2-(4-methylsulfonylphenyl)pyrimidines as prospective radioligands for imaging cyclooxygenase-2 with PET—Synthesis, triage, and radiolabeling

Article abstract of DOI:10.3390/molecules23112850

Cyclooxygenase 2 (COX-2) is an inducible enzyme responsible for the conversion of arachidonic acid into the prostaglandins, PGG2 and PGH2. Expression of this enzyme increases in inflammation. Therefore, the development of probes for imaging COX-2 with positron emission tomography (PET) has gained interest because they could be useful for the study of inflammation in vivo, and for aiding anti-inflammatory drug development targeting COX-2. Nonetheless, effective PET radioligands are still lacking. We synthesized eleven COX-2 inhibitors based on a 2(4-methylsulfonylphenyl)pyrimidine core from which we selected three as prospective PET radioligands based on desirable factors, such as high inhibitory potency for COX-2, very low inhibitory potency for COX-1, moderate lipophilicity, and amenability to labeling with a positronemitter. These inhibitors, namely 6-methoxy-2-(4-(methylsulfonyl)phenyl-N-(thiophen-2ylmethyl)pyrimidin-4-amine (17), the 6-fluoromethyl analogue (20), and the 6-(2-fluoroethoxy) analogue (27), were labeled in useful yields and with high molar activities by treating the 6-hydroxy analogue (26) with [¹¹C]iodomethane, [¹⁸F]2-fluorobromoethane, and [d₂-¹⁸F]fluorobromomethane, respectively. [¹¹C]17, [¹⁸F]20, and [d₂-¹⁸F]27 were readily purified with HPLC and formulated for intravenous injection. These methods allow these radioligands to be produced for comparative evaluation as PET radioligands for measuring COX-2 in healthy rhesus monkey and for assessing their abilities to detect inflammation.

Full text of DOI:10.3390/molecules23112850

molecules
Article
2-(4-Methylsulfonylphenyl)pyrimidines as
Prospective Radioligands for Imaging
Cyclooxygenase-2 with PET—Synthesis, Triage,
and Radiolabeling
Michelle Y. Cortes-Salva , Stal Shrestha, Prachi Singh, Cheryl L. Morse, Kimberly J. Jenko,
Jose A. Montero Santamaria, Sami S. Zoghbi, Robert B. Innis and Victor W. Pike *
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, 10 Center Drive,
Bethesda, MD 20892, USA; michelle.cortes@nih.gov (M.Y.C.-S.); stal.shrestha@med.miami.edu (S.S.);
prachi1singh1@gmail.com (P.S.); morsec@mail.nih.gov (C.L.M.); kimjojenko@gmail.com (K.J.J.);
jose.monterosantamaria@nih.gov (J.A.M.S.); zoghbis@mail.nih.gov (S.S.Z.) innisr@mail.nih.gov (R.B.I.)
* Correspondence: pikev@mail.nih.gov; Tel.: +301-594-5986; Fax: +301-480-5112
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Anne Roivainen
Received: 11 October 2018; Accepted: 27 October 2018; Published: 2 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Cyclooxygenase 2 (COX-2) is an inducible enzyme responsible for the conversion of
arachidonic acid into the prostaglandins, PGG2 and PGH2. Expression of this enzyme increases
in inflammation. Therefore, the development of probes for imaging COX-2 with positron emission
tomography (PET) has gained interest because they could be useful for the study of inflammation
in vivo, and for aiding anti-inflammatory drug development targeting COX-2. Nonetheless,
effective PET radioligands are still lacking. We synthesized eleven COX-2 inhibitors based on
a 2(4-methylsulfonylphenyl)pyrimidine core from which we selected three as prospective PET
radioligands based on desirable factors, such as high inhibitory potency for COX-2, very low inhibitory
potency for COX-1, moderate lipophilicity, and amenability to labeling with a positron-emitter. These
inhibitors, namely 6-methoxy-2-(4-(methylsulfonyl)phenyl-N-(thiophen-2ylmethyl)pyrimidin-4-amine
(17), the 6-fluoromethyl analogue (20), and the 6-(2-fluoroethoxy) analogue (27), were labeled
in useful yields and with high molar activities by treating the 6-hydroxy analogue (26) with
[11C]iodomethane, [18F]2-fluorobromoethane, and [d2-18F]fluorobromomethane, respectively. [11C]17
[18F]20, and [d2-18F]27 were readily purified with HPLC and formulated for intravenous injection.
These methods allow these radioligands to be produced for comparative evaluation as PET
radioligands for measuring COX-2 in healthy rhesus monkey and for assessing their abilities to
detect inflammation.
,
Keywords: COX-2; radioligand; carbon-11; fluorine-18
1. Introduction
Cyclooxygenase has two isoforms, COX-1 and COX-2, that exist in endoplasmic reticulum and
produce prostaglandins G2 and H2 from arachidonic acid. Prostaglandins are important mediators
of symptoms of inflammation, such as fever and pain [1]. COX-1 has been regarded as a widespread
constitutive enzyme that is responsible for homeostatic prostaglandin synthesis. By contrast, COX-2
has been regarded as an inducible enzyme whose overexpression is implicated in the progression of
many pathological and neurological disorders [
cerebral ischemia [ ], Alzheimer’s disease [
2
–
4
], including cancers [
5
], rheumatoid arthritis [
6],
7
2–
4
,8
], and Parkinson’s disease [2–4,9
]. Consequently,
COX-2 could prove to be useful as a biomarker [1] for the early detection of inflammation in periphery
Molecules 2018, 23, 2850; doi:10.3390/molecules23112850
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2850
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and brain with positron emission tomography (PET), provided that effective PET radioligands can
be developed.
Human COX-1 and COX-2 are 60% structurally similar with 70% amino acid homology and with
very similar active site architecture [10]. For this reason, early searches for potent COX-2-selective
inhibitors faced a considerable challenge [11]. Importantly, however, COX-2 has a hydrophobic pocket
at its active site that is absent in COX-1 [12–14]. Ligand binding interactions within this pocket have
been exploited to confer COX-2 inhibition selectivity. From these efforts, the COXIB’s have emerged as
potent and COX-2 selective inhibitors that have served as a new class of nonsteroidal anti-inflammatory
drug
(Bextra) [17].
Numerous COX-2 inhibitors have been labeled with a positron-emitter, either carbon-11
(t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.8 min), and tested for imaging COX-2 in vivo with PET.
Examples include [11C]celecoxib ([11C]1) [18 19], [18F]celecoxib ([18F]1) [20], and related compounds,
such as [11C]2 21] and [18F]3 22], [11C]rofecoxib ([11C]4) [19
23 24], a radiofluorinated valdecoxib
(NSAID). These drugs include celecoxib (Celebrex) [15], rofecoxib (Vioxx) [16], and valdecoxib
,
[
[
, ,
analog ([18F]5) [25], a diarylimidazole ([11C]6) [26], a diarylindole ([11C]7) [26], a diarylcyclopentene
([11C]8) [27], and [18F]desbromo-DuP-697 ([18F]9) [28,29]. All these compounds possess a pentacyclic
core. Exceptionally, [18F]pyricoxib ([18F]10) [30] has a heterohexacyclic core (Chart 1). [18F]10 has
shown some promise for the detection of colorectal cancer [30] but no other effective radioligand has
yet emerged for imaging COX-2 [31].
Successful PET radioligands for imaging proteins in living subjects must meet many requirements,
among which high affinity and selectivity for the target protein, and moderate lipophilicity are the
most critical [32]. For imaging in the brain, ability to penetrate the blood–brain barrier is further
critical [33]. These requirements have not been well satisfied in most previous candidate COX-2 PET
radioligands. Especially, few previous candidate radioligands have shown the ability to traverse the
blood–brain barrier. For example, several contain an acidic primary or secondary arylsulfonamido
group with a pKa expected to be in the range 7 to 9. These groups contribute strongly to high-affinity
COX-2 binding but their high degree of ionization at physiological pH (7.4) counters brain entry.
Nearly all the previously tested radioligands show COX-2 inhibitory potencies with IC50’s exceeding
5 nM whereas much higher affinity (indexed by 1/IC50) may be required for successful PET imaging.
We aim to develop a PET radioligand for imaging and quantifying human COX-2 under
inflammatory conditions, as a tool for biomedical research and drug development targeting COX-2 [34].
In selecting leads for this purpose, we noticed 2-(4-methylsulfonylphenyl)pyrimidines as a class of
compounds that contains some of the most potent and selective COX-2 inhibitors yet known [35],
but which has not been investigated for leads to candidate PET radioligands. The structures of these
compounds resemble those in the [18F]pyricoxib class in being based on a substituted pyrimidinyl ring
(Chart 1), except that the ring substitution pattern is altered. Thus, the trifluoro group is replaced with
another non-aryl group and the positioning of the aryl and substituted amino groups is reversed.
We prepared a set of compounds from the 2-(4-methylsulfonylphenyl)pyrimidine class, some
already known, in a search for leads to PET radioligands that might fulfill requirements for imaging
COX-2. We intended to use rhesus monkey as a preclinical model for radioligand evaluation. Therefore,
we sought compounds with very high potency for inhibiting both monkey and human blood COX-2,
and very high selectivity (>100-fold) for inhibiting COX-2 over monkey and human COX-1 based on
enzymatic immunoassays. We also sought inhibitors that would show lipophilicity and other properties
compatible with ability to traverse the blood–brain barrier (logD 2–3). From our set of prepared
2-(4-methylsulfonylphenyl)pyrimidines, we ultimately selected and produced three candidate PET
radioligands, [11C]17
,
[18F]20, and [d2-18F]27. We concluded that these radioligands merit further
investigation in non-human primate neuroinflammation and peripheral inflammation models.

