3-Substituted 1,5-Diaryl-1H-1,2,4-triazoles as Prospective PET Radioligands for Imaging Brain COX-1 in Monkey. Part 1: Synthesis and Pharmacology

Article abstract of DOI:10.1021/acschemneuro.8b00102

Cyclooxygenase-1 (COX-1) is a key enzyme in the biosynthesis of proinflammatory thromboxanes and prostaglandins and is found in glial and neuronal cells within brain. COX-1 expression is implicated in numerous neuroinflammatory states. We aim to find a direct-acting PET radioligand for imaging COX-1 in human brain as a potential biomarker of neuroinflammation and for serving as a tool in drug development. Seventeen 3-substituted 1,5-diaryl-1H-1,2,4-triazoles were prepared as prospective COX-1 PET radioligands. From this set, three 1,5 (4-methoxyphenyl)-1H-1,2,4-triazoles, carrying a 3-methoxy (5), 3 (1,1,1 trifluoroethoxy) (20), or 3-fluoromethoxy substituent (6), were selected for radioligand development, based mainly on their high affinities and selectivities for inhibiting human COX-1, absence of carboxyl group, moderate computed lipophilicities, and scope for radiolabeling with carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.8 min). Methods were developed for producing [11C]5, [11C]20, and [d2-18F]6 from hydroxy precursors in a form ready for intravenous injection for prospective evaluation in monkey with PET.

Full text of DOI:10.1021/acschemneuro.8b00102

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3-Substituted 1,5-Diaryl-1H-1,2,4-triazoles as Prospective PET Radioligands
for Imaging Brain COX-1 in Monkey. Part 1: Synthesis and Pharmacology
Prachi Singh, Stal Shrestra, Michelle Y Cortes-Salva, Kimberly J Jenko,
Sami S. Zoghbi, Cheryl L Morse, Robert B. Innis, and Victor W Pike
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00102 • Publication Date (Web): 20 Apr 2018
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3-Substituted 1,5-Diaryl-1H-1,2,4-triazoles as Prospective PET
Radioligands for Imaging Brain COX-1 in Monkey. Part 1:
Synthesis and Pharmacology
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Prachi Singh,† Stal Shrestha,† Michelle Y. CortesꢀSalva,† Kimberly J. Jenko,† Sami
S. Zoghbi,† Cheryl L. Morse,† Robert B. Innis,† and Victor W. Pike†*
†Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health,
Building 10, Room B3 C346A, 10 Center Drive, Bethesda, Maryland 20892, United States
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3-Substituted 1,5-Diaryl-1H-1,2,4-triazoles as Prospective PET Radioligands for Imaging Brain COX-1 in Monkey.
Part 1: Synthesis and Pharmacology
Prachi Singh, Stal Shrestha, Michelle Y. CortesꢀSalva, Kimberly J. Jenko, Sami S. Zoghbi, Cheryl L. Morse, Robert
B. Innis, and Victor W. Pike
(For Table of Contents Graphic Only)
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ABSTRACT:
Cyclooxygenaseꢀ1 (COXꢀ1) is a key enzyme in the biosynthesis of
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proinflammatory thromboxanes and prostaglandins and is found in glial and neuronal cells
within brain. COXꢀ1 expression is implicated in numerous neuroinflammatory states. We aim
to find a directꢀacting PET radioligand for imaging COXꢀ1 in human brain as a potential
biomarker of neuroinflammation and for serving as a tool in drug development. Seventeen 3ꢀ
substituted 1,5ꢀdiarylꢀ1Hꢀ1,2,4ꢀtriazoles were prepared as prospective COXꢀ1 PET radioligands.
From this set, three 1,5ꢀ(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,4ꢀtriazoles, carrying a 3ꢀmethoxy (5),
3ꢀ(1,1,1ꢀtrifluoroethoxy) (20), or 3ꢀfluoromethoxy substituent (6), were selected for radioligand
development, based mainly on their high affinities and selectivities for inhibiting human COXꢀ1,
absence of carboxyl group, moderate computed lipophilicities, and scope for radiolabeling with
carbonꢀ11 (t1/2 = 20.4 min) or fluorineꢀ18 (t1/2 = 109.8 min). Methods were developed for
producing [11C]5, [11C]20, and [d2ꢀ18F]6 from hydroxy precursors in a form ready for intravenous
injection for prospective evaluation in monkey with PET.
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Keywords: COXꢀ1, PET, radioligand, brain, carbonꢀ11, fluorineꢀ18
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INTRODUCTION
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Cyclooxygenases (COX’s), also known as prostaglandin endoperoxide synthases, occur as two
isoforms, COXꢀ1 and COXꢀ2, that play a rateꢀlimiting step in the syntheses of proinflammatory
thromboxanes and prostaglandins from arachidonic acid.1 Nonꢀsteroidal antiꢀinflammatory
drugs (NSAID’s), such as aspirin and ibuprofen, inhibit both COXꢀ1 and COXꢀ2. Inhibition of
the COX’s by NSAID’s attenuates both central and peripheral inflammation.2 Each COX
isoform is considered to play a distinct role3,4 in these inflammatory processes.
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Although the role of COX’s in peripheral inflammation has been investigated extensively,
very little is known about the roles of the COX’s in central inflammation. Neuroinflammatory
processes are now associated with several neurodegenerative and psychiatric illnesses, such as
Alzheimer disease, Parkinson disease, depression, schizophrenia, autism, and addiction.5
Although COXꢀ2 plays a much larger role than COXꢀ1 in peripheral inflammation, COXꢀ1 is
considered to be a key contributor to neuroinflammation.3 COXꢀ1 is constitutively expressed in
most tissues and organs, including brain.6 In healthy brain, COXꢀ1 is almost exclusively
localized to resting microglia. In response to various types of inflammatory stimuli, microglia
become activated and COXꢀ1 expression increases. Consequently, proinflammatory mediators
are increased, generating oxidative stress and processes that ultimately lead to cytotoxicity and
neuronal loss.
Genetic modulations in rodents7,8,9 and neuroinflammation models in animals8,10 have
unveiled some details of the role of COXꢀ1 in the neuroinflammatory process. For instance,
genetic knockout of COXꢀ1 in mice reduced oxidative stress and neuronal damage compared to
those in wildꢀtype mice.9 In a monkey neuroinflammation model, COXꢀ1 expression increased
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globally in activated microglia and macrophages.10 Taken together, these findings suggest that
COXꢀ1 could be a promising biomarker for studying neuroinflammation.
