Rh-based 11C-labelled urea syntheses, retained t◦he
conversion whilst losing product selectivity [43]. On the other hand, heating the reaction to 150 C
improved the product selectivity but, because of the lower conversion, the yield was in the same ra◦nge
as in entry 1 (41% in entry 8 and 42% in entry 1). The reaction temperature was therefore kept at 120 C.
Table 3. Optimisation of reaction conditions for synthesis of unsymmetrical 11C-labelled urea.
Product Selectivity
◦
RCY d (%)
Entry
Catalyst
T ( C)
6 (Equiv.)
Conversion a (%)
2:7 c
b
(%)
1
2
3
Pd(Xantphos)Cl2
Pd(PPh3)2Cl2
120
120
120
120
120
120
80
150
120
120
120
120
1
1
1
1
1
1
1
1
2
5
1
1
53 ± 5.6
69 ± 4.1
95 ± 3.5
75 ± 2.5
67 ± 9
79 ± 2.9
46 ± 3.6
13 ± 3.5
21 ± 0.5
10 ± 1
12:88
16:84
11:89
23:77
23:77
16:84
9:91
9:91
9:91
2:98
7:93
-
42 ± 5.9 (3)
32 ± 3.2 (3)
12 ± 4.0 (2)
16 ± 0.5 (2)
7 ± 1.5 (2)
21 ± 1.6 (3)
26 ± 8.5 (3)
41 ± 6.2 (4)
29 ± 3.3 (3)
42 ± 3.1 (3)
60 ± 3.4 (3)
-
Pd(OAc)2 + dppf
Pd(OAc)2 + dppp
Pd(OAc)2 + Xantphos
Pd(Xantphos)Cl2
Pd(Xantphos)Cl2
Pd(Xantphos)Cl2
Pd(Xantphos)Cl2
Pd(Xantphos)Cl2
Pd(Xantphos)Cl2
Pd(PPh3)4
4
5
6 e
7
43 ± 1.7
57 ± 9.2
44 ± 11
46 ± 4.3
58 ± 1.7
67 ± 1.7
93
49 ± 3.6
44 ± 6.8
87 ± 4.3
63 ± 2.2
71 ± 3.6
89 ± 3.3
-
8
9
10
11 f
12 g
Conditions:
1
(30
µ
mol),
2
(30
µ
mol), catalyst ([Pd] 4
µ
mol + ligand 4
µ
mol), THF (400 µL). 5 min reaction time
a
unless otherwise stated. Percentage of [11C]CO converted to non-volatile products, after purge. Decay-corrected.
b
c
Percentage of product formed, assessed by analytical HPLC of crude reaction mixture, after purge. Product
d
ratio of
2
to
7, assessed by analytical HPLC of crude reaction mixture. Radiochemical yield, calculated from the
e
f
conversion and product selectivity. Number of experiments in brackets. DMF as solvent. 10 min reaction time.
g
Single experiment.
Next, increasing the amount of piperidine (
using five equivalents of
gave almost sole formation of unsymmetrical 11C-labelled urea
whereas having two equivalents of did not improve the product ratio to the same extent (entry 9).
6) was investigated (entries 9 and 10). Not surprisingly,
6
7
(entry 10)
6
The RCY was markedly lower in entry 9 (29%) compared to entry 1 and entry 10 (both 42%), because
of both lower conversion and inferior product selectivity. As no improvement in yield was gained
by using five equivalents of
enhanced the conversion, the product selectivity and the ratio of
11). A final experiment, with Pd(PPh3)4, supported the reaction to be Pd(II)-mediated as neither
formed with the Pd(0)-source. The conditions in entry 11 were continued with for investigation of
the scope for synthesis of unsymmetrical 11C-labelled ureas, including sEH inhibitor 19 (Table 4).
6
, the reaction time was altered next. Heating the reaction for 10 min
to formed, with a 60% RCY (entry
nor
2
7
2
7

Molecules 2017, 22, 1688
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Compounds
7
–
19 were isolated by semi-preparative HPLC and the radiochemical yields are based
on the amount of [11C]CO transferred to the reaction vial [58]. Aliphatic amines were isolated in good
RCYs ranging from 12% to 41% (7–10). Another demonstration of the gain in measured radioactivity
of the product with a prolonged reaction time is seen for
7, where a 5 min reaction time led to a RCY
of 17% and 0.15 GBq isolated 37 min from EOB, whilst the 10 min reaction time gave 41% in RCY and
0.47–0.57 GBq isolated after 36–45 min from EOB. 2-(2-Aminoethyl)pyridine proved to be a challenging
substrate and
product . The byproduct was not observed when amine 6 was removed. In the synthesis of 10, steric
9
was isolated in 12% RCY. Here, an unknown 11C-labelled byproduct formed along with
9
hindrance was introduced in the primary amine, which resulted in a lower RCY compared to 7 and 8.
Table 4. Scope for unsymmetrical 11C-labelled ureas.
Compound
7
11C-Labelled Urea
Conversion a (%)
RCP c (%)
RCY b (%)
65 ± 0
39 d
41 ± 6
17 d
98 ± 1
59 e
31 e
55 ± 4
23 ± 1
8
9
>99
>99
60 e
14 e
20 ± 0
35 ± 6
12 ± 1
14 ± 4
10
>99
60 ± 3 f
59 ± 1
70 e
7 ± 2 f
12 ± 0 g
6 e
80 ± 7 f
97 ± 2 g
88 e
11
12
66 ± 3
9 ± 1
99 ± 1
>99
74 e
21 e
67 ± 9 f
8 ± 1 f
13
14
88 e
28 e
63 ± 0
5 ± 1
>99
83 e
6 e

Molecules 2017, 22, 1688
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Table 4. Cont.
Compound
15
11C-Labelled Urea
Conversion a (%)
RCP c (%)
RCY b (%)
27 ± 1
1 ± 0
90 ± 10
90 e
Trace e
-
16
17
12 ± 2
51 ± 0
Trace
Trace
-
-
18
19
66 ± 8
-
-
41 ± 7 f
99 ± 0 f
74
Conditions as in entry 11, Table 2. All experiments were performed in duplicate unless otherwise stated. a Percentage
b
of [11C]CO converted to non-volatile products. Decay-corrected. Radiochemical yield. Based on the 11C-labelled
product obtained after semi-preparative HPLC and amount of [11C]CO collected in the reaction vial. Decay-corrected.
c Radiochemical purity. Determined by analytical HPLC of the isolated 11C-labelled product. d 5 min reaction time, one
experiment. e 10 equiv. of 1-butanol added, single experiment. f Average of three experiments. g 3 equiv. of aniline used.