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Chart 1. Some features of former candidate COX-2 positron emission tomography (PET) radioligands.
Data are from the literature cited in the text, except for clogD values which were computed from
2-dimensional structure with Pallas software (see Experimental).

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2. Results and Discussion
2.1. Target Inhibitor Selection for Radioligand Development
We identified five 2-(4-methylsulfonylphenyl)pyrimidines (17
–19
,
22, and 25) from the report of
Orjales et al. [35] that presented: (i) high potency for inhibition of human COX-2 from human whole
blood (IC50 5.1 nM); (ii) very low potency for inhibition of the isoform COX-1 from human whole
≤
blood (IC50 > 100 nM); (iii) computed lipophilicity in an acceptable range for a PET radioligand (clogD
2–3, ideally about 2.2); and (iv) at least one site that was expected to be amenable to labeling with either
carbon-11 or fluorine-18. In addition to moderate lipophilicity, we sought other properties in these
inhibitors that would be consistent with ability to traverse the blood–brain barrier, such as molecular
weight of less than 500 Da, computed total polar surface area (tPSA) of less than 90 Å2, low hydrogen
bond donor number (HBD), less than 9 heteroatoms, pKa less than 9.5, and absence of acidic ionizable
groups such as sulfonamido and carboxyl groups [32] (Table 1). We designed another five previously
unknown 2-(4-methylsulfonylphenyl)pyrimidines (20, 21, 27, 29, and 30) as potentially new and potent
COX-2 inhibitors with other ostensibly favorable properties for PET radioligand development (Table 1).
Each of these compounds carried a group that was expected to be readily amenable for labeling with
either carbon-11 or fluorine-18 (i.e., in Table 1 the R1 group with carbon-11 for 21 and with fluorine-18
for 20,
27, and 29; and the R3 group with carbon-11 for 30). We also prepared two hydroxy compounds
with low COX inhibitory potency [35] as possible labeling precursors, namely 24 and 26.
2.2. Chemistry
Scheme 1 summarizes the chemistry used to prepare known COX-2 inhibitors, potentially new
inhibitors, and precursors for radiolabeling. A key synthesis material, 4,6-dichloro-2-(4(methylsulfonyl)
phenyl)pyrimidine (11), was prepared in five steps, essentially as already described [35],
starting with a Pinner reaction [36] on 4-(methylthio)benzonitrile, followed by amination,
cyclization, chlorination, and oxidation. Compound 11 was readily converted into three known
inhibitors [35], namely the two ethers, 4-chloro-6-methoxy-2-(4-(methylsulfonyl)phenyl)pyrimidine
(12) and 4-chloro-6-ethoxy-2-(4-(methylsulfonyl)phenyl)pyrimidine (13), plus the thioether
4-chloro-6-(ethylthio)-2-(4-(methylsulfonyl)phenyl)pyrimidine (14), each by treatment with the
respective alkoxide. The analogous 4-chloro-6-(2-fluoroethoxy) (15) and 4-chloro-6-thiomethyl
ethers (16) were prepared similarly in high (80%) and moderate (48%) yield, respectively.
Treatment of this set of five ethers with thiophen-2-ylmethanamine under lengthy conditions
(120 h) [35], or with our newly devised and very brief (25 min) microwave method, gave
the N-(thiophen-2-ylmethyl)pyrimidin-4-amines 17
–21 as some of the known and prospective
inhibitor targets. In two cases, the yields from the non-optimized microwave-method
matched (20) or surpassed those from the non-microwave method (21). Yields of the five
N-(thiophen-2-ylmethyl)pyrimidin-4-amines by either method ranged from moderately low (22%)
to high (80%). Treatment of 12 with benzylamine instead of thiophen-2-ylmethanamine under
either set of reaction conditions gave 33% yield of target inhibitor 22. Treatment of the dichloro
compound 11 with benzylamine under the non-microwave conditions gave inhibitor 23 in high yield
(76%). The microwave method was not tested for this compound. Likewise, treatment of 11 with
thiophen-2ylmethanamine gave ligand 25 in high yield (74%) by either method.
Treatment of 11 with potassium fluoride [37] replaced both chloro groups to give the difluoro
analogue 28 in 30% yield. Treatment of 28 with thiophen-2-ylmethanamine under microwave
conditions then gave the fluoro compound 29 in 53% yield, a slightly higher yield than obtained
under the slower non-microwave conditions (47%).
The 4-chloro compounds 23 and 25 were readily converted with cesium hydroxide under
palladium(II)-mediated conditions [38] into the respective 4-hydroxy analogues, 24 and 26, in low
but useful yields (20% and 22%, respectively). Treatment of 26 with fluoroiodomethane under