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An effective radioligand for the selective imaging of COXꢀ1 in the brains of living animal
and human subjects with positron emission tomography (PET) would be a useful tool for
increasing our understanding the roles of COXꢀ1 in neuroinflammation and the progression of
neuropsychiatric disorders. An effective radioligand could also assist in the development of
drugs targeting COXꢀ1.
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Only two candidates have been evaluated as potential directꢀacting PET radioligands for
imaging COXꢀ1 within brain, but neither has been found successful. These radioligands, [18F]1
([18F]SC63217)11 and [11C]2,12 are both 1,5ꢀdiarylpyrazoles (Chart 1), and they failed because
of low enzyme affinity, low brain uptake, and high nonspecific binding. SꢀKetoprofen (4) is a
selective inhibitor of human COXꢀ113, but has poor ability to cross the bloodꢀbrain barrier
because of almost complete ionization of its carboxyl group at physiological pH. To circumvent
this issue with 4, [11C](S)ꢀketoprofen methyl ester ([11C]3; Scheme 1)14 has been studied as a
‘proꢀdrug’ type PET radioligand. After intravenous administration, [11C]3 readily enters brain
where it rapidly undergoes hydrolysis to [11C](S)ꢀketoprofen ([11C]4; Scheme 1). [11C]3 has
been successful for imaging COXꢀ1 in brains of mice14 and rats15 that have been treated with
lipopolysaccharide to induce neuroinflammation with accompanying activated microglia.
Although [11C]3 also enters human brain,16 it does not detect neuroinflammation in Alzheimer’s
disease.17 [11C]4 likely has too low an affinity for human COXꢀ1 (IC50 of 47 nM)13 to be
effective for detection of COXꢀ1 in human brain. Such a low affinity would require very high
concentrations of COXꢀ1 (> 250 nM) to exist in one or more brain regions to provide sufficient
binding potential (Bmax/KD >5)18 for measurement with PET using [11C]4. In general, successful
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radioligands for imaging enzymes in brain have low nanomolar or subꢀnanomolar affinities.19
COXꢀ1 is known to be widely expressed in human brain, primarily in frontal, temporal and
cerebellar cortices, hippocampus (CA2, CA3, and CA4), and caudate, but the absolute
concentrations (Bmax’s values) are unknown.20 Another drawback of using a proꢀdrug type
radioligand, such as [11C]3, is that satisfactory biomathematical modeling of the PET data may
be difficult to achieve because of the extra requirement to consider the kinetics of radioligand
hydrolysis. Hitherto, there have been no directꢀacting radioligands to image COXꢀ1 in human
brain with PET.
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Chart 1. Former candidate PET radioligands for COXꢀ1 based on 1,5ꢀdiarylpyrazoles.
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Scheme 1. Structure of Proꢀdrug Type Radioligand [11C](S)ꢀKetoprofen Methyl Ester
([11C]3) and Hydrolysis of [11C]3 to COXꢀ1 Radioligand [11C]4
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Our eventual aim is to find a directꢀacting PET radioligand to image COXꢀ1 in human brain.
For this purpose, we searched the literature to find compounds that showed properties19,21,22 in
vitro that are desirable for PET radioligand development, such as high potency and selectivity for
inhibiting human COXꢀ1, absence of a carboxyl group, moderate computed lipophilicity (clogD),
and scope for labeling with either carbonꢀ11 (t1/2 = 20 min) or fluorineꢀ18 (t1/2 = 110 min). We
found that the 1,5ꢀdiarylꢀ1,2,4ꢀtriazoleꢀbased COXꢀ1 inhibitor, 5 (FK881) (IC50 = 4.9 nM; clogD
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= 3.72)23 (Chart 2), met these prima facie requirements.
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Chart 2. Structure of 5 (FK881)
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Several patented analogues of 5, having various substituents on the 1Hꢀ1,2,4ꢀtriazole ring,
also have high inhibitory potencies for human COXꢀ1 (IC50 < 10 nM) with appreciable
selectivities (>10ꢀfold) over inhibitory potencies for human COXꢀ2.24,25 On this basis, we
decided to prepare 5 and several analogues that might present even better properties for PET
radioligand development. Altogether, seventeen compounds were prepared and tested for COXꢀ
1 and COXꢀ2 inhibitory potencies in enzymatic immunoassays on rat, monkey, and human
blood. cLogD values were also calculated. Three COXꢀ1 inhibitors emerged as meriting
labeling with a positronꢀemitter for subsequent evaluation of radioligand behavior with PET in
monkey.
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RESULTS AND DISCUSSION
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Chemistry. All the prepared inhibitors feature a common 3ꢀsubstituted 1,5ꢀbis(aryl)ꢀ1Hꢀ
1,2,4ꢀtriazole scaffold. Compounds 5−14 (Table 1) were chosen for synthesis based on
reported24 high potency for inhibiting human COXꢀ1 (IC50 < 10 nM) and much lower potency for
inhibiting human COXꢀ2 (IC50 >100 nM), plus our observations of i) a clogD value of less than
4, ii) fewer than 8 heteroatoms, iii) a predicted pKa of less than 9, iv) a calculated tPSA of less
than 80 Å, v) a molecular weight of less than 500 Da, and vi) the presence of at least one group
amenable to labeling with carbonꢀ11 or fluorineꢀ18 by radioalkylation with either
[11C]iodomethane or [d2ꢀ18F]fluorobromomethane (Table 1). Such properties are generally
considered desirable in candidate PET radioligands.19,19 Analogues (24, 26−28, 31, and 32) of
these inhibitors, with unknown inhibitory potencies for COXꢀ1 or COXꢀ2 but otherwise
desirable radioligand properties, were also chosen for synthesis. These included a 2ꢀfluoroꢀ
pyridyl compound (24) and four fluoromethyl compounds (26−28, 32) with potential to be
labeled with fluorineꢀ18 or with carbonꢀ11. All the inhibitors, except for 14, carried a relatively
small substituent in the 3ꢀposition. In some inhibitors (10, 12, 13, 31, and 32), the pendant 1ꢀaryl
or 5ꢀaryl substituent, or both, differed from a more usual 4ꢀmethoxyphenyl substituent.
