Synthesis of three 18F-labelled cyclooxygenase-2 (COX-2) inhibitors based on a pyrimidine scaffold

Article abstract of DOI:10.1039/c3ob41935e

Cyclooxygenase (COX) is the key enzyme within the complex conversion of arachidonic acid into prostaglandins (PGs). Inhibitors of this enzyme represent a particularly promising class of compounds for chemoprevention and cancer therapy. The experimental da

Full text of DOI:10.1039/c3ob41935e

Organic &
Biomolecular Chemistry
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Synthesis of three 18F-labelled cyclooxygenase-2
(COX-2) inhibitors based on a pyrimidine scaffold
Cite this: Org. Biomol. Chem., 2013, 11,
8052
Ole Tietz,a Sai Kiran Sharma,a,b Jatinder Kaur,a Jenilee Way,a Alison Marshall,a
Melinda Wuesta and Frank Wuest*a,b
Cyclooxygenase (COX) is the key enzyme within the complex conversion of arachidonic acid into prosta-
glandins (PGs). Inhibitors of this enzyme represent a particularly promising class of compounds for
chemoprevention and cancer therapy. The experimental data on the involvement of COX isoform COX-2
in tumour development and progression, as well as the observed overexpression of COX-2 in a variety of
human cancers provide the rationale for targeting COX-2 for molecular imaging and therapy of cancer. A
series of trifluoromethyl-substituted pyrimidines was prepared as a novel class of selective COX-2 inhibi-
tors, based on the lead structure 1a. All compounds were tested in cyclooxygenase (COX) assays in vitro
to determine COX-1 and COX-2 inhibitory potency and selectivity. Molecular docking studies using the
catalytic site of COX-1 and COX-2, respectively, provided complementary theoretical support for the
obtained experimental biological structure–activity relationship data of three highly potent and selective
fluorobenzyl-containing COX-2 inhibitors. Selected fluorobenzyl-substituted pyrimidine derivatives were
further developed as 18F-labelled radiotracers ([18F]1a, [18F]2a, [18F]3a). Radiotracers [18F]1a and [18F]2a
were radiolabelled using 4-[18F]fluorobenzylamine ([18F]FBA) as a building block. Radiotracer [18F]3a was
radiofluorinated directly using a nucleophilic aromatic substitution reaction with no-carrier-added (n.c.a.)
Received 23rd September 2013,
Accepted 26th September 2013
DOI: 10.1039/c3ob41935e
www.rsc.org/obc
[
18F]fluoride on an iodylaryl compound as a labelling precursor.
The conformations of the two COX isoforms are very
similar, as are the amino acid sequences that constitute the
Introduction
Cyclooxygenases (COXs) control the complex conversion of substrate-binding pockets.3 Given these similarities, the devel-
arachidonic acid (AA) into prostaglandins and thromboxanes, opment of COX-2 selective inhibitors has been a great chal-
which function as locally active messenger molecules and lenge.4 Since the identification of the COX-2 isoform in the
trigger important physiological and pathophysiological pro- early 1990s, a number of selective inhibitors have been develo-
cesses. The COX enzyme family consists of a constitutively ped. A selection of these molecules has been used extensively
expressed isoform (COX-1) and an inducible isoform (COX-2). in clinical application. In 2005, most of these drugs were with-
Recently, a third isoform, believed to be a COX-1 splice drawn from the market following concerns over their cardiac
variant, has been reported as COX-3.1 Both COX-1 and COX-2 safety profile.
convert arachidonic acid to prostaglandin H2. In the first step
Among the most widely used selective COX-2 inhibitors
of the reaction COXs cyclise AA to prostaglandin G2 which (coxibs) were celecoxib, rofecoxib and valdecoxib.5 More
then is rapidly converted into PGH2 by a peroxidase reaction at recently, the underlining biochemical mechanisms of the
a second catalytic site in the same enzyme. Most COX inhibi- cardiac toxicity of the coxib compound class are starting to be
tors derive their therapeutic potential by blocking the first unravelled.6,7
catalytic site of the COX enzymes. PGH2 is converted to a
COX-1 functions as a housekeeping enzyme and is
variety of different prostaglandins by a number of downstream expressed in most resting tissues; its responsibilities include
enzymes. These prostaglandins mediate various physiological the maintenance of gastric and renal integrity.8 COX-2 is
processes by binding to G-protein coupled receptors.2
expressed in the resting tissue of the brain and the kidney, but
virtually absent in all other tissue types.9,10 COX-2 expression
is induced in response to various acute and chronic inflamma-
tory conditions. Expression can be triggered in fibroblast, epi-
thelial, endothelial, macrophage, and smooth muscle cells in
response to growth factors, cytokines, and pro-inflammatory
stimuli.11 Several studies have suggested that COX-2 is also
aDepartment of Oncology, University of Alberta, Edmonton, Canada.
E-mail: wuest@ualberta.ca
bFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton,
Canada
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phenyl)-6-(trifluoromethyl)pyrimidine 6 as a labelling pre-
cursor.
The synthetic strategy for the preparation of compound 6
follows the route outlined by Swarbrick et al.35 (Fig. 2). Claisen
condensation of 1-(4-(methylthio)-phenyl)ethanone with ethyl
trifluoroacetate yielded trifluoro-substituted dione 4 in a high
yield of 92%. Pyrimidine ring formation using condensation
with S-methylisothiourea afforded compound 5 in almost
quantitative yield. Methylthioether groups in 5 were oxidized
using Oxone to afford compound 6 in overall yield of 58% for
the three step reaction sequence.
Fig. 1 Structures of radiotracers [18F]1a, [18F]2a and [18F]3a.
COX-2 inhibitors 1a–p were prepared by heating corres-
ponding primary amines and in the presence of compound 6
in acetonitrile in sealed vials at 140 °C. Methylsulfonyl group
in the 2-position of the pyrimidine ring acts as a good leaving
group upon attack with primary amines. Upon completion of
the reaction, excess of compound 6 and amine could easily be
removed by diluting the mixture with hydrochloric acid. The
desired product precipitated and was collected by filtration.
Impurities were removed using purification with column
chromatography. Syntheses using benzylamine hydrochloride
salts, such as 4-nitrobenzylamine, were carried out by adding
triethylamine as the auxiliary base.
To synthesize compound 3a, it was necessary to replace the
secondary amine linker with an ether moiety. To this end,
4-fluorobenzyl alcohol and sodium hydride were dissolved in
dry THF under a nitrogen atmosphere, and the mixture was
cooled to 0 °C. Addition of compound 6 allowed for the for-
mation of fluorobenzyl ether compound 3a (Fig. 2). This com-
pound has been reported in a patent by the Glaxo Group.36
However, to the best of our knowledge, COX-2 potency and
selectivity of this substance has not been published to date.
The synthesis of methylsulfones 1a–j, 1k–p, and 3a are
depicted in Fig. 2.
involved in neurodegenerative diseases such as Parkinson’s
and Alzheimer’s.12 Elevated COX-2 expression has furthermore
been found in a variety of human cancers, most prominently
colorectal, gastric, and breast cancers.13–15 Some of the key sig-
nalling pathways involving effects of COX-2 in inflammation
and tumorigenesis have been dissected.16 There are, however,
discrepancies between potent anticancer effects of COX-2
inhibitors in vitro and their failure in the majority of clinical
trials.17
The role that the COX-2 enzyme plays in the development
and progression of various diseases appears to be very complex
and requires more basic research on COX-2 pharmacology.
An exact and accurate assessment of COX-2 expression
levels and activity in tissues under different disease conditions
is of vital importance to these efforts. To date, an exact assess-
ment of COX-2 expression can only be achieved by ex vivo ana-
lysis. This type of analysis is relatively complex due to the
instability of COX-2 mRNA ex vivo.18
Non-invasive monitoring of COX-2 expression in vivo would
further advance efforts into elucidating basic pharmacology of
the enzyme. Over the past decade a number of COX-2 inhibi-
tors have been radiolabelled with 11C, 18F, 99mTc, 123I and125
I
Many selective COX-2 inhibitors carry a methylsulfonyl
group as a common COX-2 pharmacophore on one of the aryl
rings. It is thought that this moiety is indispensable for high
binding potency and selectively.2 However, sulfonamide moi-
eties are also frequently used as COX-2 pharmacophores.37,38
They are thought to yield compounds with comparable or even
higher potency. Prominent examples of sulfonamide carrying
selective COX-2 inhibitors include celecoxib and valdecoxib.
Replacing a methylsulfonyl group with a sulfonamide can have
significant effects on the pharmacokinetic profile of the drug.
We identified sulfonamide 2a as a potential candidate for 18F
radiolabelling in addition to two methylsulfonyl group-con-
taining compounds 1a and 3a. The synthesis of sulfonamide-
containing structures generally requires protection of the
amine group to enable efficient and high yielding synthesis.