Molecules 2018, 23, 2850
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basic conditions gave the 4-fluoromethyl compound 27 in 56% yield. Finally, treatment of 25 with
iodomethane with sodium hydride in DMF gave the N-methyl derivative 30 in 46% yield.
Table 1. Physicochemical, structural and previously reported pharmacological parameters for selected
COX-2 inhibitors.
tPSA
(Å2)
Hetero- M.Wt.
HWB a IC50
Selectivity
#
R1
R2
R3
clogD
HBD
Atoms
(Da)
(nM)
vs. COX-1 b
17
18
19
20
21
22
25
27
29
30
OMe
OEt
SEt
O(CH2)2F
SMe
OMe
Cl
OCH2F
F
2-Thi c
2-Thi
2-Thi
2-Thi
2-Thi
Ph
2-Thi
2-Thi
2-Thi
2-Thi
H
H
H
H
H
H
H
H
H
2.80
2.88
3.47
3.09
3.34
2.71
2.74
2.72
2.91
3.47
2–3
80.1
80.1
70.9
80.1
70.9
80.1
70.9
80.2
70.9
62.1
<90
1
1
1
1
1
1
1
1
1
8
8
8
9
8
7
8
9
8
375
389
405
407
391
369
379
393
363
394
<500
0.4
0.3
1.2
>22,004
>33,333
>8403
5.1
1.2
>19,646
>81,300
Cl
Me
0
low
8
<9
Optimal parameter space
low nM
>100
a
b
c
HWB = Human whole blood. Data from reference [35]. 2-Thi = 2-thienyl. SEM’s of values are <15%.
2.3. COX-1 and COX-2 Inhibition Assays
Species differences in the structures of COXs are well documented, especially between rodent
and human [39]. Therefore, the choice of pre-clinical species for evaluation of prospective PET
radioligands must be made with care. We aimed to use rhesus monkey if inhibitors from the
2-(4-methylsulfonylphenyl)pyrimidine class showed very similar COX inhibitory potency between
rhesus monkey and human. The 2-(4-methylsulfonylphenyl)pyrimidines of interest were tested
for COX-1 and COX-2 inhibitory potencies using fresh monkey and human blood (Table 2). For
compounds whose potencies for inhibiting human COX-1 and COX-2 had already been reported
(17–19, 22, 24, 25) [35], our assay results were in fair agreement for rank order of inhibitory potency,
except that we obtained somewhat higher IC50 values for human COX-2 inhibitory potency. None of
the tested compounds showed monkey or human COX-1 inhibitory IC50’s below 1 nM, in accord with
the observations of Orjales et al. [35] for all compounds in this class. These compounds appear to be
too bulky for strong binding to the COX-1 isoform.
The thioethyl compound 19 and the hydroxy compound 26 showed much lower inhibitory
potency for monkey COX-2 than that reported for human COX-2 [35]. Nevertheless, in several other
cases (17, 20–22, 24, 27) inhibitory potencies for the monkey COX enzymes were found to be very close
to those for the human enzymes. Therefore, we concluded that monkey could indeed serve as a useful
preclinical model for testing candidate PET radioligands for imaging COX-2.

Molecules 2018, 23, 2850
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Scheme 1. Syntheses of known and potential inhibitors, and precursors for radiolabeling. Reagents
and conditions: (i) for 12 13, and 15, RX, NaH, THF, RT, 3 h; for 14 (R = SEt) and 16 (R = SMe): RNa,
,
◦
THF 50 C, 3 h. (ii) thiophen-2-ylmethanamine, NEt3, MeCN, 5 d. (iii) thiophen-2ylmethanamine,
Cs2CO3, DMF, 160 C, 100 W, 25 min. (iv) benzylamine, NEt3, MeCN, 5 d. (v) benzylamine, Cs2CO3,
DMF, 160 C, 100 W, 25 min. (vi) BippyPhos (8 mol %), Pd2dba3 (2 mol %), CsOH
20 h. (vii) FCH2I, K2CO3, DMF, 40 C, 4 h. (viii) NaH, DMF, MeI, RT, 24 h. (ix) KF, 18-crown-6, MeCN,
RT, 24 h.
◦
◦
◦
·
H2O, 100 C, dioxane,
◦
Among the N-(thiophen-2-ylmethyl)pyrimidin-4-amines, monkey and human COX-2 inhibitory
potency depended on compound structure in the following manner. Compounds containing an
electron-donating group in the R1 position of the general structure shown in Table 2, such as OMe (17),
OEt (18), SEt (19), OCH2CH2F (20), SMe (21), or OCH2F (27), showed high COX-2 inhibitory potencies
with IC50’s between 1 and 5 nM. Compounds containing relatively small electron withdrawing groups,
such as Cl (25) or F (29), in the R1 position also showed quite high COX-2 inhibitory potency (IC50,
3–4 nM). N-Methylation of 25 effectively abolished inhibitory potency at human COX-2. Replacement

Molecules 2018, 23, 2850
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of the 2-thienyl group of 17 with a phenyl group as in 22 resulted in decreased COX-2 inhibitory
potency. Compounds 17 20, and 27 showed the highest inhibitory potencies for COX-2 in both species.
,
The phenols, 24 and 26, which were potentially useful as precursors for radiolabeling, showed low
inhibitory potency for COX-2 [35].
2.4. Pharmacological Screening
At 10
µM concentration, inhibitors 17, 18, 27, and 30 showed less than 50% inhibition of
radioligand binding at all tested receptors, transporters, and binding sites, except 17 (50, 50.6 and
53.7% inhibition at 5-HT1B, benzodiazepine (BzP), and DAT (dopamine transporter), respectively),
and 27 (50.8% inhibition at BzP). Therefore, this class of COX-2 inhibitor was generally devoid of very
high binding affinity at any of the tested off-target sites, as is desirable for an effective PET radioligand.
2.5. Selection of Candidates for Radiolabeling
Inhibitors 17, 20, and 27 were selected from the prepared set (Table 2) for radiolabeling because
they presented the highest inhibitory potencies for human and monkey COX-2, very low inhibitory
potency for human and monkey COX-1, computed lipophilicities in a range generally acceptable for
PET radioligands, and readily accessible positions for labeling with 11C (17) or 18F (20 and 27).
Table 2. Structures and IC50’s of inhibitors 17
–
27,
29, and 30 for COX-1 and COX-2 in monkey and
human whole blood (WB).
IC50 (nM) a
R1
R2
R3
#
COX-1
COX-2
Monkey
WB
Human
WB
Monkey
WB
Human
WB
>1000
>8800 c
>10,000 d
>105 c
2.89 ± 0.15
2-Thi b
2-Thi
2-Thi
17
18
19
OMe
OEt
SEt
H
H
H
>1000
2.58 ± 0.23
0.4 c
3 d
0.3 c
>1000
3.52 ± 0.43
>1000
52.3 ± 0.01
>10,000 c
1.2 c
20
21
O(CH2)2F
SMe
2-Thi
2-Thi
H
H
1.28 e
1.9 e
>1000
>1000
>1000
>1000
>105 c
4.83 ± 2.31
14.6 ± 10.1
2.65 ± 0.45
7.35 ± 1.35
5.1 c
22
23
24
OMe
Cl
Ph
Ph
Ph
H
H
H
10.9c
>105 c
>1000 d
49.3 c
>1000
> 1000 d
OH
>1000
>1000
>105 c
>10,000 d
>105 c
4 d
25
Cl
2-Thi
H
1.2 c
79.4 c
>105 c
10 d
2.26 ± 0.45
26
27
29
30
OH
OCH2F
F
2-Thi
2-Thi
2-Thi
2-Thi
H
H
H
>1000
2.26 ± 0.63
2.5 d
> 108 d
Cl
Me
>1000
a
b
Data are for mean
±
SD from triplicate measurements, unless otherwise stated. 2-Thi = 2-thienyl c Data from
d
e
reference [35]. SE of values were <15%. n = 1. n = 2.