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’
Table 1. Structures, IC50 s and cLogD Values for COXꢀ1 Inhibitors
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IC50 (nM)a,b
Inhibitor
R1
R2
R3
clogD
COXꢀ1
Human
COXꢀ2
Monkey
40 ± 17
4.6 ± 1.3
3.6 ± 1.0
10.0 ± 0.5
11 ± 1
Monkey
400 ± 113
> 1000
>1000
n.d.c
>1000
n.d.
200 ± 45
n.d.
Human
264 ± 110
>1000
841 ± 102
>1000
2.76
3.72
3.84
4.01
2.99
2.88
3.62
3.72
2.75
2.74
20 ± 7
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OMe
OCH2F
SMe
SO2Me
SOMe
OMe
CF3
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
Ph
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀCNC6H4
5.0 ± 5.2
4.0 ± 2.0
1.0 ± 3.6
6.0 ± 0.2
2.0 ± 0.9
1.0 ± 1.4
3.0 ± 3.2
4.0 ± 5.9
6.0 ± 3.0
>1000
>1000
n.d.
3.6 ± 1.8
n.d.
5.0 ± 3.0
5.1 ± 1.5
n.d.
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13
4ꢀMeOC6H4
4ꢀMeOC6H4
2ꢀMeOꢀpyridinꢀ5ꢀyl
4ꢀMeOC6H4
2ꢀMeOꢀpyridinꢀ5ꢀyl
4ꢀMeOC6H4
247 ± 614
>1000
CF3
CF3
125 ± 34
n.d.
776 ± 71
4ꢀMeOC6H4
4ꢀMeOC6H4
3.71
125 ± 2
42 ± 8
n.d.
>1000
14
OCH2CF3
CF3
SCH2F
SOCH2F
SO2CH2F
OMe
4ꢀMeOC6H4
2ꢀFꢀpyridinꢀ5ꢀyl
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
Ph
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
4ꢀMeOC6H4
3.59
3.38
3.56
2.33
3.17
3.76
3.96
1.0 ± 0.3
10 ± 3.0
6.4 ± 0.8
5.0 ± 2.0
25 ± 3
1.0 ± 0.2
2.0 ± 0.1
5.0 ± 3.9
3.0 ± 1.7
3.0 ± 1.2
3.00 ± 0.06
1.7 ± 0.6
>1000
n.d.
n.d.
545 ± 102
n.d.
>1000
>1000
>1000
>1000
>1000
>1000
100 ± 25
20
24
26
27
28
31
32
3.0 ± 2.5
100
n.d.
n.d.
OCH2F
Ph
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2.80
>1000
>1000
1.0 ± 0.5
1.0 ± 0.1
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a All data are mean ± SD (n = 3).
b COXꢀ1 rat whole blood IC50’s for 4, 5, 6, and 20, were 130, 418, 280, and 15 nM, respectively.
c n.d. = not determined.
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Inhibitors 5−14 were prepared as described.24 The known inhibitor 20 was prepared by a
different route to that reported.24 Thus, 2ꢀ(4ꢀmethoxyphenyl)hydrazine carboxamide (15)24 was
treated with 4ꢀ(benzyloxy)benzoyl chloride to give 2ꢀ(4ꢀ(benzyloxy)benzoyl)ꢀ2ꢀ(4ꢀ
methoxyphenyl)hydrazine carboxamide (16). Cyclization of 16 under basic conditions gave 5ꢀ
(4ꢀ(benzyloxy)phenyl)ꢀ1ꢀ(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀol (17). Alkylation of 17 with
2ꢀchloroꢀ1,1,1ꢀtrifluoroethane gave 18. Treatment of 18 with boron trichloride selectively
removed the Oꢀbenzyl group to give the phenol 19, which later served as a precursor for
radiolabeling. Inhibitor 20 was then obtained by treatment of 19 with iodomethane under basic
conditions (Scheme 2). All steps proceeded in at least moderately high yields.
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Scheme 2. Syntheses of Labeling Precursor 19 and COXꢀ1 Inhibitor 20a
a Reagents and conditions: (i) 4ꢀ(benzyloxy)benzoyl chloride, toluene, pyridine, 110 °C, 1 h; (ii)
10% aq KOH, EtOH, 60 °C, 1.5 h; (iii) 2ꢀchloroꢀ1,1,1ꢀtrifluoroethane, KI, K2CO3, DMF, room
temperature, 24 h; (iv) 1 M BCl3, CH2Cl2, − 20 °C, 3 h, then 0 °C for 1 h; (v) MeI, K2CO3, DMF,
room temperature, 16 h.
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Inhibitors 24 and 26−28 are new. Inhibitor 24 was obtained in three steps starting with the
addition of 2,2,2ꢀtrifluoroacetimidamide to (4ꢀmethoxyphenyl)hydrazine hydrochloride (21) to
give crude (Z)ꢀ2,2,2ꢀtrifluoroꢀN'ꢀ(4ꢀmethoxyphenyl)acetohydrazonamide (22). Crude 22 was
then treated with 6ꢀchloronicotinoyl chloride to effect cyclization to the chloro intermediate 23 in
good yield. Fluoro for chloro exchange in 23 then gave 24 in moderate yield (Scheme 3).
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Scheme 3. Synthesis of COXꢀ1 Inhibitor 24a
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a Reagents and conditions: (i) 2,2,2ꢀtrifluoroacetimidamide, Et3N, MeOH, room temperature, 18
h; (ii) 6ꢀchloronicotinoyl chloride, pyridine, 1,4ꢀdioxane, 101 °C, 4 h; (iii) KF, 18ꢀcrownꢀ6,
DMSO, 100 °C, 24 h.
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Treatment
of
1,5ꢀbis(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀthiol
(25)24
with
fluoroiodomethane in the presence of sodium hydride gave the fluoromethyl thioether inhibitor
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26 in good yield (Scheme 4). Timeꢀ and stoichiometryꢀcontrolled oxidations of 26 then gave the
sulfoxide 27, and finally the sulfone 28 in high yields (Scheme 4).
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Scheme 4. Syntheses of COXꢀ1 Inhibitors 26−28a
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a Reagents and conditions: (i) ICH2F, K2CO3, DMF, 100 °C, 3.5 h; (ii) mCPBA (1.5 equiv),
CH2Cl2, room temperature, 3 h; (iii) mCPBA (4 equiv), CH2Cl2, room temperature, 5 h.