A set of aniline derivatives were also synthesised (11–14) and, similar to 5, were found to be less
reactive than their aliphatic counterparts and were isolated in RCYs varying from 5% to 12%. In the
reaction with unfunctionalised aniline, an impurity was present after the purification and 11 could only
be isolated with 80% RCP. When three equivalents of aniline were used, the RCY increased from 7% to
12% and the RCP reached 97%. Substitution in the 4-position of the aromatic ring was explored and
methoxy- and fluoro-substituents were equally tolerated (12 and 13, 8–9%), whereas the nitro group
gave a slightly lower RCY of 5% (14). It should be noted that the conversion for the reactions with
aniline derivatives were in the range of 60–67% and the product selectivities varied from 33% to 71%.
The true outcome of the reaction only became fully apparent after isolation of the respective product.
Thus, the radiochemical yields estimated for a non-isolated 11C-labelled product should be interpreted
with care as it may not fully correspond to the radiochemical yield of the isolated product. Next, cyclic
11C-labelled urea 15 was synthesised but isolated in a very low RCY (1%). An unknown 11C-labelled
byproduct formed during the reaction. This byproduct was not formed when the reference was
synthesised (even when using Pd(Xantphos)Cl2). 11C-Labelled sulfonylureas have been synthesised
from the corresponding sulfonyl azide in a Rh(I)-mediated reaction but here, synthesis of 11C-labelled
sulfonyl ureas 16 and 17 did not result any in isolable product [43]. To probe whether the trace
formation of 16 was related to the poor nucleophilicity of aniline, the amine was changed to
6 to aid in

Molecules 2017, 22, 1688
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the plausible 11C-sulfonyl isocyanate formation and subsequent product formation to sulphonylurea
17. However, as seen in Table 4, this change did not improve the reaction outcome.
Ethanol has been found to increase the conversion of [11C]phosgene-derived ammonium
[11C]isocyanate to [11C]urea [31]. Similarly, different alcoholic additives were found to accelerate
the conversion of ammonium cyanate to urea [62]. Therefore, 10 equivalents of 1-butanol were added
to the reaction mixture of
(21% and 28%) whereas the RCY of
not improved (31% and 14%). Hence, the addition of 1-butanol could be most beneficial when a poorly
7,
8
and 11
–
15. Compounds 12 and 13, were isolated with improved RCY
7
and , synthesised from an aliphatic primary amine and , were
8
6
nucleophilic primary amine is used. However, the RCY of 11, 14 and 15 were not improved thus
making the hypothesis of nucleophilicity less certain. Of note is that the 11C-labelled byproduct formed
during the synthesis of 15, was isolated in a RCY of 72% when 1-butanol was added.
Lactams has previously been synthesised under Pd(II)-catalysed conditions, however, with
the conditions employed here, in particular the short reaction time and small amount of [11C]CO,
11C-labelled lactam 18 was not formed [63]. A 11C-labelled byproduct was formed, which in part
accounts for the relatively high [11C]CO-conversion (66%). The byproduct was hypothesised from
LC/MS to be [11C]N-benzyl-N-ethylbenzamide, formed by scrambling of a phenyl group from the
Pd-ligand. A subsequent synthesis of N-benzyl-N-ethylbenzamide from benzoic acid and N-ethyl
benzylamine and matching of LC retention times confirmed the identity. When the reaction mixture
was preheated, to aid in dissolution of reactants, [11C]N-benzyl-N-ethylbenzamide was formed in
25% RCY, whereas the RCY dropped to 4% when the reaction mixture was not preheated. Lastly, sEH
inhibitor 19 was synthesised in a good 41% RCY [23]. To further demonstrate the utility of the method,
with respect to molar activity, was the Am determined for 19 in two experiments. When starting from
17.8 GBq of [11C]CO, product 19 was isolated with 1.9 GBq and in a Am of 247 GBq/
µmol, 43 min after
EOB. In the second experiment, when starting with 12.8 GBq of [11C]CO, 20 could be isolated with
2.1 GBq and a Am of 319 GBq/µmol, 41 min from EOB.
In Scheme 1, hypothetical paths
A–E through which the reaction can proceed are presented.
A carbamoylpalladium species has been proposed by Hiwatari et al. to form as an intermediate
in the reaction [64]. Thus, hypothesising the same species will form in the present reaction with
[11C]CO, 11C-labelled carbamoylpalladium species 20 can generate 11C-labelled isocyanate 21 through
the deprotonation of 20 by an external base such as in path
-elimination to give a hydridopalladium(II) complex 23, which subsequently can reductively
eliminate HCl to yield Pd(0)-complex 22 (path ). The elimination depicted in path has found
A. Alternatively, 21 can be formed by
a
β
B
A
experimental support in kinetic studies where the disappearance of a carbamoylpalladium complex
was found to have a first-order dependence on the concentration of a tertiary amine [64]. Attack of a
second amine on 21 will furnish product 24. Carbamoyl chloride 25 is proposed to form in path
a reductive elimination from 20. As in and , attack of another amine gives 24. In paths
C
and E,
after
A
B
D
the second amine reacts directly with 20, either through initial coordination to Pd (complex 26, path
or through a direct attack on the carbonyl (path E).
D)
Path
cannot deprotonate 20 and/or is slow to react with 21. However, the low RCYs of 11
with ease should be able to both deprotonate 20 and subsequently trap 21 to form product cannot
be rationalised in the same way. Although paths are feasible on paper, previous experimental
studies support an isocyanate intermediate, i.e., path or , on the basis that secondary amines
does not furnish the corresponding tetra-substituted urea under the conditions explored [63 65].