A very elegant method published by Mahalingam et al.39
described the protection of the sulfonamide groups via tert-
butyl groups, which can easily be removed under mild
conditions using catalytic amounts of Lewis acid scandium
triflate. To synthesize the tert-butyl protected precursor 8, we
to assess COX-2 expression in vivo using positron emission
tomography (PET) and single photon emission computed
tomography (SPECT). Among the radiolabelled compounds
were celecoxib,19–27 rofecoxib28–30 and a number of novel
structures.31–34 However, despite the large number of radio-
labelled COX-2 inhibitors reported in the literature, COX-2-
mediated uptake of a radiotracer in an in vivo model has not
yet been convincingly demonstrated, mainly due to insufficient
metabolic stability and high degree of non-specific binding.
The aim of this study is to develop a novel class of highly
potent and selective 18F-labelled COX-2 inhibitors as radiotra-
cers for molecular imaging of COX-2 expression in vivo (Fig. 1).
The lead structure for this investigation is compound 1a,
which was originally reported by Swarbrick and co-workers.35
Results and discussion
Chemistry
An important compound within the synthesis route of com- first attempted to reproduce the synthetic template that
pounds 1a and 3a is 2-(methylsulfonyl)-4-(4-(methylsulfonyl)- was used for the preparation of compound 6. Reaction of
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Fig. 2 Synthesis of compounds 1a–1p, 3a.
4-acetylbenzene sulfonylchloride with tert-butylamine gave In vitro COX-1 and COX-2 enzyme inhibition
tert-butyl-protected sulfonamide. However, subsequent Claisen
Compounds 1a–p, 2a–e and 3a were evaluated for their COX-2
condensation with ethyl trifluoroacetate failed presumably due
to the basic reaction conditions. Basic reaction conditions led
to the abstraction of the acidic sulfonamide proton leading to
the formation of poorly soluble salt complexes.
inhibitory potency and selectivity profile. The determined
enzyme inhibition data are summarized in Tables 1–4. Cele-
coxib was included in all assays as an internal reference com-
pound for comparison. Celecoxib showed IC50 values of 40 nM
against COX-2 and 15 μM against COX-1, which is in good
agreement with previously reported literature values.40 The
lead structure 1a displayed excellent COX-2 inhibitory potency
(IC50 (COX-2) = 7 nM) and did not show COX-1 inhibition in
the concentration range tested. This makes structure 1a more
potent and many times more selective than celecoxib. In the
original report, compound 1a displayed an IC50 (COX-2) of
0.28 nM,35 which is an order of magnitude lower than the
value we have obtained. This is likely due to differences in
the inhibition assay used to determine the IC50 values.
Compounds 1b to 1j are representative of our effort to
To omit Claisen condensation, we applied the Suzuki coup-
ling reaction between commercially available 4-chloro-2-
(methylsulphonyl)-6-(trifluoromethyl)pyrimidine with tert-
butyl 4-boronobenzenesulfonamide to form compound 7. The
methyl–sulfonyl group in compound 7 was oxidized using
Oxone to afford compound 8. Removal of the tert-butyl group
was achieved through treatment of compound 8 with scan-
dium triflate to give compound 9 in a total yield of 53% for
the three steps. Treatment of sulfonamide 9 with various
primary amines gave compounds 2a–e in 57–91% yield. The
synthesis of sulfonamides 2a–e is summarized in Fig. 3.
Fig. 3 Synthesis of compounds 2a–2e.
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Table 1 IC50 (COX-1) and IC50 (COX-2) values for compounds 1a–1j
Table 3 IC50 (COX-1) and IC50 (COX-2) values for compound 3a
IC50 a (μM)
IC50 a (μM)
R1
COX-1
COX-2
R3
F
COX-1
COX-2
Celecoxib
15
0.040
0.007
0.006
0.018
0.017
0.016
0.005
>10
Celecoxib
3a
15
>100
0.040
0.020
1a
1b
1c
1d
1e
1f
1g
1h
1i
F
Cl
Br
CF3
H
Me
Ph
t-Bu
OMe
NO2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
a Values are means of two determinations.
Table 4 IC50 (COX-1) and IC50 (COX-2) values for compound 2a–2e
>10
0.007
0.086
1j
a Values are means of two determinations.
IC50 a (μM)
Table 2 IC50 (COX-1) and IC50 (COX-2) values for compounds 1k–1o
R4
COX-1
COX-2
Celecoxib
15
0.040
0.039
0.034
0.028
0.014
0.031
2a
2b
2c
2d
2e
N-(4-Fluorobenzyl)
N-Benzyl
N-(4-Methylbenzyl)
N-(4-Methoxybenzyl)
N-Butyl
>100
>100
>100
>100
>100
IC50 a (μM)
a Values are means of two determinations.
R2
COX-1
COX-2
Celecoxib
1k
1l
1m
1n
1o
15
0.040
0.30
0.080
0.050
2.0
N-(4-Pyridyl)-CH2–
N-(3-Pyridyl)-CH2–
N-(2-Pyridyl)-CH2–
N-(2-Fluoroethyl)
N-Butyl
>100
>100
>100
>100
>100
the strongly electron withdrawing nature and modest steric
bulk of the nitro group, which has a negative effect on binding
potency. Compound 1i was found to have similar COX-2
inhibitory potency and selectivity as 1a (IC50 = 7 nM). Com-
pound 1i carries a methoxy group, which makes this com-
pound a good candidate for 11C labelling. 11C labelling can be
carried out via O-methylation of free alcohols using 11CH3I or
0.020
a Values are means of two determinations.
determine how structural changes to the second aryl ring [11C]methyltriflate.32 Compound 1i would be a good radio-
effect inhibitory potency and selectivity (Table 1).
labelling candidate for a 11C-based COX-2 radiotracer study.
We found that changes at the 4-position of the aryl ring are
We found the lead compound 1a to be relatively lipophilic
usually well tolerated. Most compounds displayed better (log P = 3.37; determined experimentally by partition in
COX-2 inhibitory potency than celecoxib with IC50 values for octanol–water using a 18F-labelled compound). High lipophili-
COX-2 inhibition below 20 nM while possessing high select- city of a PET radiotracer is likely to cause high non-specific
ivity over COX-1. The 4-bromine substituted compound (1c) is binding and high intestine uptake. Compounds 1k to 1m as
especially notable. 1c Shows good COX-2 inhibitory potency displayed in Table 2 are representative of our efforts to syn-
(IC50 = 18 nM), despite the considerable steric bulk of bromine thesize structures that are less lipophilic by substitution of the
as a substituent. In contrast, more bulky substituents, such as second aryl ring.
tert-butyl and phenyl (1h, 1g) show no COX-2 inhibition at the
The most promising candidate in this library is pyridine
concentrations tested. It can be concluded that too much compound 1m. Compound 1m was found to have a COX-2
steric bulk at 4-position of the second aryl ring is detrimental inhibitory potency (IC50 = 50 nM) comparable to that of cele-
to COX-2 binding potency. Despite the relatively modest steric coxib, but a much better COX-2 selectivity. Compound 1m is
bulk of the 4-position of the nitro-substituted compound (1j), more water soluble than 1a and might be developed as a 18F
we found its potency decreased by an order of magnitude in radiotracer by including fluorine-18 at the 4-position of the
comparison to 1a. This is likely to be due to a combination of pyridine ring. Various 18F-labelled fluoropyridines have been
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reported as highly efficient radiotracers and radiopharmaceuti-
cals.51,52 Efforts to synthesize alkyl substituted compounds
resulted in only one high potency compound (1o). Compound
1o displayed COX-2 inhibitory potency that exceeded that of
celecoxibs (IC50 = 20 nM).
Oxygen-containing compound 3a (IC50 = 20 nM) was found
to be slightly less potent than the corresponding amine com-
pound 1a (Table 3). Thus, replacing nitrogen with oxygen
reduces slightly binding potency, but binding potency of com-
pound 3a is still high when compared with an internal refer-
ence compound celecoxib.
Inhibitory potencies of sulfonamide compounds 2a to 2e
are summarized in Table 4. All five compounds were slightly
less potent than their corresponding methylsulfone counter-
parts. We can conclude that for the particular structural
pyrimidine-based backbone, methylsulfone-containing com-
pounds seem to display higher COX-2 inhibitory potency than
the corresponding sulfonamides. Nonetheless, the potential
candidate for 18F radiolabelling 2a, displayed inhibitory
potency similar to that of celecoxib (IC50 = 39 nM) while
showing higher COX-2 selectivity. Compound 2e, similar to 1i,
carries a methoxy group and might therefore serve as a good
11C radiolabelling candidate. The COX-2 inhibitory potency of
2e is comparable to that of 1i (IC50 = 14 nM).
Fig. 4 (Left) Molecular docking of compound 1a (carbon atoms in green) posi-
tioned in the binding site of COX-2 (PDB ID: 6COX; Eintermolecular = −10.70 kcal
mol−1) and (right) COX-1 (PDB ID: 1EQG; Eintermolecular = −8.03 kcal mol−1).
Hydrogen atoms of amino acid residues have been removed for clarity.