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Inhibitors 31 and 32 were obtained in three steps, each starting from
2ꢀ(4ꢀmethoxyphenyl)hydrazinecarboxamide (15) (Scheme 5). Treatment of 15 with benzoyl
chloride gave the amide 29 in good yield. Cyclization of 29 with alcoholic potassium hydroxide
gave the hydroxy intermediate 1ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀol (30) in high
yield. Treatment of 30 with iodomethane or fluoroiodomethane under basic conditions gave the
methoxy and fluoromethoxy ethers 31 and 32, respectively, in moderate yields.
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Scheme 5. Syntheses of COXꢀ1 Inhibitors 31 and 32a
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a Reagents and conditions: (i) benzoyl chloride, pyridine, toluene, 110 °C, 4 h; (ii) 10% KOH,
EtOH, 60 °C, 2 h; (iii) MeI, K2CO3, DMF, room temperature, 16 h; (iv) ICH2F, K2CO3, DMF,
100 °C, 3.5 h.
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COXꢀ1 and COXꢀ2 Inhibition Assays. The patent24 first describing the synthesis of COXꢀ1
inhibitors 6−14 had not specified human COXꢀ1 inhibitory potencies beyond stating that the
IC50’s were less than 10 nM. Because PET radioligand binding potential (Bmax/KD) is directly
proportional to radioligand affinity (1/KD) and low nanomolar affinities are often required in
successful PET radioligands19,19, more precise measures of human COXꢀ1 inhibitory potency
were needed. Moreover, because radioligands must be evaluated with PET in animals preceding
any evaluation in human subjects, we also needed to measure COXꢀ1 inhibitory potencies in a
species available to us for PET imaging, namely rat or rhesus monkey. We also needed to be
sure that inhibitory potencies in the selected preclinical species would be close to those for
human. In addition to high affinity for the imaging target, PET radioligands should show much
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lower affinity for offꢀtarget sites. Of main concern was that many COX inhibitors do not show
selectivity for binding to either isoform because of their close structural homology.26 Therefore,
in selecting candidate PET radioligands, we sought very low potency for inhibiting human COXꢀ
2, tantamount to over 100ꢀfold greater potency for inhibition of COXꢀ1 over COXꢀ2.
Consequently, we also assayed our set of compounds for inhibitory potency on monkey and
human blood COXꢀ2. The assay kit for COXꢀ2 could not however be used for rat blood.
Results from the testing of 4, the seventeen triazoleꢀbased inhibitors, and the COXꢀ2 selective
inhibitor 34 for inhibitory potencies in COXꢀ1 and COXꢀ2 enzymatic immunoassays13 using
fresh blood from human and monkey are shown in Table 1. Compound 4, a nonꢀtriazole,
showed only moderate potency for inhibition of human COXꢀ1, as previously reported,13 and
only moderate selectivity for inhibition of this isoform over human COXꢀ2. The results that we
obtained for inhibition of human whole blood COXꢀ1 (IC50 = 5 nM) and human whole blood
COXꢀ2 (IC50 > 1000 nM) by the triazole 5 (FK881) (Table 1) also agreed well with those
already reported (4.9 and 3200 nM, respectively).2323 All the other prepared triazoles, except 14,
showed subꢀ10ꢀnM potency for inhibition of human COXꢀ1 and above 100ꢀnM potency for
inhibition of human COXꢀ2, in accord with the patent report2424 for inhibitors 6−14 and 20.
Among all the tested inhibitors, 14 has the bulkiest 3ꢀsubstituent and was found to have the
lowest COXꢀ1 inhibitory potency (IC50 , 42 nM). The nonꢀtriazole 3427 was confirmed to be a
highly potent and selective COXꢀ2 inhibitor. Generally, the potencies of the triazoles for
inhibiting human COXꢀ1 resembled their potencies for inhibiting rhesus monkey COXꢀ1; in just
a few cases were somewhat higher (7, 8, 24, and 28). Therefore, monkey appeared to be an
excellent animal model for assisting the selection of candidate radioligands that might be later
evaluated in human.
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Inhibitors 4−6 and 20 were also tested in a COXꢀ1 enzymatic immunoassay using fresh rat
blood. However, these compounds showed much less potency for inhibiting COXꢀ1 from rat
than for inhibiting COXꢀ1 from monkey or human (Table 1). Therefore, we concluded that rat
was not useful for the preꢀclinical evaluation of candidate COXꢀ1 radioligands with PET.
Selection of compounds for radiolabeling was based on various considerations. First,
compounds with monkey and/or human COXꢀ1 IC50’s greater than 5 nM were omitted from
consideration (8, 12−14, 24, 26, 28, 32), except 7, which showed equally highest inhibitory
potency for human COXꢀ1 (IC50 ~ 1 nM). Compound 11 was omitted from consideration
because of less than 100ꢀfold selectivity for inhibiting human COXꢀ1 over human COXꢀ2. Two
compounds, 6 and 27, with potential for labeling with fluorineꢀ18, presented quite similar
inhibitory potencies. Compound 6 was preferred because of its potential to be labeled in a
singleꢀstep with [d2ꢀ18F]fluorobromomethane as labeling agent, whereas the labeling of 27 would
have required an extra step of thioether oxidation. Compounds 5, 7, 9, 10, 20, and 31 showed
high inhibitory potency for human COXꢀ1 with 5, 7, 9, 20, and 31 showing well over 100ꢀfold
less inhibitory potency for human COXꢀ2. From this group, we selected 5 and 20 for labeling
with carbonꢀ11 after we had considered other factors, such as access to labeling precursor.
Pharmacological Screening. COXꢀ1 inhibitors 5−9, 11−14, 20, 24, and 26−28 were found
to have Ki values exceeding 10 µM for a wide range of human recombinant receptors and
binding sites (5ꢀHT1A, 5ꢀHT1B, 5ꢀHT1D, 5ꢀHT1E, 5ꢀHT2A, 5ꢀHT2B, 5ꢀHT2C, 5ꢀHT3, 5ꢀHT5A, 5ꢀHT6
and 5ꢀHT7, α1A, α1B, α1D, α2A, α2B, β1, β 2, β 3, D1, D2, D4, D5, DAT, DOR, GABA, H1, H2, H3, H4,
KOR, M1, M2, M4, M5, MOR, NET, PBR, SERT, σ1, σ2, σ2 PC12, and the rat brain
benzodiazepine site). These results attest to the general specificity of this class of compounds for
inhibition of human COXꢀ1 versus interactions with other binding sites and receptors.