Further support of path or to unsymmetrical
is that the ratio of symmetric 11C-labelled urea
11C-labelled urea
was here found to be approximately 1:9 and the same ratio was found when and
were allowed to react with benzyl isocyanate. Furthermore, when performing the reaction with only
4-fluoroaniline and Pd(Xantphos)Cl2, was the presence of [11C]4-fluorophenyl isocyanate confirmed by
analytical HPLC and co-injection with 4-fluorophenyl isocyanate. Amine was thereafter added and
the reaction was heated for another 10 min. A second analysis revealed that 13 had formed. However,
A
, and path
B
to some extent, could explain the low RCY of
5
if the weakly basic aniline
–
14 where
6
C–E
A
B
–
A
B
2
7
1
6
6

Molecules 2017, 22, 1688
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the mere presence of the 11C-labelled isocyanate does not preclude the reaction from proceeding
through an alternative mechanism.
To lend further support to path
A in Scheme 1, DFT-calculations were performed for the urea
formation from CO and N-methyl amine. Although a solvent model for THF was used, the accuracy of
the energies will suffer from the variation in charge for the palladium complexes on the reaction path.
However, it was reasoned that the calculations would be accurate enough to give a rough estimate of
the stability of intermediates along the reaction path (Scheme 2).
Scheme 1. Hypothetical reaction paths for 11C-labelled urea formation.
Scheme 2. Calculated free energies (kcal/mol) of intermediates along path
A in Scheme 1 showing
that the path is energetically feasible.

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As shown in Scheme 2, addition of CO to the initial palladium dichloro complex A1 does not
involve any dramatic energy changes and addition of a primary amine is also feasible. Deprotonation
of the palladium-bound amine is not likely, however. Instead, the amine can attack the carbonyl directly
forming a labile N-protonated carbamoyl species. In the gas-phase, the C–N bond varies between 2.07
Å and 2.70 Å depending on whether the cation can be stabilised by the phenylphosphine or not. Upon
addition of a stabilising amine to A8, the C–N bond shortens to 1.63 Å. The carbamoyl species A9
formed by deprotonation of A8, is the most stable intermediate on the reaction path. Base-assisted
deprotonation of this species leads to the formation of an isocyanate and the palladium(0)-complex
A14 in accordance with the first-order dependence on the concentration of a tertiary amine reported
by Hiwatari et al. [64].
,
3. Materials and Methods
3.1. General Chemistry Information
Reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Fischer
(Pittsburgh, PA, USA) and used without further purification. Thin layer chromatography (TLC) was
performed on aluminium sheets pre-coated with silica gel 60 F254 (0.2 mm, Merck KGaA, Darmstadt,
Germany). Column chromatography was performed using silica gel 60 (40–63
µm, Merck KGaA,
Darmstadt, Germany). Carbon-11 was prepared by the 14N(p, )11C nuclear reaction using 17 MeV
α
protons produced by a Scanditronix MC-17 Cyclotron at PET Centre, Uppsala University Hospital,
and obtained as [11C]carbon dioxide. The target gas used was nitrogen (AGA Nitrogen 6.0) containing
0.05% oxygen (AGA Oxygen 4.8). The [11C]CO2 was transferred to the low-pressure xenon system in a
◦
stream of helium gas and concentrated before reduction to [11C]CO, over zinc heated to 400 C [22
,54].
A column with Ascarite was used to trap residual [11C]CO2. Before transferring the concentrated
[11C]CO into the reaction vial, through a transfer needle placed in the capped reaction vial, the carrier
gas was changed from helium to xenon (>99.9%, 1.5 mL/min). No venting needle was required during
the transfer and, when completed, the transfer needle was removed and the reaction vial was lowered
into a heating block. Analytical reversed phase HPLC-MS was performed on a Dionex Ultimate 3000
system using 0.05% HCOOH in water and 0.05% HCOOH in acetonitrile as mobile phase with MS
detection, equipped with a C18 (Phenomenex Kinetex SB-C18 (4.8
diode array detector. Purity determinations were performed using C18 (Phenomenex Kinetex SB-C18
(4.8 50 mm)) column and Biphenyl (Phenomenex Kinetex Biphenyl (2.6 m, 4.6 50 mm) column,
×
50 mm)) column using a UV
×
µ
×
with UV detection at 214 nm or 254 nm. Semi-preparative reversed phase HPLC was performed on
a Gilson–Finnigan ThermoQuest AQA system equipped with Gilson GX-271 system equipped with
a C18 (Macherey-Nagel Nucleodur HTec (5
µ
m, 125
×
21 mm)) using 0.1% TFA in water and 0.1%
TFA in acetonitrile as eluent. Purifications of 11C-labelled ureas were performed on a VWR La Prep
Sigma system with a LP1200 pump, 40D UV detector, a Bioscan flowcount radiodetector. The identities,
concentration and radiochemical purities of the purified 11C-labeled ureas were determined with
either a VWR Hitachi Elite LaChrom system (L-2130 pump, L-2200 autosampler, L-2300 column oven,
L-2450 diode array detector in series with a Bioscan
VWR International (LaPrep P206 pump, an Elite LaChrom L-2400 UV detector in series with a Bioscan
+-flowcount detector), 8.1 mM ammonium carbonate (aq.) and acetonitrile mobile phase and Merck
Chromolith Performance RP-18e column (4.6 100 mm) using isotopically unmodified compounds
as references. NMR spectra were recorded on a Varian Mercury plus spectrometer (1H at 399.8 MHz,
13C at 100.5 MHz and 19F at 376.5 MHz) at ambient temperature. Chemical shifts (
) are reported in
ppm, indirectly referenced to tetrametylsilane (TMS) via the residual solvent signal (1H: CHCl3
CD2HOD 3.31, (CHD2)(CD3)SO 2.50, (CHD2)(CD3)CO 77.2, CD3OD
2.05 13C: CDCl3
(CHD2)(CD3)SO δ 39.5, (CD3)2CO δ 29.8, 206.3.
β
+-flowcount radiodetector) or an Elite LaChrom
β
×
δ
δ
δ
7.26,
49.0,
δ
δ
δ
δ

Molecules 2017, 22, 1688
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3.2. Synthesis of Starting Materials
3.2.1. tert-Butyl 4-hydroxypiperidine-1-carboxylate [66] CAS: 109384-19-2
4-Hydroxypiperdine (3.1 mmol) was dissolved in THF (10 mL), to which 10% NaOH (aq., 1.5 mL
was added. Di-tert-butyl dicarbonate (1.2 equiv.) was slowly added to the reaction mixture.
After completion of the reaction, the reaction mixture was extracted with 3 15 mL chloroform.