Molecules 2018, 23, 2850
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2.6. Radiochemistry
Three radioligands, [11C]17
,
[18F]20, and [d2-18F]27, were successfully prepared from the phenol
precursor 26 (Scheme 2). For [11C]17, the labeling agent, [11C]iodomethane was rapidly prepared from
cyclotron-produced NCA (no-carrier-added) [11C]carbon dioxide by reduction to [11C]methane and
then high temperature iodination in a commercial apparatus, as previously described [40]. Treatment
of 26 with [11C]iodomethane under basic conditions in DMF at RT for 5 min gave [11C]17 in yields
of 1.85–3.11 GBq and with molar activities of 204–492 GBq/µmol (n = 4) after HPLC purification and
formulation for sterile intravenous injection.
For [18F]20, the labeling agent [18F]2-fluoro-1-bromoethane was prepared from 2-bromoethyl
tosylate and NCA [18F]fluoride ion [41]. Treatment of 26 with [18F]2-fluoro-1-bromoethane under basic
conditions at 110 ◦C for 15 min enabled formulated [18F]20 to be obtained in 8.4% yield from starting
[18F]fluoride ion with a molar activity of 60 GBq/µmol.
For [d2-18F]27
,
the labeling agent, [d2-18F]fluorobromomethane, was prepared from
[d2]dibromomethane and NCA cyclotron-produced [18F]fluoride ion, as previously described [42].
This labeling agent was double deuterated so that any derived radioligand might resist extensive
radiodefluorination in ◦vivo
[
43]. Treatment of 26 with [d2-18F]fluorobromomethane, Cs2CO3
,
and 18crown-6 at 110 C for 15 min gave [d2-18F]27 in 27% yield after HPLC purification and
formulation for sterile intravenous injection from starting [18F]fluoride ion, and with a molar activity
of 66 GBq/µmol.
The radiochemical purities of all three radioligands exceeded 99% (Figure 1). Formulated
radioligands were free of appreciable amounts of labeling precursor (26) or derived impurities
(Figure 1). Each formulated radioligand was radiochemically stable for more than 1 h as measured
by HPLC.
2.7. Calculated and Measured LogD for Prepared Radioligands
The measured lipophilicity values (logD’s) for [11C]17 and [d2-18F]27 were higher than the
computed values by 0.94 and 0.98, respectively (Table 3). The measured value for [18F]20 was almost
the same as the computed value. All the measured values were within the logD range (2–4) observed
for many effective PET radioligands for imaging proteins in brain [44], but rather higher than might be
considered ideal.
Scheme 2. Radiosyntheses of [11C]17, [18F]20, and [18F]27. Reagents and conditions: (i) [11C]CH3I,
DMF, TBAH, RT, 5 min. (ii) [18F]FCH2CH2Br, DMF, Cs2CO3, 18-crown-6, 110 ◦C, 15 min. (iii)
◦
18F]FCD2Br, Cs2CO3, 18-crown-6, 110 C, 15 min.
[

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Figure 1. HPLC radiochromatograms for the separation of the radioligands
and [d218F]27 (panels
, and , respectively) and the respective chromatograms for HPLC analyses
of the formulated radioligands (panels B, D, and F, respectively).
[ , [ ,
11C]17 18F]20
A
,
C
E
Table 3. Computed and measured logD values for radioligands.
LogD
(n = 6)
Radioligand
cLogD
[
[
11C]17
2.80
3.09
2.72
3.74 ± 0.02
2.94 ± 0.32
3.70 ± 0.12
18F]20
[d2-18F]27
3. Experimental
3.1. General
Solvents (analytical grade) and reagents were purchased from Sigma-Aldrich (St. Louis, MO,
USA) unless stated otherwise. The products were isolated with flash chromatography on a CombiFlash
Rf+ system (Teledyne ISCO; Lincoln NE) equipped with a silica gel cartridge (12 g, RediSep®Rf; Isco:
Lincoln, NE, USA) using hexane and EtOAc as mobile phase (general method A). If necessary to achieve
purity exceeding 95%, COX-2 inhibitors were subjected to reversed phase HPLC (Beckman Coulter,

Molecules 2018, 23, 2850
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Fullerton, CA, USA) on an XBridge C18 column (19
×
150 mm, 10
µ
m; Waters; Milford, MA, USA)
eluted at 6 mL/min with a gradient mobile phase composed of aq. ammonium hydroxide (0.25 mM)
(A) and acetonitrile (B) (general method B), with eluate monitored for absorbance at 254 nm. Melting
points were determined on an SMP20 apparatus (Stuart; Staffordshire, UK). 1H (400.13 MHz), 13C NMR
(100.62 MHz), and 19F NMR (376.46 MHz) spectra were recorded on an Avance 400 instrument (Bruker;
1
Billerica, MA, USA) (see Supporting Information). H and 13C-NMR chemical shifts are reported in
δ
units (ppm) downfield relative to the chemical shift for tetramethylsilane. 19F-NMR chemical shifts
were determined using the chemical shift of trifluorotoluene ( 63.72 ppm) as a reference standard.
−
Abbreviations bs, d, m, s, and t denote broad singlet, doublet, multiplet, singlet, and triplet, respectively.
LC-MS (ESI) was performed on a Velos Pro instrument (Thermo Scientific, San Jose, CA,
USA) using reversed phase chromatography on a Luna C18 column (50
×
2 mm, 3 µm;
Phenomenex: Torrance, CA, USA) eluted at 200 L/min with a gradient mobile phase composed of
µ
water-methanolacetic acid (90:10:0.5 by vol.; A) and methanol-acetic acid (100:0.5, v/v; B), starting
with A:B at 80:20 for 0.25 min, changed linearly to 10:90 over 3 min, and then held at 10:90 for
3.5 min (method 1). HRMS data were obtained at the Mass Spectrometry Laboratory, School of
Chemical Sciences, 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), or from the Laboratory of Bioorganic Chemistry, National Institute of Diabetes
and Digestive and Kidney Diseases (Bethesda, MD, USA).
The chemical purities of all prepared inhibitors were shown to be >95% with analytical reversed
phase HPLC on a Luna C18 (2) column (250
isocratically with ammonium formate (10 mM)
Eluate was monitored for absorbance at 254 nm (see Supporting Information).
cLogD values and pKa values were computed with Pallas for Windows software version 3.8
in default option (CompuDrug International; Bal Harbor, FL, USA). tPSA’s were estimated with
ChemDraw version 10 (CambridgeSoft, Cambridge, MA, USA).
×
4.6 mm, 10
µm; Phenomenex; Torrance, CA, USA) eluted
−
acetonitrile (50:50) at 2 mL/min (general method C).
3.2. Synthesis of Target Compounds and Precursors for Radiolabeling
3.2.1. Ether Derivatives of 4,6-Dichloro-2-(4-methylsulfonylphenyl)pyrimidine
4,6-Dichloro-2-(4-methylsulfonylphenyl)pyrimidine
(
11
)
was prepared in five steps from
4-(methylthio)benzonitrile and converted into the ethers 4-chloro-6-methoxy-2-(4-(methylsulfonyl)
phenyl)pyrimidine (12) and 4-chloro-6-ethoxy-2-(4-(methylsulfonyl)phenyl)pyrimidine (13), and the
thioether 4-chloro-6-(ethylthio)-2-(4(methylsulfonyl)phenyl)pyrimidine (14), all according to literature
methods [35].
4-Chloro-6-(2-fluoroethoxy)-2-(4-(methylsulfonyl)phenyl)pyrimidine (15).
0.659 mmol), sodium hydride (25 mg, 1.04 mmol) and 2-fluoroethanol (41.5
Compound 11 (200 mg,
L, 0.715 mmol) in
µ
THF (1.5 mL) was added to an oven-dried pressure tube and kept at RT (room temperature) for 3 h.
The solvent was evaporated off under reduced pressure. The residue was quenched with water (5 mL)
and extracted with EtOAc (5 mL
×
3). The organic layers were combined, washed with brine-water
(1:1 v/v: 15 mL 2), dried (MgSO4) and concentrated under reduced pressure. The residue was
×
purified with flash chromatography (general method A) to give crude product (176 mg, 80%). This
product was further purified with reversed phase HPLC (general method B) using a gradient mobile
phase composed of aq. ammonium hydroxide (0.25 mM) (C) and acetonitrile (D), starting at 20% D
and rising linearly to 95% D at 60 min to give 15 as an off-white solid; mp: 158.5–160.1 ◦C; 1H NMR
(CDCl3):
δ 3.11 (s, 3H), 4.82 (s, 2H), 4.84 (m, 1H), 4.88 (m, 1H), 6.83 (s, 1H), 8.04 (d, J = 8.65 Hz, 2H), 8.59
(d, J = 8.68 Hz, 2H); 13C NMR (CDCl3):
δ
170.02, 162.56, 161.59, 142.75, 140.90, 129.37, 127.59, 127.12,
106.76, 82.01, 80.30, 66.29, 66.08, 55.40, 44.47; 19F NMR (CDCl3): δ −174.89; HRMS (ESI) m/z [M + H]+:
calcd. for: C13H12ClFN2O3S: 331.0319, found 331.0321 (Supplementary Material).