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Radiochemistry. [11C]5 was prepared by methylation of the hydroxy triazole 33 with noꢀ
carrierꢀadded (NCA) [11C]iodomethane in DMF under basic conditions at room temperature for 5
min. [d2ꢀ18F]6 was obtained by treating 33 with NCA [d2ꢀ18F]fluorobromomethane, cesium
carbonate, and 18ꢀcrownꢀ6 at 80 °C for 10 min. [11C]20 was obtained by treating the phenol
precursor 19 with NCA [11C]iodomethane in DMSO under basic conditions at 70 °C for 3 min.
(Scheme 6). These labeling methods were fully automated and extended to include first pass
HPLC separation and formulation for intravenous injection. Each radioligand was readily
obtained in activities sufficient for subsequent PET experiments in monkey. The formulated
radioligands had high radiochemical purities, moderately high molar activities, and negligible
chemical impurities (see Supporting Information).
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Scheme 6. Syntheses of Radioligands [11C]5, [d2ꢀ18F]6, and [11C]20
a Reagents and conditions: (i) [11C]iodomethane, DMF, TBAH, room temperature, 5 min; (ii)
[18F]FCD2Br, Cs2CO3, 18ꢀcrownꢀ6, 80 °C, 10 min; (iii) [11C]iodomethane, DMSO, KOH, 70 °C,
3 min.
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CONCLUSIONS
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Among the evaluated 3ꢀsubstituted 1,5ꢀdiarylꢀ1Hꢀ1,2,4ꢀtriazoles, inhibitors 5, 6, and 20 showed
the most favorable pharmacology for further evaluation as potential PET radioligands for
imaging brain COXꢀ1 in monkey and human. Each of these inhibitors was readily labeled with
either carbonꢀ11 or fluorineꢀ18 in moderately high molar activity and was readily purified and
formulated for intravenous injection. Part 2 describes the evaluation of these three candidate
radioligands in monkey with PET for the quantitative imaging of COXꢀ1 in brain.28
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EXPERIMENTAL SECTION
Materials and Methods. (S)ꢀKetoprofen methyl ester (3) was prepared29 from (S)ꢀ
ketoprofen (4), purchased from Sigma Aldrich (St. Louis, MO). The following compounds were
prepared
as
previously
described:
inhibitors
5−14;24
starting
materials
2ꢀ(4ꢀmethoxyphenyl)hydrazinecarboxamide
(15)25
and (4ꢀmethoxyphenyl)hydrazine
hydrochloride (21);24 and the labeling precursors 1,5ꢀbis(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀ
24
thiol
(25)25
and
1,5ꢀbis(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀol
(33)
.
[2ꢀ(4ꢀMethanesulfonylphenyl)ꢀ6ꢀmethoxypyrimidinꢀ4ꢀyl]thiophenꢀ2ꢀylmethylamine (34), which
is highly selective for inhibiting human COXꢀ2 over human COXꢀ1, was prepared as previously
described.27
COXꢀ1 and COXꢀ2 enzymatic immunoassay kits were purchased from Cayman Chemical
(Ann Arbor, MI). IC50 values were obtained with a detection platform spectrophotometer
(SpectraMax i3x MultiꢀMode; Molecular Devices LLC; CA).
Reagents and solvents were purchased from Sigma Aldrich (St Louis, MO) and used without
further treatment. Airꢀsensitive reagents were stored under nitrogen in a glovebox. Precoated
silica gel layers (0.2 mm; Grace; Deerfield, IL) were used for TLC and compounds were
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observed under 254 nm light. Synthesized compounds were isolated with a Teledyne Rf+
chromatography apparatus using silica gel cartridges (24 g, RediSep®Rf; Isco: Lincoln, NE) with
hexane and ethyl acetate as mobile phases (general method A). To achieve greater than 95%
purity, some compounds were purified with preparative reversed phase HPLC (Beckman
Coulter; Fullerton, CA) on an XBridge C18 column (19 × 150 mm, 10 µm; Waters; Milford MA)
eluted at 6 mL/min with a gradient mobile phase composed of aq. ammonium hydroxide (0.25
mM) (A) and acetonitrile (B) starting at 20% B rising linearly to 80% B at 30 min (general
method B). Eluate was monitored for absorbance at 254 nm. Melting points were determined on
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a SMP20 apparatus (Stuart; Staffordshire, UK). H (400.13 MHz), 13C NMR (100.62 MHz), and
19F NMR (376.46 MHz) spectra were recorded on an Avance 400 instrument (Bruker; Billerica,
1
13
MA). H and CꢀNMR chemical shifts are reported in δ units (ppm) downfield relative to the
chemical shift for tetramethylsilane. Abbreviations bs, d, m, s, and t denote broad singlet,
doublet, multiplet, singlet, and triplet, respectively. LCꢀMS (ESI) was performed on a Velos Pro
instrument (Thermo Scientific, San Jose, CA) using reversed phase chromatography on a Luna
C18 column (50 × 2 mm, 3 µm; Phenomenex: Torrance, CA) eluted at 200 µL/min with a
gradient mobile phase composed of waterꢀmethanolꢀacetic acid (90:10:0.5 by vol.; C) and
methanolꢀacetic acid (100:0.5, v/v; D), starting with C:D at 80:20 for 0.25 min, changed linearly
to 20:80 over 3 min, and then held at 20:80 for 3.5 min (method 1) or starting with C:D 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 2).
HRMS data were obtained at the Mass Spectrometry Laboratory, School of Chemical Sciences,
University of Illinois at UrbanaꢀChampaign (Urbana, IL) under electron ionization conditions
using a doubleꢀfocusing highꢀresolution mass spectrometer (Micromass, Waters; Milford, MA).
The chemical purities of all prepared inhibitors were shown to be > 95% with analytical reversed
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phase HPLC on a Luna C18 (2) column (250 × 4.6 mm, 10 µm; Phenomenex; Torrance, CA)
eluted isocratically with ammonium formate (10 mM)−acetonitrile (50:50) at 2 mL/min (general
method C). Eluate was monitored for absorbance at 254 nm (see Supporting Information).