)
×
The combined organic phases were washed with 15 mL Brine, dried over Na2SO4, filtered and
concentrated in vacuo. Flash column chromatography followed, using 15% ethanol in ethyl acetate
as eluent. Isolated as a white solid in 95% purity and used without further purification.1H-NMR
(400 MHz, CDCl3) δ 3.91–3.76 (m, 3H), 3.07–2.96 (m, 2H), 1.89–1.80 (m, 2H), 1.68 (d, J = 4.2 Hz, 1H),
1.50–1.46 (m, 1H), 1.45 (s, 9H), 1.43–1.39 (m, 1H).
3.2.2. tert-Butyl 4-phenoxypiperidine-1-carboxylate [66] CAS: 155989-69-8
tert-Butyl 4-hydroxypiperidine-1-carboxylate (3.1 mmol), phenol (1.0 equiv.) and PPh3 (1.0 equiv.)
were dissolved in anhydrous THF (16 mL) before addition of diisopropyl azodicarboxylate (1.0 equiv.).
The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated
before purification with flash column chromatography, using 7:1 i-hexane:ethyl acetate as eluent.
1
Isolated as a white solid (498 mg, 59%). Rf = 0.47 (7:1 i-hexane:ethyl acetate). H-NMR (400 MHz,
CDCl3)
δ 7.31–7.27 (m, 2H), 6.99–6.88 (m, 3H), 4.46 (hept, J = 3.5 Hz, 1H), 3.75–3.64 (m, 2H), 3.39–3.26
(m, 2H), 1.98–1.86 (m, 2H), 1.83–1.69 (m, 2H), 1.47 (s, 9H). 13C-NMR (101 MHz, CDCl3)
δ 157.3, 155.0,
129.7, 121.2, 116.3, 79.7, 72.3, 40.9, 30.7, 28.5. MS-EI m/z 277.1.
3.2.3. 4-Phenoxypiperidine [66] CAS: 3202-33-3
tert-Butyl 4-phenoxypiperidine-1-carboxylate (453 mg) was dissolved in dichloromethane (10 mL).
TFA (1.4 mL) was added and the mixture was stirred at room temperature for 2.5 h. The reaction
mixture was concentrated and re-dissolved in dichloromethane (20 mL), followed by washing
with 1.5 M NaOH (20 mL). The water phase was extracted with 2
×
20 mL DCM. The combined
organic phases were dried over Na2SO4, filtered and concentrated. Purification with flash column
chromatography using 5% MeOH and 1% triethylamine (TEA) in DCM Product was isolated as a
white solid (290 mg, 96%). Rf = 0.18 (5% MeOH, 1% TEA in DCM). 1H-NMR (400 MHz, CDCl3)
δ
7.30–7.25 (m, 2H), 6.96–6.89 (m, 3H), 4.39 (hept, J = 3.9 Hz, 1H), 3.20–3.12 (m, 2H), 2.80–2.72 (m, 2H),
2.24 (br. s, 1H), 2.08–1.97 (m, 2H), 1.75–1.65 (m, 2H). 13C-NMR (101 MHz, CDCl3)
116.3 73.1, 43.8, 32.2. HPLC purity > 99%.
δ 157.4, 129.6, 121.0,
3.3. Synthesis of Reference Compounds
3.3.1. General Procedure for Synthesis of Reference Compounds via an Oxidative Carbonylation
The reaction was performed in a double-chamber system [67 68]. Amine/amines, Pd(OAc)2
,
(0.05 equiv.) and Cu(OAc)2 (0.5 equiv.) were added to the reaction chamber and dissolved in
1,4-dioxane (2 mL). In the CO-chamber was Mo(CO)6 (200 mg) dissolved in 1,4-dioxane (2 mL).
After capping of the system, 1,8-diazabicyclo[5.4.0]undec-7-ene was added to the CO-chamber.
The double-chamber system was positioned in a Dry–Syn heating block and heated to 95 ◦C. After
completion of the reaction, the reaction mixture was filtered through a short silica plug before flash
column chromatography.
3.3.2. N,N0-Dibenzylurea [69] CAS: 1466-67-7
Synthesised according to the general procedure from benzylamine (1 mmol). Purified with flash
column chromatography, using 1% MeOH and 1% AcOH in DCM as eluent. Isolated as a white powder
(74 mg, 31%). Rf = 0.23 (1% MeOH and 1% acetic acid in DCM). 1H-NMR (400 MHz, DMSO-d6)
δ

Molecules 2017, 22, 1688
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7.34–7.18 (m, 10H), 6.46 (t, J = 6.1 Hz, 2H), 4.23–4.20 (m, 4H). 13C-NMR (101 MHz, CDCl3 and MeOD)
δ 159.1, 139.3, 128.4, 127.2, 127.0, 44.0. MS-ESI [M + H+] = m/z 241.3. HPLC purity > 99%.
3.3.3. N-(2-(Pyridin-2-yl)ethyl)piperidine-1-carboxamide CAS: 1710806-84-0
Synthesised according to the general procedure from 2-(2-aminoethyl)pyridine (1.0 mmol) and
piperidine (3 equiv.). Purified with flash column chromatography, using 1% MeOH and 1% AcOH in
DCM as eluent. Isolated as a solid (49 mg, 21%). Rf = 0.16 (ethyl acetate + 1% TEA). 1H-NMR (400 MHz,
(CD3)2CO)
1H), 6.04 (br. s, 1H), 3.54
1.49
1.41 (m, 4H). 13C-NMR (101 MHz, (CD3)2CO)
δ
8.51
−
8.47 (m, 1H), 7.67 (td, J = 7.7, 1.9 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.20
3.47 (m, 2H), 3.33 3.27 (m, 4H), 2.94 (t, J = 7.0 Hz, 2H), 1.60 1.53 (m, 2H),
161.2, 158.3, 150.0, 137.1, 124.0, 122.1, 45.4, 41.3,
−
7.15 (m,
−
−
−
−
δ
41.2, 39.1, 39.0, 26.5, 25.4. HRMS calc: 234.1606 found: 234.1614. HPLC purity > 99%.