Molecules 2018, 23, 2850
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4-Chloro-6-(methylthio)-2-(4-(methylsulfonyl)phenyl)pyrimidine) (16). Sodium methanethiolate (36.4 mg,
0.519 mmol) and THF (6.0 mL) were loaded into a Schlenk tube (25-mL volume) under argon. A solution
of 11 (166 mg, 0.547 mmol) in THF (1.0 mL) was then added dropwise. The Schlenk tube was sealed
◦
and heated at 50 C for 3 h. The mixture was then quenched with water (10 mL), extracted with
EtOAc (10 mL
×
3) and washed with aq. NaHCO3 (0.1 M; 10 mL). The product was dried (MgSO4)
and purified with flash chromatography (general method A; 15–80% ethyl acetate in hexane) to give
16 as a white solid (82.8 mg, 48%); mp: 160.2–161.9 C; 1H NMR (CDCl3):
δ
= 2.69 (s, 3H), 3.11 (s,
= 173.07,
◦
3H), 7.18 (s, 1H), 8.05 (d, J = 8.52 Hz, 2H), 8.64 (d, J = 8.52 Hz, 2H); 13C NMR (CDCl3):
δ
159.75, 142.66, 143.58, 141.11, 129.44, 127.60, 116.51, 44.49, 12.95; HRMS (ESI): m/z [M + H]+: calcd. for
C12H11ClN2O2S 315.0029, found 315.0025.
3.2.2. Conversion of 4-Halo-2-(4-(methylsulfonyl)phenyl)-pyrimidine Derivatives into
4-Alkylamino Derivatives
General procedure A (non-microwave method) (GP1). This method was based on that in reference [35],
as follows. A mixture of the appropriate 4-halo-2-(4-(methylsulfonyl)phenyl)-pyrimidine derivative (1
mmol) with Et3N (2–4 mmol) and the corresponding amine (2–4 mmol) in MeCN (7 mL) was stirred at
RT for 5 d. Solvent was then removed under vacuum and the resulting residue purified with flash
chromatography (general method A; 20–80% ethyl acetate in hexane). If needed, compounds were
further purified to >95% purity with HPLC.
General procedure B (fast microwave method) (GP2). The appropriate 4-halo-2-(4-(methylsulfonyl)
phenyl)-pyrimidine (1 mmol), and corresponding amine (0.2–1 mmol), Cs2CO3 (30 mmol %), and DMF
(1.00 mL) were added to a 10-mL microw◦ave vial (Discover; CEM, NC) containing a stirrer bar.
The sealed vial was irradiated (100 W) at 160 C for 25 min. The vial contents were extracted with water
(10 mL) and EtOAc (10 mL
×
3), and then washed with aq. NaHCO3 (0.1 M; 10 mL). The combined
organic layers were dried (silica gel) and the product isolated with flash chromatography (general
method A; 20–80% ethyl acetate in hexane). If needed, compounds were further purified to >95% purity
with reversed phase HPLC (general method B) on an XBridge C18 column using a gradient mobile
phase composed of aq. ammonium hydroxide (0.25 mM) (C) and acetonitrile (D), starting at 20% D
rising linearly to 95% D at 60 min.
6-Methoxy-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (17). GP2 on 12
(0.209 mmol; 62.6 mg) and thiophen-2-ylmethanamine gave 17 as an off-white solid (22 mg; 29%); mp
139.0–141.0 ◦C; lit. mp 120.0–124.1 ◦C [35]; 1H and 13C-NMR data matched those in the literature [35];
HPLC (general method C; tR = 11.5 min; purity 99.4%). From GP1 (55%).
6-Ethoxy-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (18). GP1 on 13 and
◦
thiophen-2-ylmethanamine gave 18 as a white solid (68.3 mg; 64%); mp 169.2–170.4 C; lit. mp
160–162.2 ◦C [35]; 1H and 13C-NMR data matched those in the literature [35]; HPLC (general method
C; tR = 12.8 min, purity 99.4%). From GP2 (22%).
6-Thioethyl-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (19). GP1◦on 14 and
thiophen-2-ylmethanamine gave 19 as a white solid (111 mg, 80%); mp: 160.0–162.1 C; lit.mp
◦
1
152.0–153.9 C [35]; H and 13C-NMR data matched those in the literature [35]; HPLC (general
method C; tR = 4.5 min; purity 99.3%). From GP2 (55%).
6-(2-Fluoroethoxy)-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (20). GP2 on 15
and thiophen-2-ylmethanamine gave 20 as a white solid (56 mg; 62%); mp: 147.7–149.2 ◦C; 1H NMR
(CDCl3):
6.96–6.99 (m, 1H), 7.04 (d, J = 2.73 Hz,1H), 7.23 (dd, J = 1.04, 4.04 Hz, 1H), 8.00 (d, J = 8.52 Hz, 2H), 8.57
(d, J = 8.49 Hz, 2H); 13C NMR (CDCl3):
= 170.02, 162.56, 161.59, 142.79, 140.90, 129.37, 127.59, 127.12,
106.76, 55.40, 44.47; 19F NMR (CDCl3): 75.78. MS (ESI): m/z (%) [M + H]+ 408.1 (100); HRMS (ESI)
δ = 3.10 (s, 3H), 4.66–4.68 (m, 1H), 44.71–4.77 (m, 4H), 4.88 (m, 1H), 5.36 (b, s, 1H), 5.78 (s, 1H),
δ
δ
=
−