γꢀRadioactivity from carbonꢀ11 or fluorineꢀ18 was measured with a calibrated dose calibrator
(Atomlab 300, Biodex Medical Systems, Shirley, NY) or an automatic γꢀcounter (Wizard 3”,
1480; PerkinElmer; Waltham, MA). Radioactivity measurements were corrected for physical
decay. All radiochemistry was performed in a leadꢀshielded hotꢀcell for personnel protection
from radiation. The molar activities30 of prepared radioligands were obtained by HPLC
measurement of carrier associated with a measured activity of analyte. The HPLC system
response (absorbance at 254 or 274 nm per mass of ligand) was calibrated by injections of
reference compound of known amounts.
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cLogD values and pKa values were computed with Pallas for Windows software version 3.8
in default option (CompuDrug International; Bal Harbor, FL). tPSA’s were estimated with
ChemDraw version 10.
Grouped data are reported as mean ± SD.
Synthesis
2-(4-(Benzyloxy)benzoyl)-2-(4-methoxyphenyl)hydrazinecarboxamide (16). A mixture of
2ꢀ(4ꢀmethoxyphenyl)hydrazinecarboxamide (15; 2.0 g, 1.1 mmol), 4ꢀ(benzyloxy)benzoyl
chloride (3.40 g, 13.8 mmol, 1.25 equiv) and pyridine (1.1 g, 13.8 mmol, 1.25 equiv) in toluene
(30 mL) was heated for 1 h at 110 ˚C. Ethyl acetate (450 mL), THF (50 mL), and water (100
mL) were added, and the reaction mixture was sonicated for 20 min. The resultant solid was
filtered off, washed with cold water, and dried under reduced pressure to give 16 as a white solid
(3.87 g; 90%). All characterization data agree with literature values.25
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5-(4-(Benzyloxy)phenyl)-1-(4-methoxyphenyl)-1H-1,2,4-triazol-3-ol (17). A mixture of 16
(0.40 g, 1.02 mmol), aq KOH solution (10% w/v; 2.7 mL) and anhydrous ethanol (99.5%; 200
proof; 1.35 mL) was heated at 65 °C for 1.5 h and then cooled to room temperature. Solvent was
removed under reduced pressure. Hydrochloric acid (2 M) was then added dropwise to the
residue until the pH became 2. The resultant white solid was filtered off, washed with cold water
and dried under vacuum to give 17 (0.30 g; 78%). All characterization data agree with literature
values.25
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5-(4-(Benzyloxy)phenyl)-1-(4-methoxyphenyl)-3-(2,2,2-trifluoroethoxy)-1H-1,2,4-triazole
(18). Compound 17 (0.5 g, 1.34 mmol), 2ꢀchloroꢀ1,1,1ꢀtrifluoroethane (0.8 g, 6.7 mmol, 5
equiv), KI (0.67 g, 4.02 mmol, 3 equiv), and K2CO3 (0.93 g, 6.7 mmol, 5 equiv) were mixed
together in DMF (5 mL) and stirred at room temperature for 24 h. Water (25 mL) and ethyl
acetate (20 mL) were poured into the reaction mixture. The organic layer was separated off,
washed with brine (25 mL × 2) and dried (MgSO4). Solvent was removed under reduced
pressure and the residue was purified with flash chromatography (20−50% ethyl acetate in
hexane; general method A) to give 18 as a white solid (0.47 g; 77%). All characterization data
agree with literature values.25
4-(1-(4-Methoxyphenyl)-3-(2,2,2-trifluoroethoxy)-1H-1,2,4-triazol-5-yl)phenol (19).
A
solution of BCl3 (1.0 M) in dichloromethane (0.5 mL) was added dropwise to a solution of 18
(100 mg, 0.22 mmol) in dichloromethane (6 mL) at −20 °C under inert atmosphere. The
resulting mixture was stirred for 3 h at −20 °C, and then for 1 h at 0 °C. Solvent and unreacted
BCl3 were removed under reduced pressure.
The residue was purified with flash
chromatography (10−20% ethyl acetate in hexane; general method A) to give 19 as a white solid
(51 mg; 64%). All characterization data agree with literature values.25
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1,5-Bis(4-methoxyphenyl)-3-(2,2,2-trifluoroethoxy)-1H-1,2,4-triazole (20). Compound 19
(1.0 g, 3.4 mmol), iodomethane (2.4 g, 17 mmol, 5 equiv), K2CO3 (1.4 g, 10.2 mmol, 3 equiv)
and DMF (5 mL) were mixed in a oneꢀneck roundꢀbottom flask and stirred at room temperature.
Reaction progress was monitored with TLC (silica gel; ethyl acetate−hexane; 1:1 v/v). After 16
h, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (40 mL ×
3). The organic layers were combined and washed with brineꢀwater (1: 1 v/v; 40 mL × 2) and
dried (MgSO4). Solvent was removed under reduced pressure, and the residue was purified with
flash chromatography (20−50% ethyl acetate in hexane; general method A), followed by
preparative HPLC (general method B) to give 20 as a white solid (0.97 g; 75%). Mp 91.4–93.2
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°C. H NMR (CD3OD, δ) 7.40–7.37 (m, 2H), 7.31–7.26 (m, 2H), 7.03–6.99 (m, 2H), 6.95–6.88
(m, 2H), 4.87 (q, J = 8.5 Hz, 2H), 3.85 (s, 3H), 3.80 (s, 3H). 13C NMR (CDCl3, δ) 166.27,
161.01, 159.86, 153.48, 131.07, 130.28, 127.05, 124.36, 121.60, 119.68, 118.84, 114.62, 113.96,
66.25, 65.89, 65.52, 65.16, 55.57, 55.31. 19F NMR (DMSOꢀd6, δ) −76.10. HPLC (tR = 7.5 min;
99% purity). HRMSꢀESI (m/z): [M+H]+ calcd for C18H16F3N3O3, 380.1222; found, 380.1221.
(Z)-2,2,2-Trifluoro-N'-(6-methoxypyridin-3-yl)acetohydrazonamide
(22).
(4ꢀ
methoxyphenyl)hydrazine hydrochloride (21, 2.52 g, 14.4 mmol) was treated with
2,2,2ꢀtrifluoroacetimidamide (1.79 g, 15.9 mmol, 1.11 equiv) in methanol (10 mL) in the
presence of triethylamine (2.8 g, 21.6 mmol, 1.5 equiv) under an inert atmosphere, at room
temperature for 18 h. The reaction mixture was then poured into a mixture of water (10 mL) and
ethyl acetate−THF (9: 1 v/v; 25 mL). The organic layer was separated off, washed with brine
(20 mL × 2) and dried (MgSO4). The solvent was removed under reduced pressure and the
resultant thick black liquid (22) was used without further purification to prepare 23.