3.3.4. 3,4-Dihydroquinazolin-2(1H)-one [70] CAS: 66655-67-2
Synthesised according to the general procedure from 2-aminobenzylamine (1.5 mmol). Purified
twice with flash column chromatography, using 5% MeOH, 1% TEA in DCM and 1% TEA in ethyl
acetate, respectively, followed by semi-preparative purification. Isolated as a white powder (26 mg,
1
12%). Rf = 0.74 (5% MeOH, 1% TEA in DCM). H-NMR (400 MHz, (CD3)2SO)
δ 8.98 (br. s, 1H),
7.12–7.04 (m, 2H), 6.84 (td, J = 7.5, 1.2 Hz, 1H), 6.79–6.73 (m, 2H), 4.29 (s, 2H). 13C-NMR (101 MHz,
(CD3)2SO)
154.6, 138.1, 127.6, 125.6, 120.9, 118.1, 113.5, 42.5. MS-ESI [M + H+] = m/z 149.1. HPLC
purity > 99%.
δ
3.3.5. 2-Ethylisoindolin-1-one [71] CAS: 23967-95-5
Synthesised according to the general procedure from N-ethylbenzylamine (1 mmol). Purified with
flash column chromatography, using 3:1 i-hexane:ethyl acetate + 1% TEA. Isolated as a yellow liquid
(49 mg, 31%). Rf = 0.25 (3:1 i-hexane:ethyl acetate + 1% TEA). 1H-NMR (400 MHz, CDCl3/CD3OD)
δ
7.77 (d, J = 7.5 Hz, 1H), 7.54–7.49 (m, 1H), 7.47–7.41 (m, 2H), 4.40 (s, 2H), 3.64 (q, J = 7.3 Hz, 2H), 1.25
(t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, CDCl3/CD3OD)
δ 141.4, 132.7, 131.7, 128.4, 123.6, 123.0, 49.8,
37.4, 13.6 (Carbonyl carbon missing). MS-ESI [M + H+] = m/z 162.1. HPLC purity > 99%.
3.3.6. N-(2,4-Dichlorobenzyl)-4-phenoxypiperidine-1-carboxamide CAS: 950645-62-2
Synthesised according to the general procedure from 2,4-dichlorobenzylamine (0.43 mmol) and
4-phenoxypiperidine (2 equiv.). Purified with flash column chromatography, using 1% MeOH and 1%
AcOH in DCM as eluent, followed by semi-preparative chromatography. Isolated as a white powder
(47 mg, 29%). Rf = 0.53 (5% MeOH, 1% TEA in DCM). 1H-NMR (400 MHz, (CD3)2CO)
δ 7.46–7.42 (m,
2H), 7.33 (dd, J = 8.2, 2.2 Hz, 1H), 7.31–7.24 (m, 2H), 7.00–6.94 (m, 2H), 6.92 (tt, J = 7.3, 1.1 Hz, 1H), 4.62
(tt, J = 7.6, 3.7 Hz, 1H), 4.45 (d, J = 5.0 Hz, 2H), 3.86–3.75 (m, 2H), 3.41–3.30 (m, 2H), 2.03–1.95 (m, 2H),
1.72–1.61 (m, 2H).13C-NMR (101 MHz, (CD3)2CO)
δ 157.6, 157.5, 137.2, 133.3, 132.5, 130.3, 129.6, 128.6,
127.1, 120.8, 116.1, 72.2, 41.6, 41.2, 30.7. HRMS calc: 379.0980 found: 379.0999. HPLC purity > 99%.
3.3.7. General Procedure for the Synthesis of Reference Compounds from an Isocyanate
The isocyanate derivative (1 mmol) was dissolved in DCM (2 mL) and cooled to
−
10 ◦C before
piperidine (1 equiv.) in DCM (2 mL) was added under N2. The reaction was stirred under N2 for
30 min and then let to warm room temperature and stir until completion. The reaction mixture was
concentrated in vacuo and recrystallised.
3.3.8. N-Benzylpiperidine-1-carboxamide [72] CAS: 39531-35-6
Synthesised according to the general procedure from benzyl isocyanate. Recrystallised in
petroleum ether and isolated as colourless crystals (87 mg, 40%). Rf = 0.33 (3:1 i-hexane:ethyl acetate +
◦
1
1% TEA). Melting point: 99–100 C [73]. H-NMR (400 MHz, (CD3)2CO)
δ 7.32–7.24 (m, 4H), 7.21–7.16

Molecules 2017, 22, 1688
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(m, 1H), 6.27 (s, 1H), 4.35 (d, J = 5.8 Hz, 2H), 3.40–3.35 (m, 4H), 1.63–1.55 (m, 2H), 1.53–1.44 (m,
4H). 13C-NMR (101 MHz, (CD3)2CO)
δ 158.3, 142.3, 128.9, 128.2, 127.3, 45.6, 44.9, 26.6, 25.4. MS-ESI
[M + H+] = m/z 219.4. HPLC purity > 99%.
3.3.9. N-Butylpiperidine-1-carboxamide CAS: 1461-79-6
Synthesised according to the general procedure from butyl isocyanate. Recrystallised in petroleum
ether and isolated as colourless crystals (150 mg, 81%). Rf = 0.15 (3:1 i-hexane:ethyl acetate + 1% TEA).
Melting point: 60–62 ◦C. 1H-NMR (400 MHz, (CD3)2CO)
δ
5.73 (s, 1H), 3.35–3.26 (m, 4H), 3.16–3.09
(m, 2H), 1.60–1.52 (m, 2H), 1.51–1.39 (m, 6H), 1.37–1.24 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13C-NMR
(101 MHz, (CD3)2CO) 158.4, 45.5, 41.0, 40.9, 33.4, 33.4, 26.5, 25.4, 20.7, 14.2. HRMS calc: 185.1654
δ
found: 185.1653. HPLC purity > 99%.