Compounds 1a, 2a and 3a displayed COX-2 inhibitory
potency and selectivity rendering all three compounds suitable
for development as 18F-labelled radiotracers.
Fig. 5 (Left) Molecular docking of compound 2a (carbon atoms in green) posi-
tioned in the binding site of COX-2 (PDB ID: 6COX; Eintermolecular = −10.55 kcal
mol−1) and (right) COX-1 (PDB ID: 1EQG; Eintermolecular = −8.39 kcal mol−1).
Hydrogen atoms of amino acid residues have been removed for clarity.
Molecular docking studies
Molecular docking experiments were performed using X-ray
crystal structure data for COX-1 and COX-2 obtained from the
protein data bank to explore possible interaction of com-
pounds 1a, 2a and 3a with the active site of COX-1 and COX-2
enzymes. High inhibitory potency of compound 1a (COX-2,
IC50 = 7 nM) suggests a favourable orientation within the
COX-2 binding site. The SO2CH3 group in compound 1a com-
pletely enters into the secondary pocket region of the COX-2
active site, where it is oriented towards Q192, R513, H90, and
A516 residues (Fig. 4, left). One of the oxygen atoms of the
SO2CH3 group undergoes hydrogen bonding interactions with
the nitrogen atom of H90 (SvO⋯N = 2.64 Å). The other
oxygen atom indicates hydrogen bonding interactions with the
nitrogen atom of Q192 amino acid residue (SvO⋯N = 2.23 Å).
Moreover, the phenyl ring bearing a fluorine atom is placed in
the vicinity of R120, A527 and V349 amino acid residues. On
the other side, docking studies of compound 1a into COX-1
enzyme indicated that compound 1a was not able to enter into
the COX-1 active site completely (Fig. 4, right). The fluorine-
containing phenyl ring is situated near A527, S530 and L531
residues while the SO2CH3 group is positioned outside the
active site of COX-1 enzyme. This finding is in good agreement
with the determined high inhibitory potency and selectivity of
compound 1a towards COX-2.
site where it is surrounded by H90, Q192, A516 and R513 resi-
dues (Fig. 5, left). The nitrogen atom of the SO2NH2 group dis-
plays hydrogen bonding to carbonyl oxygen of Q192 (N⋯OvC
= 2.85 Å), and to carbonyl oxygen of L352 (N⋯OvC = 2.78 Å).
One oxygen atom of the SO2NH2 group is hydrogen bonded to
the nitrogen atom of H90 residue (SvO⋯N = 2.57 Å).
The 4-fluoro phenyl ring of compound 2a is positioned in
the vicinity of R120, V349, V116, L531 and A527 amino acid
residues. The CF3 group of compound 2a is positioned at the
entrance of COX-2 hydrophobic pocket constituted by W387,
Y385 and F518 residues. Compound 2a shows only partial
entry into the COX-1 enzyme active site and did therefore not
exhibit significant interactions with the COX-1 active site resi-
dues (Fig. 5, right). This is also in agreement with the deter-
mined COX-2 inhibitory potency and selectivity of compound
2a.
The top scored docking pose of compound 3a (COX-2 IC50
=
20 nM) displays a favorable orientation into the COX-2 active
site wherein the SO2CH3 group is sloping towards the second-
ary pocket region of COX-2 active site lined by H90, R513, S353
and L352 residues (Fig. 6, left). One of the oxygen atoms of the
SO2CH3 group is hydrogen bonded to the nitrogen atom of
H90 residue (SvO⋯N = 2.64 Å), and the other oxygen atom
shows hydrogen bonding interactions with nitrogen atom of
Docking studies with compound 2a (COX-2, IC50 = 39 nM)
indicated that the phenyl ring bearing the SO2NH2 group is
inserted into the secondary pocket region of the COX-2 active
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Fig. 8 Radiosynthesis of [18F]1a (R = CH3) and [18F]2a (R = NH2).
Fig. 6 (Left) Molecular modeling (docking) of compound 3a (carbon atoms in
green) positioned in the binding site of COX-2 (PDB ID: 6COX; Eintermolecular
=
−10.12 kcal mol−1) and (right) COX-1 (PDB ID: 1EQG; Eintermolecular = −9.03 kcal
95 min. Decay-corrected radiochemical yield based on [18F]FBA
was 27 11%. In a typical experiment, starting from 1 GBq of
[18F]FBA prepared on an automated synthesis unit provided
160 MBq of radiotracer [18F]1a. Specific activity of [18F]1a
at the end of synthesis was determined to be greater than
mol−1). Hydrogen atoms of amino acid residues have been removed for clarity.
R513 amino acid residue (SvO⋯N = 2.79 Å). The CF3 group of
compound 3a is located in close proximity of the S530 residue
where the measured distance between one of the F atom of the
CF3 group and the –OH group of S530 is 1.36 Å. The 4-fluoro
phenyl ring of compound 3a is positioned in the vicinity of
L359, V116 and R120 amino acid residues. However, docking
of compound 3a within the COX-1 active site clearly indicates
that the SO2CH3 pharmacophore, which probably contributes
to potent COX-2 inhibition, is not able to enter into the COX-1
active site, whereas the F-phenyl ring is sandwiched between
the Y385 and W387 amino acid residues of the hydrophobic
region.
40 GBq μmol−1
.
Synthesis of radiotracer [18F]2a was accomplished by the
same methodology described for [18F]1a but using labelling
precursor 9 as the starting material. Radiosynthesis was
carried out within 110 min with a decay-corrected radiochemi-
cal yield of 23% 1%. Specific activity of [18F]2a was greater
than 40 GBq μmol−1 at the end of synthesis. Radiosyntheses of
compounds of [18F]2a and of [18F]2b are depicted in Fig. 8.
We also attempted to synthesize radiotracer [18F]3a by
expanding the concept of nucleophilic substitution reactions
with compound 6 as the labelling precursor. To replace the
nitrogen present in compound [18F]1a with an oxygen, we per-
formed radiosyntheses with 4-[18F]fluorobenzyl alcohol ([18F]-
FBAlc) as a building block. This strategy would allow the exten-
sion of feasible radiochemistry as exemplified for compounds
[18F]1a and [18F]2a to the readily available 18F building block
[18F]FBAlc.
Radiochemistry
Radiotracers [18F]1a and [18F]2a were synthesized using a
4-[18F]fluorobenzylamine ([18F]FBA) as a building block. [18F]-
FBA was synthesized using a method recently described by our
group41,42 (Fig. 7). 4-Cyano-N,N,N-trimethylanilinium trifluoro-
methansulfonate as a labelling precursor was radio-fluorinated
using nucleophilic no-carrier-added (n.c.a.) [18F]KF in the pres-
ence of Kryptofix K222 in dry DMSO at elevated temperature.
The resulting 4-[18F]fluorobenzonitrile ([18F]FBN) was
reduced to [18F]FBA using transition metal-assisted NaBH4
reduction. A fully automated [18F]FBA synthesis was recently
developed by Way et al.42 This work also describes the useful-
ness of [18F]FBA as a building block for the synthesis of a
variety of 18F-labelled compounds like prosthetic groups for
peptide labelling or built-up synthesis of complex molecules
as 18F-labelled Hsp90 inhibitor geldanamycin.
As described in the chemistry section, we successfully syn-
thesized reference compound 3a by the reaction of 4-fluoro-
benzyl alcohol with compound 6 in the presence of sodium
hydride. Radiosynthesis of [18F]FBAlc as a 18F building block
was accomplished following a protocol developed by Donohue
et al.43 (4-Trimethylamino)benzaldehyde trifluoromethane-
sulfonate was treated with a powerful radiofluorination agent
[18F]KF in the presence of Kryptofix K222 in dry acetonitrile
at elevated temperature to yield 4-[18F]fluorobenzaldehyde.
4-[18F]Fluorobenzaldehyde was reduced to [18F]FBAlc using
NaBH4. NaBH4 was dissolved in water and passed through
Radiotracer [18F]1a was prepared through the reaction of
compound 6 with [18F]FBA. The reaction was carried out in
THF at 140 °C for 20 min (Fig. 5). Total synthesis time for the
preparation of [18F]1a, including HPLC purification, was
a
solid phase extraction cartridge containing 4-[18F]-
fluorobenzaldehyde.
[18F]FBAlc was purified using HPLC, and the solvent was
evaporated under reduced pressure. Dried [18F]FBAlc was re-
dissolved in dry solvent to be used in subsequent reaction
steps. Despite extensive efforts to promote the substitution
reaction between [18F]FBAlc and labelling precursor 6, it was
not possible to obtain reasonable amounts of the desired
product [18F]3a. Radiochemical yields were below 1% as indi-
cated by radio-TLC analysis of the reaction mixture.
Fig. 7 Radiosynthesis of 4-[18F]fluorobenzylamine [18F]FBA.