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2-Chloro-5-(1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-1,2,4-triazol-5-yl)pyridine (23).
Compound 22 (1.0 g, 4.3 mmol) and pyridine (0.5 mL) were mixed in 1,4ꢀdioxane (11 mL) in a
twoꢀnecked roundꢀbottom flask. 6ꢀChloronicotinoyl chloride (0.83 g, 4.7 mmol, 1.1 equiv) was
dissolved separately in 1,4ꢀdioxane (11 mL) and added dropwise to the reaction mixture. The
resulted mixture was refluxed at 101 °C for 4 h. After cooling the mixture to room temperature,
hydrochloric acid (0.1 M; 6.4 mL) was added with stirring to neutralize the unreacted base. This
mixture was then poured into a mixture of ethyl acetate (50 mL) and water (50 mL). The organic
layer was separated off, washed with brine, and dried (MgSO4). Solvent was then removed
under reduced pressure. The residue was purified with flash chromatography (30−60% ethyl
acetate in hexane; general method A) to give 23 as a light brown solid (1.06 g; 70%). Mp
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103.0−105.3 °C. H NMR (CDCl3, δ): 8.48 (dd, J = 2.5, 0.8 Hz, 1H), 7.89 (dd, J = 8.4, 2.5 Hz,
1H), 7.37 (dd, J = 8.4, 0.8 Hz, 1H), 7.35–7.27 (m, 2H), 7.05–6.96 (m, 2H), 3.88 (s, 3H). 13C
NMR (CDCl3 δ): 161.04, 154.32, 153.92, 153.58, 152.23, 149.33, 138.71, 129.42, 127.01,
124.44, 121.97, 120.40, 115.25, 55.71. 19F NMR (DMSOꢀd6, δ) −63.72. HRMSꢀESI (m/z):
[M+H]+ calcd for C14H9ClF3N4O, 355.0573; found, 355.0573.
2-Fluoro-5-(1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-1,2,4-triazol-5-yl)pyridine
(24).
Compound 23 (0.50 g, 1.4 mmol), potassium fluoride (0.12 g, 2.1 mmol, 1.5 equiv), 18ꢀcrownꢀ6
(0.37 g, 1.4 mmol) and DMSO (2 mL) were mixed together under an inert atmosphere in a
pressure tube and heated at 100 °C for 24 h. Water (20 mL) and ethyl acetate (20 mL) were
added. The organic layer was separated off, washed with brine (10 mL × 2), dried (MgSO4), and
concentrated to give the crude product, which was purified with preparative HPLC (general
1
method B) to afford 24 as a white solid (0.02 g; 43%). Mp 176.7–178.0 °C. H NMR (CD3OD,
δ) 8.39–8.33 (m, 1H), 8.03 (ddd, J = 8.6, 7.4, 2.5 Hz, 1H), 7.46–7.36 (m, 2H), 7.13 (ddd, J = 8.6,
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2.8, 0.7 Hz, 1H), 7.11–7.06 (m, 2H), 3.87 (s, 3H). 13C NMR (CD3OD, δ) 166.91, 164.49,
162.62, 154.21, 149.51, 149.35, 143.61, 143.52, 130.86, 128.57, 122.97, 122.92, 116.11, 111.16,
110.79, 56.24. HPLC (general method C; tR = 8.68 min; 98.6% purity). HRMSꢀESI (m/z):
[M+H]+ calcd for C15H10F4N4O, 366.1063; found, 366.1063.
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3-(Fluoromethylthio)-1,5-bis(4-methoxyphenyl)-1H-1,2,4-triazole
(26).
1,5ꢀBis(4ꢀ
methoxyphenyl)ꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀthiol (25; 0.53 g, 1.68 mmol) was heated at 100 °C for 3.5 h
with fluoroiodomethane (1.3 g, 8.4 mmol, 5 equiv) and potassium carbonate (0.7 g, 5.04 mmol, 3
equiv) in DMF (5 mL) and cooled. After 3.5 h, the reaction mixture was cooled to room
temperature. Water (25 mL) and ethyl acetate (20 mL) were poured into the reaction mixture.
The organic layer was separated off, washed with brine (20 mL × 2), and dried (MgSO4).
Solvent was removed under reduced pressure and the residue was purified with flash
chromatography (30ꢀ60% ethyl acetate in hexane; (general method A) followed by preparative
HPLC (general method B) to give 26 as a white solid (0.43 g; 74%). Mp 93.4−95.6 °C. 1H
NMR (CDCl3, δ) = 7.45–7.41 (m, 2H), 7.29–7.25 (m, 2H), 6.94–6.90 (m, 2H), 6.85–6.81 (m,
2H), 6.17 (s, 1H), 6.04 (s, 1H), 3.84 (s, 3H), 3.80 (s, 3H). 13C NMR (CD3OD, δ) = 162.94,
161.90, 158.60, 158.57, 157.02, 131.94, 131.59, 128.44, 120.27, 115.78, 115.19, 87.08, 84.91,
56.18, 55.95. 19F NMR (DMSOꢀd6, δ) = −188.39. HPLC (general method C; tR = 10.4 min;
99.8% purity). HRMSꢀESI (m/z): [M+H]+: calcd for C17H16FN3O2S, 346.1026; found, 346.1019
3-(Fluoromethylsulfinyl)-1,5-bis(4-methoxyphenyl)-1H-1,2,4-triazole (27). A mixture of 26
(100 mg, 0.31 mmol) and mCPBA (76 mg, 0.46 mmol, 1.5 equiv) in dichloromethane (1 mL)
was stirred at room temperature for 3 h. Dichloromethane (20 mL) and saturated sodium
bicarbonate solution (10 mL) were poured into the mixture. The organic layer was separated off
and dried (MgSO4). The solvent was removed under reduced pressure, and the residue was
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purified with preparative HPLC (general method B) to give 27 as a light yellow oil, which
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solidified on storage at 4 °C (98 mg; 88%). Mp 116.6−118.4 °C. H NMR (CDCl3, δ) 7.50–7.45
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(m, 2H), 7.32–7.28 (m, 2H), 6.98–6.94 (m, 2H), 6.86–6.84 (m, 2H), 5.82–5.77 (m, 1H), 5.71–
5.65 (m, 1H), 3.86 (s, 3H), 3.82 (s, 3H). 13C NMR (CDCl3, δ) 161.50, 161.18, 161.08, 160.44,
156.33, 130.56, 130.42, 126.95, 118.68, 114.77, 114.17, 97.65, 95.50, 55.63, 55.39. 19F NMR
(DMSOꢀd6, δ) −183.07. HPLC (general method C; tR = 12.2 min; 99.2% purity). HRMSꢀESI
(m/z): [M+H]+, calcd for C17H16FN3O3S, 362.0975; found, 362.0969.