3.3.10. N-Isopropylpiperidine-1-carboxamide CAS: 10581-04-1
Synthesised according to the general procedure from isopropyl isocyanate. Recrystallised in
petroleum ether and isolated as white crystals (100 mg, 58%). Melting point: 114–121 ◦C. 1H-NMR
(400 MHz, (CD3)2SO)
δ 6.02 (d, J = 7.6 Hz, 1H), 3.80–3.66 (m, 1H), 3.25–3.20 (m, 4H), 1.56–1.46 (m, 2H),
1.44–1.34 (m, 4H), 1.03 (d, J = 6.6 Hz, 6H). 13C-NMR (101 MHz, (CD3)2CO)
δ 156.7, 44.2, 41.6, 25.3, 24.2,
23.0. HRMS calc: 171.1497 found: 171.1504. HPLC purity > 99%.
3.3.11. N-Phenylpiperidine-1-carboxamide [72] CAS: 2645-36-5
Synthesised according to the general procedure from phenyl isocyanate. Recrystallised in ethyl
acetate and isolated as colorless crystals (173 mg, 84%). Rf = 0.37 (3:1 i-hexane:ethyl acetate + 1%
◦
1
TEA). Melting point: 168–171 C. H-NMR (400 MHz, (CD3)2CO)
2H), 7.23–7.17 (m, 2H), 6.91 (tt, J = 7.4, 1.2 Hz, 1H), 3.50–3.44 (m, 4H), 1.67–1.59 (m, 2H), 1.58–1.49
(m, 4H). 13C-NMR (101 MHz, (CD3)2CO)
155.7, 141.9, 129.1, 122.4, 120.2, 45.8, 26.6, 25.3. MS-ESI
[M + H+] = m/z 205.4. HPLC purity > 99%.
δ 7.84 (br. s, 1H), 7.54–7.49 (m,
δ
3.3.12. N-(4-Methoxyphenyl)piperidine-1-carboxamide CAS: 2645-37-6
Synthesised according to the general procedure from 4-methoxyphenyl isocyanate. Recrystallised
in petroleum ether and isolated as colourless crystals (183 mg, 78%). Rf = 0.49 (3:1 i-hexane:ethyl acetate
+ 1% TEA). Melting point: 124–125 ◦C. 1H-NMR (400 MHz, (CD3)2CO)
δ
7.68 (s, 1H), 7.43–7.37 (m,
2H), 6.82–6.76 (m, 2H), 3.73 (s, 3H), 3.50–3.39 (m, 4H), 1.65–1.58 (m, 2H), 1.57–1.49 (m, 4H). 13C-NMR
(101 MHz, (CD3)2CO) 155.9, 135.0, 122.1, 122.0, 114.3, 55.6, 45.8, 26.6, 25.3. HRMS calc: 235.1447
δ
found: 235.1450. HPLC purity > 99%.
3.3.13. N-(4-Fluorophenyl)piperidine-1-carboxamide CAS: 60465-12-5
Synthesised according to the general procedure from 4-fluorophenyl isocyanate. Recrystallised
in ethyl acetate and isolated as colourless crystals (188 mg, 84%). Rf = 0.39 (3:1 i-hexane:ethyl acetate
+ 1% TEA). Melting point: 179–184 ◦C. 1H-NMR (400 MHz, (CD3)2CO)
δ
7.90 (br. s, 1H), 7.55–7.48
(m, 2H), 7.02–6.94 (m, 2H), 3.50–3.42 (m, 4H), 1.67–1.58 (m, 2H), 1.57–1.48 (m, 4H). 13C-NMR (101
MHz, (CD3)2CO)
δ 158.8 (d, J = 238.2 Hz), 155.8, 138.2 (d, J = 2.6 Hz), 121.9 (d, J = 7.5 Hz), 115.4 (d,
J = 22.3 Hz), 45.7, 26.6, 25.3. 19F-NMR (376 MHz, (CD3)2CO)
δ -119.3 – -130.5. HRMS calc: 223.1247
found: 223.1245. HPLC purity > 99%.
3.3.14. N-(4-Nitrophenyl)piperidine-1-carboxamide [63] CAS: 2589-20-0
Synthesised according to the general procedure from 4-nitrophenyl isocyanate. Recrystallised
in petroleum ether and ethyl acetate and isolated as yellow◦crystals (223 mg, 89%). Rf = 0.39 (3:1
1
i-hexane:ethyl acetate + 1% TEA). Melting point: 159–162 C. H-NMR (400 MHz, (CD3)2CO)
δ
8.51–8.47 (m, 1H), 7.67 (td, J = 7.7, 1.9 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.20–7.15 (m, 1H), 6.04 (br. s,

Molecules 2017, 22, 1688
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1H), 3.54–3.47 (m, 2H), 3.33–3.27 (m, 4H), 2.94 (t, J = 7.0 Hz, 2H), 1.60–1.53 (m, 2H), 1.49–1.41 (m, 4H).
13C-NMR (101 MHz, (CD3)2CO)
δ 161.2, 158.3, 150.0, 137.1, 124.0, 122.1, 45.4, 41.3, 41.2, 39.1, 39.0, 26.5,
25.4. MS-ESI [M + H+] = m/z 250.2. HPLC purity > 99%.
3.3.15. N-Tosylpiperidine-1-carboxamide CAS: 23730-08-7
Synthesised according to the general procedure from p-toluenesulfonyl isocyanate. Recrystallised
in petroleum ether and isolated as white crystals (144 mg, 51%). Rf = 0.85 (5% MeOH, 1% TEA in DCM).
Melting point: 133–137 ◦C. 1H-NMR (400 MHz, (CD3)2SO)
δ
10.75 (br. s, 1H), 7.76 (d, J = 8.2 Hz, 2H),
7.36 (d, J = 8.1 Hz, 2H), 3.29–3.23 (m, 4H), 2.38 (s, 3H), 1.54–1.46 (m, 2H), 1.43–1.35 (m, 4H). 13C-NMR
(101 MHz, (CD3)2CO) 151.4, 143.4, 138.7, 129.0, 128.2, 44.9, 25.6, 24.1, 20.6. HRMS calc: 283.1116
δ
found: 283.1103. HPLC purity > 99%.
3.4. General Procedure for the Synthesis and Analysis of 11C-Labelled Ureas
Pd(Xantphos)Cl2 (4 µmol) and amine/amines (30 µmol) were added to an oven-dried, conical
glass vial followed by freshly distilled tetrahydrofuran (400 µ
L). [11C]CO was transferred to the capped
reaction vial and the radioactivity was measured to determine the starting amount of [11C]CO. The
◦
reaction was heated at 120 C for 10 min. When finished, the radioactivity was measured to confirm that
no radioactive material had escaped during heating. The vial was purged with N2 to remove unreacted
[11C]CO and, possibly, volatile labelled compounds formed during the reaction. The radioactivity was
measured after the purge followed by either analytical HPLC for product selectivity determination or
semi-preparative HPLC purification for 11C-labelled product isolation. Purification was performed
using either column C1 = Phenomenex Kinetex C18 (5
µ
m, 150 × 10.0 mm) column, C2 = Reprosil–Pur
Basic C18 (5 m, 150 10.0 mm) column or C3 = Gemini NX C18 (5
µ
×
µ
m, 250 10.0 mm) column
×
with ammonium formate buffer 50 mM (pH 3.5) (A) and acetonitrile (B) as eluents. Run time was
20 min with flow 5 mL/min followed by flushing the column with 100% B. After isolation and a
final radioactivity measurement of the 11C-labelled product, an aliquot was analysed to determine
radiochemical purity and the identity of the 11C-labelled product was confirmed using the isotopically
unmodified product as reference. The analytical method was the same for all compounds, 10–90%
acetonitrile in 10 min (flow 2 mL/min). Molar activity determinations were based on a calibration
curve, constructed with isotopically unmodified 20. For full definitions and calculations, see the
supporting materials.