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Radiosynthesis of [18F]3a based on the reaction between
Organic & Biomolecular Chemistry
[18F]FBAlc and compound 6 was performed using different
reaction conditions. This included variation of reaction temp-
erature (0 °C to 180 °C) use of different bases (no base, triethyl-
amine, potassium tert-butoxide, sodium hydride), and the use
of different solvents (THF, CH3CN, DMF). To our disappoint-
ment, no product formation of 3a was achieved as confirmed
by radio-TLC and radio-HPLC analyses. We hypothesize that
the ratio of [18F]FBAlc to a base and a precursor is crucial
Fig. 10 Radiosynthesis of [18F]3a from precursor 11.
for the success of the reaction. The reaction does not proceed heated. Dilution of the reaction mixture with water and collec-
in the absence of a base. During the radiosynthesis only trace tion of the formed precipitate by filtration yielded iodyl com-
amounts of [18F]FBAlc are present in the reaction mixture, pound 15. The product also contained small amounts of
therefore any added base will be in large stoichiometric excess. starting material 11 and the mono-oxidized iodine intermedi-
We believe that the excess of base interferes with the reaction. ate. Both small impurities could be removed by column
Moreover, the reaction tended to be very water sensitive. chromatography to give pure iodyl compound 11 in 38% yield.
Although most residual water can be removed during the
Radiolabelling with n.c.a. [18F]fluoride was achieved by
radiosynthesis, even trace amounts of water seem to have a heating 1 mg of iodylaryl compound 11 in 300 μl of dry DMF
detrimental effect on the reaction using tracer concentrations in the presence of Kryptofix K222 at 180 °C for 20 min (Fig. 10).
of 4-fluorobenzyl alcohol.
A range of different solvents (DMF, DMSO, NMP), tempera-
As a result, it seems to be very challenging to fine-tune the tures (80 °C to 180 °C) and different precursor concentrations
amount of base required for successful radiosynthesis of com- were tested. It was found that optimal radiochemical yields of
pound [18F]3a based on the reaction of labelling precursor 6 5 to 10% (as determined by radio-TLC) could be achieved by
with [18F]FBAlc.
using 1 mg of iodyl compound 11 in DMF at 180 °C. To
The difficulties to prepare compound [18F]3a according to prepare [18F]3a in radiopharmaceutical quality for in vitro and
the synthesis method of cold reference compound 3a (Fig. 2) in vivo work, the reaction mixture was diluted in 0.1 M NaOAc
prompted us to envisage an alternative synthesis route. Direct buffer (pH 5.3) and passed through a solid phase extraction
nucleophilic aromatic radiofluorination has been reported cartridge. The cartridge was washed with water and product
using iodylbenzene derivatives as labelling precursors. Iodyl- [18F]3a was eluted with CH3CN. The radiotracer was further
benzene derivatives substituted with electron donating as well purified using HPLC. After evaporation of the solvent, the
as electron withdrawing groups on the aromatic ring were radiotracer was redissolved in 10% EtOH–saline for further
shown to readily undergo radiofluorination reaction with radiopharmacological evaluation. Total synthesis was accom-
n.c.a. [18F]fluoride as exemplified for various compounds plished in 120 min. A starting activity of 300 MBq [18F]fluoride
described in a recent patent publication.44 Synthesis of the typically yielded 3–5 MBq of purified product [18F]3a. The
iodyl group-containing compound 11 as a labelling precursor identity of the radiotracer was confirmed by HPLC co-injection
for the preparation of radiotracer [18F]3a is given in Fig. 9.
Synthesis of iodylaryl compound 11 was achieved by the
with the non-radiolabelled reference compound.
Upon further reaction optimization of the reaction con-
reaction of compound 6 with 4-iodobenzyl alcohol and sodium ditions, the iodylaryl-based direct radiofluorination approach
hydride, applying similar reaction conditions described for the could prove to be a versatile strategy for the site specific radio-
synthesis of 3a to form compound 10 in 71% yield. 4-Iodo- labelling of more complex drug-like molecules in sufficient
benzyl compound 10 was oxidized to iodyl compound 11 using radiochemical yields.
Oxone in a mixture of water and methanol, while being gently
Other direct radiolabelling strategies using nucleophilic
aromatic substitution reactions with [18F]KF on non-activated
aromatic systems involve iodonium and sulfonium salts.
However, syntheses of iodonium and sulfonium salts of struc-
turally more complex compounds tend to be challenging,
making them generally less versatile with respect to their
incorporation of n.c.a. fluorine-18 at specific sites in a given
molecule.45–47
Summary and conclusion
We have prepared a series of novel COX-2 inhibitors based on
a pyrimidine scaffold in continuation to the original work
reported by Swarbrick and co-workers.35 In vitro COX-1 and
COX-2 enzyme inhibition studies revealed the great potential
of pyrimidine-based compounds as highly potent and selective
COX-2 inhibitors. Except for bulky substituents such as phenyl
Fig. 9 Synthesis of iodylaryl compound 11.
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and tert-butyl groups attached to the para position of the
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 4.72 (s,
benzyl ring (compounds 1g and 1h), COX-2 enzyme seems to 2H, CH2); 5.85 (s, 1H, N–H); 7.04 (m, J = 8.8 Hz, 2H, Ar–H);
accept a broad variety of electron-donating (Me, OMe,) and 7.33 (s, 1H, Ar–H); 7.37 (m, J = 5.27 Hz, J = 3.22 Hz, 2H, Ar–H);
electron-withdrawing (F, Cl, Br, CF3, NO2) groups. Molecular 8.07 (m, J = 8.8 Hz, 2H, Ar–H); 8.22 (m, J = 8.8 Hz, 2H, Ar–H).
docking studies confirmed the determined high COX-2 inhibi- 13C-NMR (150 MHz, CDCl3): 44.5; 45.1; 102.6; 115.7 (d, 2J(C–F)
tory potency and selectivity of fluorine-containing compounds = 21 Hz); 122.0 (q, 1J(C–F) = 275 Hz); 128.0; 128.2; 129.2 (d,
4
1a, 2a and 3a. The high COX-2 inhibitory potency and selec- 3J(C–F) = 8 Hz); 134.1 (d, J(C–F) = 3 Hz); 141.4; 142.8; 162.2
1
2
tivity of fluorine-containing compounds 1a, 2a and 3a make (d, J(C–F) = 245 Hz); 162.7 (q, J(C–F) = 36 Hz); 165.4; 165.5.
them interesting compounds for the development of corres- 19F-NMR (375 MHz, CDCl3): −70.78 (s, 3F, CF3); −114.83 (m,
ponding 18F-labelled radiotracers. Radiolabelling was achieved 1F, Ar–F). LR-MS: 448.1 [M + Na].
through two different routes, using the indirect labelling
N-(4-Chlorobenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro-
method with 4-[18F]fluoro-benzylamine as the building block, methyl)pyrimidin-2-amine (1b). Compound 1b (48.4 mg, 84%
and the direct radiolabelling method with iodylaryl derivative yield) was obtained as a pale yellow solid.
11 as the labelling precursor. All radiotracers could be pre-
1H-NMR (400 MHz, CDCl3): 3.04 (s, 3H, SO2CH3); 4.67 (s,
pared in radiochemical yields and radiopharmaceutical quality 2H, CH2); 5.82 (s, 1H, N–H); 7.20 (s, 1H, Ar–H); 7.27 (m, 4H,
suitable for subsequent radiopharmacological evaluation. First Ar–H); 8.00 (m, J = 8.8 Hz, 2H, Ar–H); 8.14 (m, J = 8.8 Hz, 2H,
results on radiopharmacological evaluation of radiotracers Ar–H). 13C-NMR (150 MHz, CDCl3): 44.5; 45.1; 102.7; 122.0
[18F]1a, [18F]2a, and [18F]3a have been reported during the 20th (q, 1J(C–F) = 275 Hz); 128.0; 128.2; 128.9; 129.5; 133.4;
International Symposium on Radiopharmaceutical Sciences 136.9; 141.4; 142.8; 162.2 (q, 2J(C–F) = 36 Hz); 165.5; 165.6.
2013 in Jeju, Korea.50
19F-NMR (375 MHz, CDCl3): −70.45 (s, 3F, CF3). LR-MS:
464.0 [M + Na].
N-(4-Bromobenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro-
methyl)pyrimidin-2-amine (1c). Compound 1c (51.6 mg, 82%
yield) was obtained as a pale yellow solid.
Experimental
Chemistry
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 4.71 (s,
2H, CH2); 5.87 (s, 1H, N–H); 7.28 (m, J = 8.5 Hz, 2H, Ar–H);
7.33 (s, 1H, Ar–H); 7.48 (m, J = 8.5 Hz, 2H, Ar–H); 8.07 (m, J =
8.8 Hz, 2H, Ar–H); 8.20 (m, J = 8.8 Hz, 2H, Ar–H). 13C-NMR
(150 MHz, CDCl3): 44.5; 45.1; 102.7; 121.4 (q, 1J(C–F) =
275 Hz); 128.0; 128.2; 129.7; 130.9; 132.5; 137.4; 141.4; 142.8;
162.4 (q, 2J(C–F) = 36 Hz); 165.5; 165.6. 19F-NMR (375 MHz,
CDCl3): −70.7 (s, 3F, CF3). LR-MS: 510.0 [M + Na].