Molecules 2018, 23, 2850
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m/z [M + H]+: calcd. for C18H18FN3O3S2: 408.0852, found 408.0856; HPLC (general method C; tR =
12.3 min; purity 99.5%). From GP1 (62%).
6-Thiomethyl-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (21). GP2 on 16 and
◦
thiophen-2-ylmethanamine gave 21 as an off-white solid (49.0 mg, 79%); mp: 159.1–160.4 C; 1H NMR
(CDCl3):
δ 2.58 (s, 3H), 3.08 (s, 3H), 4.81 (d, J = 5.45 Hz, 2H), 5.24 (br, s, 1H) 6.21 (s, 1H), 6.97–6.98 (m, 1H),
7.03–7.04 (m, 1H), 7.24 (dd, J = 1.2, 5.12 Hz, 1H), 8.00 (d, J = 8.6 Hz, 2H), 8.62 (d, J = 4.28 Hz, 2H); 13
C
NMR (CDCl3):
δ 169.10, 161.23, 161.11, 143.12, 141.08, 129.12, 127.26, 126.94, 125.87, 125.31, 44.56, 40.36,
30.94, 12.71; HRMS (ESI): m/z [M + H]+ calcd. for C17H17N3O2S3: 392.0561, found 392.0559; HPLC
(general method C; tR = 15.1 min, purity 99.2%). From GP1 (56%).
N-Benzyl-6-methoxy-2-(4-(methylsulfonyl)phenyl)pyrimidin-4-amine (22). GP2 on 12 and benzylamine
gave 22 as a white solid (33% yield): mp 133.4–134.6 ◦C; lit. mp 124.0–126.5 ◦C [35]; 1H and 13C-NMR
data matched those in the literature [35]; HPLC (general method C; tR = 13.6 min; purity 99.5%). From
GP1 (33%).
N-Benzyl-6-chloro-2-(4-(methylsulfonyl)phenyl)pyrimidin-4-amine (23). GP1 on 11 and benzylamine gave
23 as a white solid (76% yield): mp 130.4–131.4 ◦C; lit. mp 144.0–145.7 ◦C [35]; 1H and 13C-NMR data
matched those in the literature [35].
General procedure for conversion of 4-chloropyrimidine derivatives into 4-hydroxypyrimidine derivatives (GP3).
The appropriate 4-chloropyrimidine derivative (1 mmol), Pd2dba3 (2 mol%), Bippyphos (8 mol%),
CsOH
·
H2O (3 mmol), 1,4-dioxane (2 mL), and a stirrer bar were loaded into an oven-dried pressure-cap
◦
vial. The vial was sealed under argon. The contents were stirred vigorously at 100 C for 20 h and then
allowed to cool. The solution was made acidic (pH 3) with hydrochloric acid (2 M), diluted with water
(5 mL), extracted with EtOAc (5 mL
×
3), and dried (MgSO4). Product was then isolated with HPLC.
6-(Benzylamino)-2-(4-(methylsulfonyl)phenyl)pyrimidin-4-ol (24). GP3 gave 24 as a white solid (20.0 mg,
20%): mp: 287.9–289.2 ◦C; lit. mp > 265 ◦C [35]; 1H NMR matched those in the literature [35]; m/z (%)
[M + H]+ 356.1 (100); HPLC (general method C; tR = 2.8 min; purity 99.1%).
6-Chloro-2-(4-(methylsulfonyl)phenyl)-N-(thio◦phen-2-ylmethyl)pyrimidin-4-amine (25). GP2 gave 25 as
◦
1
a white solid (50 g; 74%); mp 170.0–171.2 C; lit. mp 167.0–169.1 C [35]; H and 13C-NMR data
matched those in the literature [35]; HPLC (general method C; tR = 12.8 min; purity 99.9%). From
GP1 (74%).
2-(4-(Methylsulfonyl)phenyl)-6-(thiophen-2-yl-methylamino)pyrimidin-4-ol (26). GP3 gave 26 as a white
◦
solid (38.3 mg, 22%): mp 261.7–263.2; lit. mp 261.0–267.4 C [35]; 1H and 13C-NMR data matched those
in the literature [35]; HPLC (general method C; tR = 2.7 min; purity 99.6%).
6-(2-Fluoromethoxy)-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-yl-methyl)pyrimidin-4-amine
Compound 26 (66.6 mg, 0.184 mmol), K2CO3 (67.5 mg, 0.221 mmol), fluoroiodomethane (23.2
(
27).
L,
µ
0.343 mmol) and DMF (1 mL) were added to an oven-dried pressure tube. The mixture was stirred at
40 ◦C for 4 h and then the solvent was evaporated off under reduced pressure. Water (10 mL) was
added to the residue and the mixture extracted with EtOAc (10 mL
were washed with brine-water (1:1 v/v: 20 mL 2), dried (MgSO4), and concentrated under reduced
pressure. Flash chromatography (general method A) of the residue gave 27 as a white solid (41.2 g,
×
3). The combined organic layers
×
◦
56%); mp: 176.6–178.2 C; 1H NMR (CDCl3):
δ = 3.06 (s, 3H), 4.81 (br, s, 2H), 5.42 (br, s, 1H), 5.84 (s,1H),
6.08 (s, 1H), 6.21 (s, 1H), 6.98 (t, J = 4.92 Hz,1H), 7.05 (d, J = 2.8 Hz,1H), 7.24 (d, J = 7.56 Hz, 1H), 8.01 (d,
J = 8.44 Hz, 2H), 8.60 (d, J = 8.36 Hz, 2H); 13C NMR (CDCl3): δ = 168.10, 164.49, 161.63, 142.48, 141.82,
129.16, 127.32, 126.98, 125.91, 125.36, 96.56, 94.37, 44.53, 40.67; 19F NMR (CDCl3):
δ
=
−
155.09; HRMS
(ESI) m/z [M + H]+ calcd. for C17H16FN3O3S2: 394.0684, found: 394.0686; HPLC (general method C;
tR = 11.9 min; purity 99.0%).