3.4.1. [carbonyl-11C]N,N0-Dibenzylurea 2
Synthesised according to the general procedure (three experiments). (1) Purification method: 40%
B. Column: C1. Starting from 3.4 GBq, 0.88 GBq was isolated at 33 min from EOB; (2) Purification
method: 35% B Column: C1. Starting from 3.4 GBq, 0.83 GBq was isolated at 36 min from EOB;
(3) Purification method: 45% B Column: C3. Starting from 2.7 GBq, 0.44 GBq was isolated at 34 min
from EOB. Analytical HPLC Rt = 5.1 min.
3.4.2. [carbonyl-11C]N,N0-Dipropylurea 3
Synthesised according to the general procedure (two experiments). (1) Purification method: 0–70%
B Column: C1. Starting from 2.2 GBq, 0.24 GBq was isolated at 42 min from EOB; (2) Purification
method: 0–70% B Column: C1. Starting from 6.7 GBq, 0.62 GBq was isolated at 53 min from EOB.
Analytical HPLC Rt = 2.6 min.
3.4.3. [carbonyl-11C]N,N0-Dicyclohexylurea 4
Synthesised according to the general procedure (two experiments). (1) Purification method: 40% B
Column: C1. Starting from 3.4 GBq, 0.53 GBq was isolated at 37 min from EOB; (2) Purification method:

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35% B Column: C1. Starting from 3.8 GBq, 0.60 GBq was isolated at 41 min from EOB. Analytical
HPLC Rt = 5.6 min.
3.4.4. [carbonyl-11C]N,N0-Diphenylurea 5
Synthesised according to the general procedure (two experiments). (1) Purification method: 35%
B Column: C1. Starting from 3.0 GBq, 0.028 GBq was isolated at 50 min from EOB; (2) Purification
method: 35% B Column: C1. Starting from 6.7 GBq, 0.059 GBq was isolated at 43 min from EOB.
Analytical HPLC Rt = 5.5 min.
3.4.5. [carbonyl-11C]N-Benzylpiperidine-1-carboxamide 7
Synthesised according to the general procedure (four experiments). (1) Purification method: 35%
B Column: C1. Starting from 4.9 GBq, 0.47 GBq was isolated at 45 min from EOB; (2) Purification
method: 35% B Column: C1. Starting from 3.0 GBq, 0.57 GBq was isolated at 36 min from EOB;
(3) 5 min reaction time. Purification method: 35% B Column: C3. Starting from 2.6 GBq, 0.15 GBq was
isolated at 37 min from EOB; (4) 10 equiv. of 1-butanol added. Purification method: 35% B Column:
C3. Starting from 3.2 GBq, 0.29 GBq was isolated at 42 min from EOB. Analytical HPLC Rt = 4.9 min.
3.4.6. [carbonyl-11C]N-Butylpiperidine-1-carboxamide 8
Synthesised according to the general procedure (three experiments). (1) Purification method:
20–50% Column: C1. Starting from 3.7 GBq, 0.25 GBq was isolated at 44 min from EOB; (2) Purification
method: 20–50% Column: C1. Starting from 2.9 GBq, 0.17 GBq was isolated at 46 min from EOB; (3) 10
equiv. of 1-butanol added. Purification method: 20–50% Column: C3. Starting from 2.8 GBq, 0.093 GBq
was isolated at 49 min from EOB. Analytical HPLC Rt = 4.5 min.
3.4.7. [carbonyl-11C]N-(2-(Pyridin-2-yl)ethyl)piperidine-1-carboxamide 9
Synthesised according to the general procedure (two experiments). (1) Purification method:
20–50% B Column: C1. Starting from 3.3 GBq, 0.14 GBq was isolated at 36 min from EOB; (2) Purification
method: 20% B Column: C1. Starting from 3.7 GBq, 0.13 GBq was isolated at 39 min from EOB.
Analytical HPLC Rt = 3.8 min.
3.4.8. [carbonyl-11C]N-Isopropylpiperidine-1-carboxamide 10
Synthesised according to the general procedure (two experiments). (1) Purification method:
15–50% B Column: C1. Starting from 3.2 GBq, 0.11 GBq was isolated at 39 min from EOB; (2) Purification
method: 15–50% B Column: C1. Starting from 3.2 GBq, 0.19 GBq was isolated at 38 min from EOB.
Analytical HPLC Rt = 3.3 min.
3.4.9. [carbonyl-11C]N-Phenylpiperidine-1-carboxamide 11
Synthesised according to the general procedure (six experiments). (1) Purification method: 35% B
Column: C1. Starting from 3.6 GBq, 0.19 GBq was isolated at 40 min from EOB; (2) Purification method:
30% B Column: C1. Starting from 3.1 GBq, 0.094 GBq was isolated at 42 min from EOB; (3) 3 equiv.
aniline. Purification method: 20–50% B Column: C1. Starting from 3.6 GBq, 0.051 GBq was isolated
at 40 min from EOB; (4) 3 equiv. aniline. Purification method: 20–50%% B Column: C1. Starting
from 3.2 GBq, 0.0.058 GBq was isolated at 40 min from EOB; (5) 3 equiv. aniline. Purification method:
20–50% B Column: C1. Starting from 4.3 GBq, 0.16 GBq was isolated at 42 min from EOB; (6) 10 equiv.
of 1-butanol. Purification method: 20–50% B Column: C1. Starting from 2.7 GBq, 0.026 GBq was
isolated at 62 min from EOB. Analytical HPLC Rt = 4.7 min.