General methods. All reagents and solvents were obtained
from Sigma-Aldrich, unless otherwise stated and used without
further purification. Nuclear magnetic resonance spectra were
recorded on a 400 MHz Varian unit and a 600 MHz Burker
unit. 1H-NMR and 13C-NMR chemical shifts are recorded in
ppm relative to tetramethylsilane (TMS). 19F-NMR chemical
shifts are recorded in ppm relative to trichlorofluoromethane.
Low resolution mass spectra were obtained using an Agilent
Technologies 6220 TOF instrument. Column chromatography
was conducted using Merck silica gel (mesh size 230–400
ASTM). Thin-layer chromatography (TLC) was performed using
Merck silica gel F-254 aluminum plates, with visualization
under UV light (254 nm). High performance liquid chromato-
graphy (HPLC) purifications and analysis were performed
using a Phenomenex LUNA® C18 column (100 Å, 250 ×
10 mm, 10 μm) using a Gilson 322 Pump module fitted with a
171 Diode Array and a radio detector. Compounds 4, 5 and 6
were prepared according to the literature procedure.35 Com-
pounds 1a, 1e and 1f have been described by Swarbrick et al.35
General procedure for the synthesis of compounds 1a–1p.
Compound 6 (50 mg, 0.13 mmol) was dissolved in CH3CN
(0.75–1.5 ml) and the corresponding amine (0.65 mmol, 5.0
eq.) was added. The reaction vessel was sealed and heated at
140 °C for 2–6 h. The reaction mixture was cooled to room
temperature, and 8 ml of 1 N HCl was added. The reaction
mixture was stirred, and the precipitating solid was filtered off.
The product was thoroughly washed with water.
N-(4-Trifluorobenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro-
methyl)pyrimidin-2-amine (1d). Compound 1d (42.3 mg, 68%
yield) was obtained as a white solid.
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 4.83 (s,
2H, CH2); 5.95 (s, 1H, N–H); 7.35 (s, 1H, Ar–H); 7.52 (m, J =
8.2 Hz, 2H, Ar–H); 7.61 (m, J = 8.2 Hz, 2H, Ar–H); 8.06 (m, J =
8.5 Hz, 2H, Ar–H); 8.19 (m, J = 8.2 Hz, 2H, Ar–H). 13C-NMR
(150 MHz, CDCl3): 44.5; 45.3; 102.9; 122.1 (q, 1J(C–F) = 275
Hz); 124.0 (q, 1J(C–F) = 271 Hz); 125.7; 127.6; 128.0; 128.2;
129.8 (q, 2J(C–F) = 32 Hz); 141.3; 142.5; 162.5 (q, 2J(C–F) =
36 Hz); 165.5; 165.6. 19F-NMR (375 MHz, CDCl3): −62.5 (s, 3F,
CF3); −70.7 (s, 3F, CF3). LR-MS: 498.1 [M + Na].
N-(Benzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-pyri-
midin-2-amine (1e). Compound 1e (42.9 mg, 81% yield) was
obtained as a white solid.
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 4.76 (s,
2H, CH2); 5.86 (s, 1H, N–H); 7.30 (m, 1H, Ar–H) 7.32 (s,
1H, Ar–H); 7.36 (m, 2H, Ar–H); 7.40 (m, 2H, Ar–H); 8.10 (m,
J = 8.8 Hz, 2H, Ar–H); 8.22 (m, J = 8.5 Hz, 2H, Ar–H).
13C-NMR (150 MHz, CDCl3): 44.5; 45.8; 102.4; 121.1 (q,
1J(C–F)
138.3; 141.5; 142.7; 162.4 (q, 2J(C–F) = 36 Hz); 165.4; 165.5.
= 275 Hz); 127.5; 127.6; 128.0; 128.2; 128.7;
N-(4-Fluorobenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro-methyl)-
pyrimidin-2-amine (1a). Compound 1a (90.6 mg, 82% yield) 19F-NMR (375 MHz, CDCl3): −70.46 (s, 3F, CF3). LR-MS: 430.1
was obtained as a pale yellow solid.
[M + Na].
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1
N-(4-Methylbenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro- (q, J(C–F) = 275 Hz); 128.0; 128.2; 129.7; 135.2; 141.6; 142.6;
158.5; 162.9 (q, 2J(C–F) = 36 Hz); 165.5; 165.6. 19F-NMR
methyl)pyrimidin-2-amine (1f). Compound 1f (42.6 mg, 78%
yield) was obtained as a white solid.
(375 MHz, CDCl3): −70.7 (s, 3F, CF3). LR-MS: 475.1 [M + Na].
N-(4-Pyridyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-
pyrimidin-2-amine (1k). Compound 1k (30.7 mg, 58% yield)
was obtained as an off-white solid.
1H-NMR (400 MHz, CDCl3): 2.35 (s, 3H, CH3–Ar); 3.10 (s,
3H, SO2CH3); 4.71 (s, 2H, CH2); 5.83 (s, 1H, N–H); 7.16 (m, J =
7.9 Hz, 2H, Ar–H); 7.29 (m, J = 8.2 Hz, 2H, Ar–H); 7.30 (s, 1H,
Ar–H); 8.06 (m, J = 8.5 Hz, 2H, Ar–H); 8.22 (m, J = 8.2 Hz, 2H,
Ar–H). 13C-NMR (150 MHz, CDCl3): 21.1; 44.5; 45.5; 102.3;
122.2 (q, 1J(C–F) = 275 Hz); 127.6; 128.0; 128.2; 129.4; 135.2;
137.3; 141.6; 142.6; 162.1 (q, 2J(C–F) = 36 Hz); 165.4; 165.5.
19F-NMR (375 MHz, CDCl3): −70.7 (s, 3F, CF3). LR-MS: 444.1
[M + Na].
1H-NMR (400 MHz, CDCl3): 3.11 (s, 3H, SO2CH3); 5.01 (s,
2H, CH2); 6.74 (s, 1H, N–H); 7.43 (s, 1H, Ar–H); 7.95 (m, J =
5.6 Hz, 2H, Ar–H); 8.07 (m, J = 8.2 Hz, 2H, Ar–H); 8.28 (m, J =
8.2 Hz, 2H, Ar–H);8.72 (m, J = 5.6 Hz, 2H, Ar–H). 13C-NMR
1
(150 MHz, CDCl3): 44.4; 45.0; 103.9; 113.8; 121.3 (q, J(C–F) =
275 Hz); 128.1; 128.2; 128.7; 134.3; 141.2; 143.1; 162.2 (q,
N-(4-Phenylbenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro- 2J(C–F) = 36 Hz); 165.3; 165.4. 19F-NMR (375 MHz, CDCl3):
methyl)pyrimidin-2-amine (1g). Compound 1g (31.0 mg, 49%
yield) was obtained as a white solid.
−70.3 (s, 3F, CF3). LR-MS: 431.1 [M + Na].
N-(3-Pyridyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-
pyrimidin-2-amine (1l). Compound 1l (23.8 mg, 45% yield) was
obtained as a pale yellow solid.
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 4.80 (s,
2H, CH2); 5.91 (s, 1H, N–H); 7.33 (s, 1H, Ar–H); 7.36 (m, J = 7.3
Hz, 1H, Ar–H); 7.46 (m, 4H, Ar–H); 7.59 (m, 4H, Ar–H); 8.07
(m, J = 8.8 Hz, 2H, Ar–H); 8.24 (m, J = 8.2 Hz, 2H, Ar–H).
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 4.92 (s,
2H, CH2); 6.91 (s, 1H, N–H); 7.36 (s, 1H, Ar–H); 7.69 (m, 1H,
Ar–H); 8.05 (m, J = 7.6 Hz, 2H, Ar–H);8.16 (m, J = 8.5 Hz, 2H,
Ar–H);8.34 (s, 1H, Ar–H); 8.62 (m, J = 5.2 Hz, 1H, Ar–H); 9.09
(m, 1H, Ar–H). 13C-NMR (150 MHz, CDCl3): 42.9; 44.5; 103.3;
111.0; 121.1; 121.3 (q, 1J(C–F) = 275 Hz); 125.1; 128.1; 128.2;
128.3; 133.3; 141.1; 142.9; 162.2 (q, 2J(C–F) = 36 Hz); 165.4;
1
13C-NMR (150 MHz, CDCl3): 44.5; 45.1; 103.2; 120.7 (q, J(C–F)
= 275 Hz); 123.9; 128.0; 128.2; 128.5; 129.0; 130.0; 130.5; 141.2;
142.9; 146.1; 146.2; 147.4; 162.5 (q, 2J(C–F) = 36 Hz); 165.2;
165.3. 19F-NMR (375 MHz, CDCl3): −70.6 (s, 3F, CF3). LR-MS:
506.1 [M + Na].