Molecules 2018, 23, 2850
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4,6-Difluoro-2-(4-(methylsulfonyl)phenyl)pyrimidine (28). Compound 11 (120 mg, 1.39 mmol), potassium
fluoride (80.5 mg, 0.39 mmol), and 18-crown-6 (366 mg, 1.39 mmol) in MeCN (2 mL) were added
to an oven-dried screw-cap tube under argon. The mixture was stirred at RT for 24 h, quenched
with water (10 mL), and extracted EtOAc (10 mL
with brine-water (1:1 v/v: 30 mL 2) and the solvent was evaporated off under reduced pressure.
The residue was purified with flash chromatography (general method A) to give 28 as an off-white solid
(32 mg, 30%); mp: 164.5–166.2 ◦C; 1H NMR (CDCl3):
= 3.11 (s, 3H), 6.57 (s, 1H), 8.08 (d, J = 6.22 Hz,
= 173.69, 171.12, 163.88, 143.58, 139.45, 129.67, 127.82,
54.91; HRMS (ESI): m/z [M + H]+ calcd. for C11H8F2N2O2S:
×
3). The combined organic layers were washed
×
δ
2H), 8.63 (d, J = 6.22 Hz, 2H); 13C NMR (CDCl3):
δ
91.50 (t), 44.41; 19F NMR (CDCl3):
271.0353, found 271.0347.
δ
=
−
6-Fluoro-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine (29). Microwave reaction
conditions in GP2 when applied to 28 with thiophen-2-ylmethanamine gave 29 as a white solid (53%);
◦
mp: 130.4–131.4 C; 1H NMR (CDCl3):
δ
= 3.09 (s, 3H), 4.85 (s, b, 2H), 5.65 (s, b, 1H), 5.91 (s, 1H),
6.98–7.01 (m, 1H), 7.05 (s, 1H), 7.25 (d, J = 2.74 Hz,1H), 8.02 (d, J = 8.52 Hz, 2H), 8.58 (d, J = 8.37
Hz, 2H); 13C NMR (CDCl3):
= 169.86, 165.53, 162.86, 162.68, 142.20, 141.73, 140.34, 129.27, 127.41,
127.05, 126.17, 125.57, 44.50, 40.73; 19F NMR (CDCl3): 65.17; HRMS (ESI): m/z [M + H]+ calcd.
δ
δ
=
−
for C16H14FN3O2S2: 364.0590, found 364.0585; HPLC (general method C; tR = 3.9 min; purity 99.9%).
From 28 (32 mg, 0.118 mmol) with GP1 (47%).
6-Chloro-N-methyl-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine
(30).
Compound 25 (270 mg, 0.071 mmol), sodium hydride, (4.3 mg, 0.178 mmol; 95% dry) and
DMF (1.0 mL) were added to a Schlenk tube (10-mL volume) under argon and left for 1 h. Iodomethane
(6.88
evaporated to dryness and water (10 mL) was added. The mixture was then extracted with EtOAc
(10 mL 2). The combined organic layers were washed with brine-water (1:1 v/v: 20 mL 2) and
dried (MgSO4). The residue was purified with flash chromatography (general method A; 20–80% ethyl
acetate in hexane; to give 30 as a white solid (13 mg, 46%); mp: 149.9–151.9 ◦C; 1H NMR (CDCl3):
3.08 (s, 3H), 3.12 (s, 3H), 5.08 (br, s 2H), 6.45 (s, 1H), 6.95–6.97 (m, 1H), 7.03 (d, 1H, J = 2.00 Hz), 7.24 (d,
1H, J = 4.96 Hz), 8.00 (d, 2H, J = 8.44 Hz), 8.63 (d, 2H, J = 8.36 Hz); 13C NMR (CDCl3):
= 162.63, 161.91,
µL, 0.111 mmol) was then added and the tube closed. After 24 h, the tube contents were
×
×
δ
=
δ
160.67, 142.16, 142.06, 139.20, 129.33, 127.35, 126.74, 126.42, 125.66, 100.27, 47.95, 44.51 35.44. HRMS
(ESI) m/z [M + H]+ calcd. for C17H16ClN3O2S2: 394.0451; found 394.0445. HPLC (general method C;
tR = 3.4 min, purity 99.9%).
3.3. COX-1 and COX-2 Inhibition Assays
Thromboxane B2 EIA kit (COX-1) and Prostaglandin E2 EIA Kit (COX-2) were purchased from
Cayman Chemicals, USA [45]. Blood from non-treated rhesus monkey and healthy human subjects
was used with these ELISA kits to establish the inhibitory potencies (IC50’s) of test compounds for
these enzymes [46]. Thromboxane, the product of COX-1, and prostaglandin E2, the product of COX2,
were measured using 96-well plates and a UV spectrophotometer. The concentration-inhibition curves
were fitted into a 4-parameter non-linear regression model in GraphPad Prism to calculate the IC50 of
each test compound.
3.4. Pharmacological Screening
Inhibitors 17
Drug Screening Program (NIMH-PDSP) for assay of binding affinities to a wide range of human
recombinant receptors (BZP, -opiate, M1, M2, M3, M4, M5, -opiate, 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1e,
5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7, α1A α1B α1D α2A α2B α2C β1 β2 β3,
, 18, 27, and 30 were submitted to the National Institute of Mental Health Psychoactive
κ
κ
,
,
,
,
,
,
,
,
and σ2), σ1 receptor (guinea pig), GABAA receptor (rat brain), transporters (DAT, noradrenalin,
and serotonin), binding sites (PBR and the rat brain benzodiazepine site). Detailed assay protocols are
available at the NIMH-PDSP web site (https://pdspdb.unc.edu/pdspWeb/).

Molecules 2018, 23, 2850
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3.5. Radiochemistry
3.5.1. Radiochemistry Materials and Methods
Radioactivity from carbon-11 or fluorine-18 was measured with a calibrated dose calibrator
(Atomlab 300, Biodex Medical Systems, Shirley, NY, USA) or an automatic -counter (Wizard 3”,
γ
1480; PerkinElmer; Waltham, MA, USA). Radioactivity measurements were corrected for physical
decay. Radiochemistry was executed in lead-shielded hot-cells for personal protection from radiation.
Radioligands were separated with reversed phase HPLC (Beckman Coulter, Fullerton, CA, USA)
on a Luna (C-18/2) column (10
were identified and measured for radiochemical purity with reversed phase HPLC (Beckman Coulter,
Fullerton, CA, USA) on a Luna C18(2) column (4.6 250 mm, 10 m). All HPLC column eluates were
×
250 mm, 10 µm; Phenomenex; Torrance, CA, USA). Radioligands
×
µ
monitored for radioactivity and absorbance at 254 nm. Absorbance responses for analytical HPLC
systems were calibrated for mass of injectates to enable molar activities [47] to be measured.
3.5.2. Production of NCA [11C]Carbon Dioxide
NCA [11C]carbon dioxide (~85 GBq) was produced with a PETtrace cyclotron (GE Medical
α
Systems; Milwaukee, WI, USA) according to the 14N(p, )11C reaction [48] by irradiation of nitrogen
gas (initial pressure, 160, psi; 75 mL volume) containing 1% oxygen with a proton beam (16.5 MeV,
45 µA) for 40 min.
3.5.3. Production of [11C]Iodomethane
[11C]Iodomethane was produced from [11C]carbon dioxide via reduction to [11C]methane
followed by vapor phase iodination [40]. Thus, at the end of proton irradiation, [11C]carbon dioxide
was delivered to a PETtrace MeI Process Module (GE Medical Systems; Severna Park, MD, USA)
through stainless tubing (OD 1/8 in, ID 1/16 in) over 3 min. The [11C]carbon dioxide was reduced
to [11C]methane with hydrogen over heated nickel (360 ◦C) and trapped on molecular sieves (13X).
Finally, the [11C]methane was recirculated over iodine at 720 ◦C to generate [11C]iodomethane, which
was trapped on Porapak Q held in the recirculation path.
3.5.4. Radiosynthesis of [11C]17
The phenol precursor 26 (1.01 mg, 2
µmol) was dissolved in DMF (80 µL) at 7 min before the
end of radionuclide production. The solution was sonicated and then TBAH in methanol (1 M,
3.0 µL) was added. This solution was loaded into the loop (constructed of stainless steel tubing) of
an AutoLoop apparatus (Bioscan; Washington, DC, USA). [11C]Iodomethane was then released into the
loop and held for 5 min at RT. [11C]17 was isolated with reversed phase HPLC on a Luna C18 column
(10
×
250 mm, 10 µm) eluted with MeCN-water (55:45 v/v) at 6 mL/min (tR = 9.9 min) (Figure 1A).
The◦product solution was taken to dryness by rotary evaporation under reduced pressure and heat
(80 C). The radioactive residue was formulated in sterile physiological saline (3 mL) containing
ethanol (1 mL) plus Tween 80 (12 mg; J.T. Baker, Phillipsburg, NJ, USA), and sterile-filtered (0.2 µm,
Millex-MP, 25 mm; Millipore, Billerica, MA, USA) into a sterile, pyrogen-free dose vial containing
saline (6 mL).
The identity of [11C]17 (tR = 5.4 min) was confirmed with reversed phase HPLC on a Luna C18
column (4.6
×
250 mm, 10 µm) eluted with MeCN-water (60:40 v/v) at 2 mL/min (Figure 1B), and with
LC-MS (ESI) (method 1) of associated carrier [tR = 4.85 min (m/z) [M + H]+: 376]. The radiochemical
purity of [11C]17 exceeded 99%. [11C]17 was obtained in yields of 1.9–3.1 GBq with a molar activity of
204–492 GBq/µmol (n = 4).