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3.4.10. [carbonyl-11C]N-(4-Methoxyphenyl)piperidine-1-carboxamide 12
Synthesised according to the general procedure (three experiments). (1) Purification method:
20–50% B Column: C1. Starting from 3.2 GBq, 0.084 GBq was isolated at 41 min from EOB;
(2) Purification method: 20–50% B Column: C1. Starting from 1.8 GBq, 0.052 GBq was isolated
at 43 min from EOB; (3) 10 equiv. 1-butanol. Purification method: 20–50% B Column: C3. Starting from
2.9 GBq, 0.16 GBq was isolated at 43 min from EOB. Analytical HPLC Rt = 4.5 min.
3.4.11. [carbonyl-11C]N-(4-Fluorophenyl)piperidine-1-carboxamide 13
Synthesised according to the general procedure (four experiments). (1) Purification method: 35%
B Column: C1. Starting from 3.7 GBq, 0.072 GBq was isolated at 46 min from EOB; (2) Purification
method: 35% B Column: C1. Starting from 3.6 GBq, 0.086 GBq was isolated at 37 min from EOB;
(3) Purification method: 35% B Column: C1. Starting from 21.7 GBq, 0.61 GBq was isolated at 37 min
from EOB; (4) 10 equiv. 1-butanol. Purification method: 35% B Column: C3. Starting from 4.3 GBq,
0.38 GBq was isolated at 40 min from EOB. Analytical HPLC Rt = 4.8 min.
3.4.12. [carbonyl-11C]N-(4-Nitrophenyl)piperidine-1-carboxamide 14
Synthesised according to the general procedure (three experiments). (1) Purification method: 40%
B Column: C1. Starting from 3.3 GBq, 0.028 GBq was isolated at 49 min from EOB; (2) Purification
method: 40% B Column: C1. Starting from 3.6 GBq, 0.059 GBq was isolated at 39 min from EOB;
(3) 10 equiv. 1-butanol. Purification method: 40% B Column: C3. Starting from 2.7 GBq, 0.007 GBq was
isolated at 96 min from EOB. Analytical HPLC Rt = 5.4 min.
3.4.13. [carbonyl-11C]3,4-Dihydroquinazolin-2(1H)-one 15
Synthesised according to the general procedure (three experiments). (1) Purification method: 20%
B Column: C1. Starting from 3.4 GBq, 0.007 GBq was isolated at 43 min from EOB; (2) Purification
method: 20% B Column: C1. Starting from 3.1 GBq, 0.018 GBq was isolated at 34 min from EOB.
Analytical HPLC Rt = 2.7 min.
3.4.14. [carbonyl-11C]N-(2,4-Dichlorobenzyl)-4-phenoxypiperidine-1-carboxamide 19
Synthesised according to the general procedure (three experiments). (1) Purification method: 60%
B Column: C1. Starting from 3.2 GBq, 0.46 GBq was isolated at 33 min from EOB; (2) Purification
method: 60% B Column: C2. Starting from 17.8 GBq, 1.9 GBq was isolated at 43 min from EOB;
(3) Purification method: 60% B Column: C3. Starting from 12.8 GBq, 2.1 GBq was isolated at 41 min
from EOB. Analytical HPLC Rt = 7.4 min.
3.5. Computational Details
The density functional theory calculations were performed in Jaguar version 9.5, release 11,
Schrodinger, Inc., New York, NY, USA, 2016 [74]. To facilitate the calculations, N-methyl amine was
used as a minimal primary amine. The geometries were optimised using the LACVP** basis set [75
]
and the B3LYP-D3 a posteriori-corrected functional [76]. Vibrational analyses were performed to
characterise the stationary points identified and to calculate zero point energies. The contributions to
the free energies were calculated at a temperature of 393.15 K (120 ◦C) using the B3LYP-D3/LACVP**
geometries. For the chloride anion, an entropy of 40.97 cal K−1 mol−1 was used [77]. Solvation
·
energies were calculated using B3LYP-D3/LACVP**+ and the standard Poisson-Boltzmann continuum
solvation model [78] with parameters suitable for THF using the B3LYP-D3/LACVP** geometries.

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4. Conclusions
An important aspect of PET tracer development is the possibility to label aspiring tracers, to enable
their preclinical and subsequent clinical evaluation. The labelling method should give the labelled
compound with high enough radioactivity and purity to enable the study. The palladium(II)-mediated
oxidative carbonylation of aliphatic and aromatic amines presented herein is a facile and simple method
for the synthesis of 11C-labelled ureas. In total, 14 symmetrical and unsymmetrical 11C-labelled ureas
were synthesised and isolated using only [11C]CO, a Pd-source and amines as reaction components.
DFT-calculations and selectivity experiments supported a reaction proceeding via a 11C-labelled
isocyanate. The reaction outcome was largely dependent on the amine nucleophilicity, with aliphatic
amines performing better than aromatic amines as substrates. However, with the addition of 1-butanol
to the reaction mixture, the radiochemical yields of two aromatic 11C-labelled ureas were improved.
Not all aniline derivatives benefitted from the alcoholic additive, but the addition presents a viable
approach when using challenging aromatic amines in 11C-urea synthesis. Finally, the advantage of
using [11C]CO to achieve high molar activity, as opposed to other 11C-labelling synthons, was apparent
as the isolation of sEH inhibitor 19 in a good 41% radiochemical yield and with high molar activity
(247 GBq/µmol–319 Gbq/µmol).
1
Supplementary Materials: The following is available online: Definitions and molar activity calculations, H
and 13C-NMR chromatograms of all synthesised reference compounds, HPLC chromatograms of isolated
11C-compounds, DFT calculated structures of intermediates on path A.
Acknowledgments: We gratefully acknowledge the financial support from the Disciplinary domain of Medicine
and Pharmacy, Uppsala University and the King Gustaf V and Queen Victoria Freemason Foundation.
Author Contributions: S.R. synthesised the reference compounds and starting material, designed the experiments,
performed labelling experiments and drafted the manuscript. P.B. performed, analysed and summarised the
DFT-calculations. P.N. designed the experiments and performed labelling experiments. L.O. designed the
experiments and contributed with reference compounds. J.E. designed the experiments and set up the purification
method. M.L. designed the experiments. All authors contributed to the critical discussion and presentation of the
results. All authors read, commented on and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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