N-(4-tert-Butylbenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro- 165.5. 19F-NMR (375 MHz, CDCl3): −70.4 (s, 3F, CF3). LR-MS:
methyl)pyrimidin-2-amine (1h). Compound 1h (23.9 mg, 40%
431.1 [M + Na].
yield) was obtained as a white solid.
N-(2-Pyridyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-
pyrimidin-2-amine (1m). Compound 1m (8.5 mg, 16% yield)
was obtained as a pale yellow solid.
1H-NMR (400 MHz, CDCl3): 1.32 (s, 9H, C(CH3)3); 3.11 (s,
3H, SO2CH3); 4.75 (s, 2H, CH2); 5.90 (s, 1H, N–H); 7.30 (s, 1H,
Ar–H); 7.35 (m, J = 7.9 Hz, 2H, Ar–H); 7.38 (m, J = 7.9 Hz, 2H,
Ar–H); 8.09 (m, J = 7.9 Hz, 2H, Ar–H); 8.26 (m, J = 7.9 Hz, 2H,
Ar–H). 13C-NMR (150 MHz, CDCl3): 31.3; 34.6; 44.5; 45.4;
102.3; 122.5 (q, 1J(C–F) = 275 Hz); 125.6; 128.0; 128.2; 128.8;
135.3; 141.6; 142.6; 150.7; 162.7 (q, 2J(C–F) = 36 Hz); 165.4;
165.5. 19F-NMR (375 MHz, CDCl3): −70.7 (s, 3F, CF3).
1H-NMR (400 MHz, CDCl3): 3.11 (s, 3H, SO2CH3); 3.13 (m,
2H, CH2); 5.17 (s, 1H, N–H); 7.36 (s, 1H, Ar–H); 7.81 (m, J =
6.4 Hz, 1H, Ar–H); 8.00 (m, J = 7.6 Hz, 2H, Ar–H); 8.14 (m,
J = 7.6 Hz, 2H, Ar–H); 8.24 (m, J = 7.3 Hz, 1H, Ar–H); 8.37 (m,
J = 7.3 Hz, 1H, Ar–H); 8.67 (m, J = 6.4 Hz, 1H, Ar–H). 13C-NMR
(150 MHz, CDCl3): 42.6; 44.5; 103.5; 113.9; 118.9; 121.5 (q,
N-(4-Methoxybenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro- 1J(C–F) = 275 Hz); 125.3; 128.0; 128.2; 128.9; 134.5; 140.8;
methyl)pyrimidin-2-amine (1i). Compound 1i (111.6 mg, 98%
143.0; 161.8 (q, 2J(C–F) = 36 Hz); 165.5; 165.6. 19F-NMR
yield) was obtained as a white solid.
(375 MHz, CDCl3): −70.4 (s, 3F, CF3). LR-MS: 431.1 [M + Na].
N-(2-Fluoroethyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-
pyrimidin-2-amine (1n). The synthesis follows the general
method described; with the exception that triethylamine
(4.9 eq.) was added to the reaction mixture. Compound 1n
(26.1 mg, 52% yield) was obtained as a silvery white solid.
1H-NMR (400 MHz, CDCl3): 3.12 (s, 3H, SO2CH3); 3.92 (m,
J = 5.5 Hz, 2H, CH2); 4.67 (m, J = 5.0 Hz, 2H, CH2); 5.78 (s, 1H,
N–H); 7.34 (s, 1H, Ar–H); 8.10 (m, J = 7.6 Hz, 2H, Ar–H); 8.25
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 3.81 (s,
3H, CH3–O); 4.68 (s, 2H, CH2); 5.80 (s, 1H, N–H); 6.89 (m, J =
8.5 Hz, 2H, Ar–H); 7.30 (s, 1H, Ar–H); 7.33 (m, J = 8.8 Hz, 2H,
Ar–H); 8.07 (m, J = 8.5 Hz, 2H, Ar–H); 8.24 (m, J = 7.6 Hz, 2H,
Ar–H). 13C-NMR (150 MHz, CDCl3): 44.5; 45.3; 55.3; 102.3;
121.5 (q, 1J(C–F) = 275 Hz); 128.0; 128.2; 129.4; 129.8; 130.3;
141.6; 142.6; 159.1; 162.1 (q, 2J(C–F) = 36 Hz); 165.4; 165.5.
19F-NMR (375 MHz, CDCl3): −70.7 (s, 3F, CF3). LR-MS: 460.1
[M + Na].
(m, J = 7.6 Hz, 2H, Ar–H). 13C-NMR (150 MHz, CDCl3): 42.1 (d,
N-(4-Nitrobenzyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)- 2J(C–F) = 20 Hz); 44.5; 82.0 (d, J(C–F) = 167 Hz); 102.7; 121.3
pyrimidin-2-amine (1j). The synthesis follows the general
method described; with the exception that triethylamine (4.9
1
(q, 1J(C–F) = 275 Hz); 128.0; 128.3; 139.5; 142.8; 162.1 (q, 2J(C–
F) = 36 Hz); 165.4; 165.5. 19F-NMR (375 MHz, CDCl3): −67.9 (s,
eq.) was added to the reaction mixture. Compound 1j
(24.1 mg, 41% yield) was obtained as a yellow solid.
1F, C–F); −70.8 (s, 3F, CF3). LR-MS: 386.1 [M + Na].
N-(Butyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-pyrimi-
din-2-amine (1o). Compound 1o (29.5 mg 61% yield) was
obtained as a pale yellow solid.
1H-NMR (400 MHz, CDCl3): 3.10 (s, 3H, SO2CH3); 4.88 (s,
2H, CH2); 6.03 (s, 1H, N–H); 7.37 (s, 1H, Ar–H); 7.57 (m, J = 8.5
Hz, 2H, Ar–H); 8.07 (m, J = 8.5 Hz, 2H, Ar–H); 8.20 (m, 4H, Ar–
H). 13C-NMR (150 MHz, CDCl3): 44.5; 45.1; 102.2; 120.1; 122.0
1H-NMR (400 MHz, CDCl3): 0.98 (m, J = 7.3 Hz, 3H, CH3);
1.47 (m, J = 7.0, 7.6 Hz, 2H, CH2); 1.67 (m, J = 7.0, 7.6 Hz, 2H,
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CH2); 3.11 (s, 3H, SO2CH3); 3.59 (m, J = 6.1, 6.4 Hz, 2H, CH2); concentrated in vacuo. The title compound 8 (1.08 g, 87%
4.83 (s, 2H, CH2); 5.48 (s, 1H, N–H); 7.26 (s, 1H, Ar–H); 8.10 yield) was obtained as a white solid.
(m, J = 8.2 Hz, 2H, Ar–H); 8.27 (m, J = 8.2 Hz, 2H, Ar–H).
1H-NMR (600 MHz, DMSO-d6): 1.18 (s, 9H, (CH3)3); 3.64 (s,
13C-NMR (150 MHz, CDCl3): 13.8; 20.0; 31.4; 41.5; 44.5; 101.7; 3H, SO2CH3); 7.87 (s, 1H, NH); 8.13 (m, J = 9.0, 2H, Ar–H); 8.69
121.5 (q, 1J(C–F) = 275 Hz); 128.2; 128.5; 140.3; 143.4; 161.2 (q, (m, J = 9.0 Hz, 2H, Ar–H); 9.04 (s, 1H, Ar–H). 13C-NMR
2J(C–F) = 36 Hz); 165.3; 165.4. 19F-NMR (375 MHz, CDCl3): (150 MHz, DMSO-d6): 30.2; 39.6; 54.0; 117.5; 121.6 (q, J(C–F)
−70.7 (s, 3F, CF3). LR-MS: 396.1 [M + Na].
2-[(4-Fluorobenzyl)oxy]-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro-
methyl)pyrimidine (3a). To a solution of 33 mg 4-fluorobenzyl
alcohol (0.26 mmol) in 3 ml of dry THF under an inert atmos-
1
= 275 Hz); 127.5; 129.5; 137.0; 148.6; 158.7 (q, 2J(C–F) = 36 Hz);
166.4; 167.2. 19F-NMR (565 MHz, DMSO-d6): −69.02 (s, 3F,
CF3). LR-MS: 460.1 [M + Na].
4-[2-(Methylsulfonyl)-6-(trifluoromethyl)pyrimidin-4-yl]benzene-
phere and at 0 °C was added 11 mg of 60% NaH suspension. sulfonamide (9). 110 mg of compound 8 (0.252 mmol) was dis-
After 5 min, 100 mg of 2-(methylsulfonyl)-4-[4-(methylsulfonyl)- solved in 4 ml of nitromethane and 50 mg of scandium triflate
phenyl]-6-(trifluoromethyl)pyrimidine
6 (0.26 mmol) was was added. The mixture was heated at 80 °C overnight and sub-
added in small portions. The reaction mixture was stirred at sequently concentrated in vacuo. The residue was partitioned
0 °C for 1 hour and quenched by adding an excess of 1 N HCl. between ethyl acetate and water; the organic phase was dried
Compound 3a was extracted in ethyl acetate. The organic layer using sodium sulfate and concentrated in vacuo. Compound 9
was washed with water, dried over sodium sulfate and solvent (85 mg, 88% yield) was obtained as a light brown solid.
removed in vacuo. Compound 3a was purified using a silica
column eluting at 1% MeOH–CH2Cl2 and obtained as a white 2H, NH2); 8.12 (m, J = 8.4, 2H, Ar–H); 8.70 (m, J = 8.4 Hz, 2H,
1H-NMR (600 MHz, DMSO-d6): 3.64 (s, 3H, SO2CH3); 7.68 (s,
solid (78.6 mg, 71% yield).