Molecules 2018, 23, 2850
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3.5.5. Production of NCA [18F]Fluoride Ion
NCA [18F]fluoride ion was produced with the 18O(p,n)18F reaction by irradiating 18O-enriched
water (95 atom %; 1.8 mL) with a proton beam (14.1 MeV; 20–25 µA) from a PETtrace cyclotron.
3.5.6. Radiosynthesis of [18F]20
For radioligand production, cyclotron-produced [18F]fluoride ion (~7.4 GBq) in [18O]water
(0.4–0.65 mL) was delivered into a glass vial containing K 2.2.2 (5.0 mg, 13.3
µmol) and potassium
carbonate (0.50 mg, 3.6 mol) in MeCN-H2O (9:1 v/v; 0.1 mL). This solution was transferred to
µ
a modified version [42] of a TRACERlab FXFN module and diluted with MeCN (2 mL). The mixture
◦
was evaporated to dryness at 90 C under reduced pressure with a nitrogen bleed. MeCN (2 mL)
was again added and then evaporated to dryness. 2-Bromoethyltosylate (30
µL, 161 µmol) in
1,2dichlorobenzene-t-butanol (9:1 v/v, 1.0 mL) was added to the dry [18F]fluoride ion-K 2.2.2-K2CO3
complex, sealed and then heated at 95 ◦C for 15 min. The reaction vessel was then cooled to 35 ◦C.
Nitrogen was used to carry the volatile [18F]2-fluorobromoethane through a silica gel SepPak Plus
cartridge (Waters; Milford, MA, USA) and into a septum-sealed vial (1 mL; Alltech, Deerfield, IL, USA)
containing phenol precursor 26 (0.5 mg, 1
µmol), Cs2CO3 (~2 mg, 6.1 µmol), and 18-crown-6 (~5 mg,
18.9
µ
mol) in DMF (0.5 mL). The vial was then heated at 110 ◦C for 15 min. [18F]20 was separated
with reversed phase HPLC on a Luna C18 column (10
×
250 mm, 10 µm) eluted with MeCN-H2O
(40:60 v/v) at 6 mL/min (tR = 49.7 min) (Figure 1C). The [18F]20 was captured on a C18 Sep-Pak
Plus cartridge (Waters), washed with water (5 mL), eluted with ethanol (1 mL) and saline (2 mL)
containing Tween 80 (12 mg; J.T. Baker), and sterile-filtered (0.2 µm, Millex-MP, 25 mm; Millipore,
Billerica, MA, USA) into a sterile, pyrogen-free dose vial containing saline (7 mL). The identity and
purity of [18F]20 (tR = 10.1 min) were confirmed with reversed phase HPLC on a Luna C18 column
(4.6
×
250 mm, 10 µ
m) eluted with MeCN-water (50:50 v/v) at 2 mL/min (Figure 1D). [18F]20 was
obtained in 8.4% yield from [18F]fluoride ion with radiochemical purity exceeding 99% and a molar
activity of 60 GBq/µmol.
3.5.7. Radiosynthesis of [d2-18F]27
For radioligand production, cyclotron-produced [18F]fluoride ion (~7.4 GBq) in [18O]water
(0.4–0.65 mL) was delivered into a glass vial containing K 2.2.2 (5.0 mg, 13.3
µmol) and potassium
carbonate (0.50 mg, 3.6 mol) in MeCN-H2O (0.1 mL, 9:1 v/v). This solution was transferred
µ
to a modified version [42] of a TRACERlab FXFN module (GE) and diluted with MeCN (2 mL).
The mixture was evaporated to dryness at 90 ◦C under reduced pressure with a nitrogen flow. MeCN
(2 mL) was again added and evaporated to dryness. The vessel was sealed. Then CD2Br2 (100 µL)
in MeCN (1.0 mL) was added to the dry [18F]fluoride ion-K 2.2.2-K2CO3 complex, which was then
heated at 95 ◦C for 15 min. The vessel was then cooled to 35 ◦C. Nitrogen was used to transfer the
volatile [d2-18F]fluorobromomethane through a series of four silica gel SepPak Plus cartridges (Waters;
Milford, MA) into a septum-sealed 1-mL vial (Alltech, Deerfield, IL, USA) containing phenol precursor
26 (0.5 mg, 1
µ
mol), Cs2CO3 (~2 mg, 6.1◦µmol) and 18-crown-6 (~5 mg, 18.9
µmol) in DMF (0.5 mL).
The vial was sealed and heated at 110 C for 15 min. The [d2-18F]27 was separated with reversed
phase HPLC on a Luna C18 column (10
×
250 mm, 10 µm) eluted with MeCN-water (40:60 v/v) at
6 mL/min (tR = 45 min) (Figure 1E). The [d2-18F]27 was captured on a C18 Sep-Pak Plus cartridge
(Waters), washed with water (5 mL), eluted with ethanol (1 mL) and saline (2 mL) containing Tween 80
(12 mg), and sterile-filtered (0.2
µm, Millex-MP, 25 mm) into a sterile, pyrogen-free dose vial containing
saline (7 mL). The identity of the [d2-18F]27 was confirmed with reversed phase HPLC on a Luna C18
column (4.6
×
250 mm) eluted with MeCN-water (50:50 v/v) at 2 mL/min (tR = 9 min) (Figure 1F).
[d2-18F]27 was obtained in 27%yield from [18F]fluoride ion with radiochemical purity exceeding 99%
and a molar activity of 66 GBq/µmol).

Molecules 2018, 23, 2850
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3.6. LogD Determinations
cLogD (at pH 7.4) values were computed with Pallas for Windows software version 3.8 in
default mode (CompuDrug International; Bal Harbor, FL, USA). The logD values of [11C]17 [18F]20
, ,
and [d2-18F]27 were measured by a method that we have described previously [49]. The radioactivity
contents in the n-octanol and sodium phosphate buffer (0.1 M; pH 7.4) phases were determined with
a γ-counter.
4. Conclusions
Three COX-2 inhibitors, 17, 20, and 27, were identified from the 2-(4-methylsulfonylphenyl)-
pyrimidine class as having promising physiochemical and pharmacological properties for development
as PET radioligands for imaging COX-2 in monkey and human in vivo. Methods were established for
satisfactorily labeling these ligands with positron-emitters for comparative PET imaging in monkey
in vivo, including a neuroinflammation model [50]. These results will be published in detail elsewhere.
Supplementary Materials: NMR spectra and HPLC analyses of synthesis intermediates and new
COX-2 inhibitors.
Author Contributions: M.Y.C.-S., P.S. and C.L.M. performed chemistry and radiochemistry. S.S., M.Y.C.-S. S.S.Z.,
K.J.J. and J.A.M.S. performed binding assays. S.S.Z. and R.B.I. provided oversight on binding assays. K.J.J. and
S.S.Z. performed logD determinations. V.W.P. conceived and supervised the project. All authors contributed to
review and writing of the paper (overseen by V.W.P.).
Funding: These studies were supported by the Intramural Research Program of the National Institute of Health
(NIH), project numbers [ZIA-MH002793] and [ZIA-MH002795].
Acknowledgments: We are grateful to the NIH Clinical Center PET Department (Chief; Peter Herscovitch) for
regular supply of carbon-11 and fluorine-18.
Conflicts of Interest: The authors declare no competing financial interest.
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Sample Availability: Samples of the compounds are all available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).