Ar–H); 9.05 (s, 1H, Ar–H). 13C-NMR (150 MHz, DMSO-d6): 39.6;
1H-NMR (600 MHz, CDCl3): 3.05 (s, 3H, SO2CH3); 5.47 (s, 117.5; 121.6 (q, 1J(C–F) = 275 Hz); 126.8; 129.5; 137.0; 148.2;
2H, CH2); 7.01 (m, J = 8.2 Hz, 2H, Ar–H); 7.46 (m, J = 8.2 Hz, 156.7 (q, 2J(C–F) = 36 Hz); 166.4; 167.3. 19F-NMR (565 MHz,
2H, Ar–H); 7.65 (s, 1H, Ar–H); 8.04 (m, J = 8.4 Hz, 2H, Ar–H); DMSO-d6): −69.00 (s, 3F, CF3). LR-MS: 404.0 [M + Na].
8.24 (m, J = 8.4 Hz, 2H, Ar–H). 13C-NMR (150 MHz, CDCl3):
General procedure for the synthesis of compounds 2a–2e.
2
1
44.4; 69.7; 107.1; 115.5 (d, J(C–F) = 21 Hz); 121.1 (q, J(C–F) = Compound 9 (50 mg, 0.13 mmol) was dissolved in 1.5 ml of
275 Hz); 128.2; 128.5; 130.7 (d, 3J(C–F) = 9 Hz); 131.3 acetonitrile and corresponding amine (0.65 mmol, 5.0 eq.) was
(d, 4J(C–F) = 3 Hz); 140.3; 143.4; 159.1 (q, 2J(C–F) = 36 Hz); added. The reaction vessel was sealed and heated at 140 °C for
1
162.7 (d, J(C–F) = 247 Hz); 165.6; 167.4. 19F-NMR (565 MHz, 2 to 6 hours. Solvent was removed on a rotary evaporator and
CDCl3): −71.1 (s, 3F, CF3); −114.1 (m, 1F, Ar–F). LR-MS: 449.1 the residue partitioned between ethyl acetate and 1 N HCl. The
[M + Na].
organic layer was washed dried over sodium sulfate and the
solvent removed in vacuo. Further impurities were removed by
purification on a silica column.
N-tert-Butyl-4-[2-(methylsulfanyl)-6-(trifluoromethyl)-pyrimidin-
4-yl]benzenesulfonamide (7). 4-Chloro-2-(methylsulfonyl)-6-(tri-
fluoromethyl)pyrimidine (Key Organics Ltd, Camelford, UK)
4-{2-[(4-Fluorobenzyl)amino]-6-(trifluoromethyl)pyrimidin-4-yl}-
(1.0 g, 4.42 mmol), tert-butyl 4-boronobenzenesulfonamide benzenesulfonamide (2a). Compound 2a (90.6 mg, 82% yield)
(Combi-Blocks Inc., San Diego, USA) (1.25 g, 4.8 mmol) and was obtained as a yellow solid.
tetrakis-triphenylphosphinepalladium(0) (100 mg) were dis-
1H-NMR (600 MHz, DMSO-d6): 4.70 (s, 2H, CH2); 7.21 (m,
solved in 70 ml of DME and 1.15 g of sodium carbonate in J = 8.8 Hz, 2H, Ar–H); 7.51 (m, J = 8.8 Hz, 2H, Ar–H); 7.57 (s,
water (11 ml) added dropwise. The reaction mixture was 2H, SO2NH2); 7.71 (s, 1H, Ar–H); 8.43 (m, J = 8.8 Hz, 2H,
heated under reflux for 15 hours. The solvent was reduced on Ar–H); 8.62 (m, J = 8.8 Hz, 2H, Ar–H). 13C-NMR (150 MHz,
a rotary evaporator and the residue partitioned between ethyl DMSO-d6): 44.1; 101.8; 115.5 (d, 2J(C–F) = 23 Hz); 121.5 (q,
3
acetate and water; the organic phase was dried using sodium 1J(C–F) = 275 Hz); 126.6; 128.4; 129.8 (d, J(C–F) = 8 Hz); 136.4
sulfate and concentrated in vacuo. The compound was purified (d, 4J(C–F) = 3 Hz); 139.2; 146.9; 157.1 (q, 2J(C–F) = 36 Hz);
1
using a silica column, eluting at 30% ethyl acetate–hexanes. 161.6 (d, J(C–F) = 243 Hz); 162.5; 166.1. 19F-NMR (565 MHz,
The title compound 11 (1.27 g, 69% yield) was obtained as a DMSO-d6): −70.03 (s, 3F, CF3); −117.08 (m, 1F, Ar–F). LR-MS:
pale yellow solid. LR-MS: 444.1 [M + Na]. Low resolution mass 449.1 [M + Na].
4-[2-(Benzylamino)-6-(trifluoromethyl)pyrimidin-4-yl]benzene-
sulfonamide (2b). Compound 2b (48.4 mg, 84% yield) was
obtained as a dark yellow solid.
spectrometry indicates that the product is sulfoxide. However,
the product can be used directly in the next step involving oxi-
dation with Oxone.
1H-NMR (600 MHz, DMSO-d6): 4.71 (s, 2H, CH2); 7.28 (m,
N-tert-Butyl-4-[2-(methylsulfonyl)-6-(trifluoromethyl)-pyrimidin-
4-yl]benzenesulfonamide (8). Compound 7 (1.2 g, 2.85 mmol) 1H, Ar–H); 7.39 (m, J = 8.8 Hz, 2H, Ar–H); 7.47 (m, J = 8.8 Hz,
was dissolved in 100 ml of dichloromethane. A solution of 2H, Ar–H); 7.57 (s, 2H, SO2NH2); 7.71 (s, 1H, Ar–H); 8.03 (m,
Oxone (4.37 g, 7.13 mmol) in 50 ml of water was added in J = 8.8 Hz, 2H, Ar–H); 8.42 (m, J = 8.8 Hz, 2H, Ar–H). 13C-NMR
1
small portions, and the mixture was stirred at room tempera- (150 MHz, DMSO-d6): 44.8; 101.7; 121.2 (q, J(C–F) = 275 Hz);
ture. After 18 h, solvent was reduced on a rotary evaporator 126.6; 127.3; 128.3; 128.8; 129.4; 139.3; 140.2; 146.9; 157.1 (q,
and the residue partitioned between ethyl acetate and water; 2J(C–F) = 36 Hz); 162.7; 166.1. 19F-NMR (565 MHz, DMSO-d6):
the organic phase was dried over sodium sulfate and −70.03 (s, 3F, CF3). LR-MS: 431.1 [M + Na].
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4-{2-[(4-Methylbenzyl)amino]-6-(trifluoromethyl)pyrimidin-4-yl}- 4.65 mmol) in 40 ml of H2O added drop-wise. The reaction
benzenesulfonamide (2c). Compound 2c (49.8 mg, 91% yield)
was obtained as a brown solid.
mixture was heated at 60 °C for 6 hours, cooled on ice and
diluted in excess water. The forming precipitate was filtered
off, washed with water and dried under vacuum. The com-
pound was purified on a silica column eluting from 10 to 50%
MeOH–CH2Cl2. Compound 11 was obtained as a white solid
(353 mg, 67% yield).
1H-NMR (600 MHz, DMSO-d6): 2.31 (s, 3H, CH3); 4.66 (s,
2H, CH2); 7.18 (m, J = 8.8 Hz, 2H, Ar–H); 7.33 (m, J = 8.8 Hz,
2H, Ar–H); 7.57 (s, 2H, SO2NH2); 7.70 (s, 1H, Ar–H); 8.03 (m,
J = 8.8 Hz, 2H, Ar–H); 8.43 (m, J = 8.8 Hz, 2H, Ar–H). 13C-NMR
(150 MHz, DMSO-d6): 38.5; 44.5; 101.6; 121.2 (q, 1J(C–F) =
275 Hz); 126.6; 127.8; 128.4; 129.3; 136.7; 137.2; 139.3; 146.9;
157.1 (q, 2J(C–F) = 36 Hz); 162.7; 166.1. 19F-NMR (565 MHz,
DMSO-d6): −70.04 (s, 3F, CF3). LR-MS: 445.1 [M + Na].
1H-NMR (600 MHz, DMSO-d6): 3.30 (s, 3H, SO2CH3); 5.67
(s, 2H, CH2); 7.76 (m, J = 8.4 Hz, 2H, Ar–H); 8.00 (m, J = 8.4 Hz,
2H, Ar–H); 8.14 (m, J = 8.4 Hz, 2H, Ar–H); 8.38 (s, 1H, Ar–H);
8.59 (m, J = 8.4 Hz, 2H, Ar–H). 13C-NMR (150 MHz, DMSO-d6):
4-{2-[(4-Methoxybenzyl)amino]-6-(trifluoromethyl)pyrimidin-4-yl}- 43.7; 69.3; 108.6; 121.7 (q, 1J(C–F) = 275 Hz); 127.2; 128.1;
benzenesulfonamide (2d). Compound 2d (32.5 mg, 57% yield)
129.3; 131.0; 139.7; 139.8; 144.2; 151.2; 158.0 (q, 2J(C–F) =
36 Hz); 165.3; 167.7. 19F-NMR (560 MHz, DMSO-d6): −68.5 (s,
was obtained as a dark yellow solid.
1H-NMR (600 MHz, DMSO-d6): 3.72 (s, 1H, CH3); 4.60 (s,
3F, CF3). LR-MS: 589.0 [M + Na].
2H, CH2); 6.89 (m, J = 8.8 Hz, 2H, Ar–H); 7.34 (m, J = 8.8 Hz,
In vitro COX inhibition assay
2H, Ar–H); 7.52 (s, 2H, SO2NH2); 7.64 (s, 1H, Ar–H); 7.97 (m,
J = 8.8 Hz, 2H, Ar–H); 8.38 (m, J = 8.8 Hz, 2H, Ar–H). 13C-NMR
The ability of celecoxib and compounds 1a–1p, 2a–2e, 3a to
inhibit ovine COX-1 and recombinant human COX-2 was deter-
(150 MHz, DMSO-d6): 44.2; 55.5; 101.6; 115.6; 121.1 (q, 1J(C–
F) = 275 Hz); 126.6; 128.8; 129.2; 132.1; 139.3; 146.9; 157.1 (q,
2J(C–F) = 36 Hz); 158.7; 162.7; 165.6. 19F-NMR (565 MHz,
DMSO-d6): −70.05 (s, 3F, CF3). LR-MS: 461.1 [M + Na].
mined using a COX fluorescence inhibitor assay (Cayman
Chemical, Ann Arbor, USA; catalog #: 700100) according to the
manufacturers protocol. Compounds were assayed in a concen-
4-[2-(Butylamino)-6-(trifluoromethyl)pyrimidin-4-yl]benzene- tration range of 10−9 –10−3 M. PRISM5 software was used to
sulfonamide (2e). Compound 2e (31.4 mg, 65%) was obtained
as a dark yellow solid.
calculate IC50 values.
1H-NMR (600 MHz, DMSO-d6): 0.98 (m, J = 7.2 Hz, 4H,
(CH2)2); 1.44 (m, J = 7.2 Hz, 3H, CH3); 1.64 (m, J = 7.2 Hz, 2H,
CH2); 7.57 (s, 2H, SO2NH2); 7.65 (s, 1H, Ar–H); 8.05 (m, J = 8.8
Hz, 2H, Ar–H); 8.43 (m, J = 8.8 Hz, 2H, Ar–H). 13C-NMR
(150 MHz, DMSO-d6): 14.2; 20.0; 31.2; 41.0; 101.6; 121.1 (q,
1J(C–F) = 275 Hz); 126.6; 128.9; 139.5; 146.8; 157.1 (q, 2J(C–F) =
36 Hz); 162.8; 165.7. 19F-NMR (565 MHz, DMSO-d6): −70.08 (s,
3F, CF3). LR-MS: 397.1 [M + Na].
Molecular docking studies
The molecular docking experiments were performed using
crystal coordinates from the X-ray crystal structure of COX-1
(ovine, 1EQG, ibuprofen bound in the active site) and COX-2
(murine, 6COX, SC558 bound in the active site) were obtained
from the protein data bank.48,49 Compounds were built using
the builder toolkit of the software package ArgusLab 4.0.1 (Mark,
A. ArgusLab, Version 4.0.1; Thompson Planaria Software LLC:
2-[(4-Iodobenzyl)oxy]-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro- Seattle, WA) and energy minimized using the semi-empirical
methyl)pyrimidine (10). 677 mg of 4-iodobenzyl alcohol
(2.89 mmol) was dissolved in 25 ml of dry THF under an inert
gas atmosphere and at 0 °C, 115 mg of 60% NaH (2.89 mmol)
was added to the stirring mixture. After 5 min, 1.0 g of com-
pound 6 (2.63 mmol) was added. The reaction mixture was
stirred at 0 °C for 1 h and quenched by adding an excess of
1 N HCl. The title compound was extracted in ethyl acetate.
The organic layer was washed with water, dried over sodium
sulfate and solvent removed in vacuo. Compound 10 was puri-
fied using a silica column eluting at 1% MeOH–CH2Cl2 and
obtained as a pale yellow solid (1.0 g, 71% yield).
quantum mechanical method PM3. The monomeric structure of
the enzyme was chosen and the active site was defined around
the ligand. The molecule to be docked in the active site of the
enzyme was inserted in the work space carrying the structure of
the enzyme. The docking program implements an efficient grid
based docking algorithm which approximates an exhaustive
search within the free volume of the binding site cavity. The con-
formational space was surveyed by the geometry optimization of
the flexible ligand (rings are treated as rigid) in combination
with the incremental construction of the ligand torsions. Thus,
docking occurred between the flexible ligand parts of the com-
pound and enzyme. The ligand orientation was determined by a
shape scoring function based on Ascore and the final positions
were ranked by lowest interaction energy values. The interaction
is the sum of the energies involved in H-bond interactions,
hydrophobic interactions and van der Waal’s interactions.
H-bond and hydrophobic interactions between the compound
and the enzyme were explored by distance measurements.
1H-NMR (600 MHz, DMSO-d6): 3.15 (s, 3H, SO2CH3); 5.55
(s, 2H, CH2); 7.32 (m, J = 8.4 Hz, 2H, Ar–H); 7.75 (s, 1H, Ar–H);
7.75 (m, J = 8.4 Hz, 2H, Ar–H); 8.14 (m, J = 8.4 Hz, 2H, Ar–H);
8.33 (m, J = 8.4 Hz, 2H, Ar–H). 13C-NMR (150 MHz, DMSO-d6):
43.7; 69.3; 95.0; 108.5; 121.7 (q, 1J(C–F) = 275 Hz); 123.6; 128.1;
129.2; 131.0; 137.2; 139.8; 143.7; 157.9 (q, 2J(C–F) = 36 Hz);
165.3; 167.7. 19F-NMR (375 MHz, DMSO-d6): −71.0 (s, 3F, CF3).
LR-MS: 557.0 [M + Na].
Radiochemistry
2-[(4-Iodylbenzyl)oxy]-4-[4-(methylsulfonyl)phenyl]-6-(tri-fluoro-
methyl)pyrimidine (11). 500 mg of compound 10 (0.93 mmol)
was dissolved in 100 ml of MeOH and Oxone (2.8 g,
No-carrier-added (n.c.a.) [18F]fluoride was produced via the
18O(p,n)18F nuclear exchange reaction from [18O]H2O (Rotem
8062 | Org. Biomol. Chem., 2013, 11, 8052–8064
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Industries, Hyox oxygen-18 enriched water) on an ACSI TR19/9 radionuclide production and excellent technical support. F.W.
Cyclotron (Advanced Cyclotron Systems Inc., Burnaby, thanks the Dianne and Irving Kipnes Foundation and the
Canada).
Natural Sciences and Engineering Research Council of Canada
[18F]fluoride was trapped on a Waters SepPak® light QMA (NSERC) for supporting this work.
anion exchange cartridge. Radiosynthesis of 4-[18F]fluoro-
benzylamine ([18F]FBA) followed the procedure published by
Way et al.42 Please refer to the publication for a detailed
description of the procedure.
Notes and references
Synthesis of [18F]1a. To 6 mg of compound 6 was added
[18F]FBA in 1 ml of THF (typically 1 GBq of [18F]FBA) in a
sealed vessel. The reaction vessel was heated at 140 °C for
30 min. The mixture was diluted in 10 ml water and passed
onto a SepPak® C18 cartridge, the cartridge was washed with
5 ml water and the title compound eluted using 3 ml of
CH3CN. The volume of the solvent was reduced on a rotary
evaporator to prepare a 1 ml 70/30 CH3CN–H2O formulation
for HPLC injection. The compound was purified using HPLC
(HPLC conditions: isocratic 70/30 CH3CN–H2O; flow rate 3 ml
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The lipophilicity of compound [18F]1a was determined by
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Acknowledgements
The authors would like to thank John Wilson, David Clenden-
ing and Blake Lazurko from the Edmonton PET Center for
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