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3-(Fluoromethylsulfonyl)-1,5-bis(4-methoxyphenyl)-1H-1,2,4-triazole (28). A mixture of
26 (100 mg, 0.31 mmol) and mCPBA (200 mg, 1.16 mmol, 4 equiv) in dichloromethane (1 mL),
was stirred at room temperature for 5 h. The solvent was then removed under reduced pressure.
The residue was purified with preparative HPLC (general method B) to give 28 as a white solid
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(91 mg; 78%). Mp 127.3−128.1 °C. H NMR (CD3OD, δ) 7.41–7.37 (m, 2H), 7.32–7.28 (m,
2H), 7.01–6.97 (m, 2H), 6.89–6.84 (m, 2H), 5.67 (s, 1H), 5.56 (s, 1H), 3.79 (s, 3H), 3.74 (s, 3H).
13C NMR (CD3OD, δ) 163.41, 162.46, 159.86, 158.28, 131.89, 131.48, 128.52, 119.52, 115.94,
115.30, 93.43, 91.27, 56.21, 55.97. 19F NMR (DMSOꢀd6, δ) −187.84. HPLC (general method C;
tR = 7.9 min, >99.9% purity). HRMSꢀESI (m/z): [M+H]+ calcd for C17H16FN3O4S, 378.0924;
found, 378.0919
2-Benzoyl-2-(4-methoxyphenyl)hydrazine-1-carboxamide (29).
A
mixture of
2ꢀ(4ꢀmethoxyphenyl)hydrazinecarboxamide (15; 1.0 g, 5.5 mmol), benzoyl chloride (0.96 g, 6.87
mmol, 1.25 equiv) and pyridine (1.55 g, 6.9 mmol, 1.25 equiv) in toluene (30 mL) was heated for
4 h at 110 ˚C. A mixture of ethyl acetate (450 mL), THF (50 mL) and water (100 mL) was
added and sonicated for 20 min. The white precipitate was filtered off, washed with cold water
and dried under reduced pressure to give 29 (1.07 g; 68%). Mp 130.3−132.1 °C. 1H NMR
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(DMSOꢀd6, δ) 8.89 (s, 1H), 7.52 (s, 2H), 7.40−7.28 (m, 5H), 6.90 (d, J = 8.02 Hz), 3.74 (s, 2H),
3.41 (bs, 2H). 13C NMR (DMSOꢀd6, δ) 157.84, 136.43, 130,21, 128.13, 126.99, 114.08, 55.74,
49.06. HRMSꢀESI (m/z): [M+H]+ calcd for C18H16F3N3O3, 308.1011; found, 308.1006.
1-(4-Methoxyphenyl)-5-phenyl-1H-1,2,4-triazol-3-ol (30). A mixture of 29 (0.62 g, 2.17
mmol), aq KOH solution (10% w/v; 8 mL) and ethanol (99.9%; 200 proof; 4 mL) was heated at
60 °C for 2 h and then cooled. Solvent was removed under reduced pressure. Hydrochloric acid
(2 M) was then added dropwise until the reaction mixture reached pH 2. The white precipitate
was filtered off, washed with cold water, and dried under vacuum to give 30 (0.48 g; 83%). Mp
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191.0−192.2 °C. H NMR (DMSOꢀd6, δ) 7.44–7.34 (m, 5H), 7.30–7.25 (m, 2H), 7.03–6.97 (m,
2H), 3.79 (s, 3H). 13C NMR (CD3OD, δ) 161.73, 131.79, 131.62, 129.92, 129.76, 128.46,
115.64, 56.09. HRMSꢀESI (m/z): [M+H]+ calcd for C15H13N3O2, 268.1086; found, 268.1094
3-Methoxy-1-(4-methoxyphenyl)-5-phenyl-1H-1,2,4-triazole (31). Compound 30 (0.9 g, 3.4
mmol), iodomethane (2.41 g, 17 mmol, 5 equiv), K2CO3 (1.4 g, 10.2 mmol, 3 equiv) and DMF (5
mL) were mixed together in a oneꢀneck roundꢀbottom flask and stirred at room temperature
for16 h. Reaction progress was monitored with TLC (silica gel; ethyl acetateꢀhexane, 1: 1 v/v).
The mixture was poured into water (50 mL) and extracted with ethyl acetate (50 mL × 2). The
combined organic layers were washed with brine−water (1: 1 v/v; 50 mL) and dried (MgSO4).
Solvent was removed under reduced pressure.
The residue was purified with flash
chromatography (general method A; 40−60% ethyl acetate in hexane) followed by preparative
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HPLC (general method B) to give 31 as a white solid (0.55 g; 57%). Mp 125.1−126.2 °C. H
NMR (CD3OD, δ) 7.44–7.38 (m, 3H), 7.36–7.30 (m, 2H), 7.27–7.22 (m, 2H), 6.99–6.93 (m, 2H),
4.01 (s, 3H), 3.80 (s, 3H). 13C NMR (CD3OD, δ) 169.26, 161.65, 154.82, 132.02, 131.50,
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129.95, 129.73, 128.59, 128.49, 115.65, 57.51, 56.14. HPLC (general method C; tR = 2.3 min,
97.2% purity). HRMSꢀESI (m/z): [M+H]+ calcd for C16H15N3O2, 282.1243; found 282.1244
3-(Fluoromethoxy)-1-(4-methoxyphenyl)-5-phenyl-1H-1,2,4-triazole (32). Compound 30
(0.45 g, 1.68 mmol) was heated at 100 °C for 3.5 h with fluoroiodomethane (1.3 g, 8.4 mmol, 5
equiv) and potassium carbonate (0.7 g, 5.04 mmol, 3 equiv) in DMF (5 mL) and then cooled.
Water (20 mL) and ethyl acetate (20 mL) were poured into the reaction mixture. The organic
layer was separated off, washed with brine (20 mL × 2) and dried (MgSO4). Solvent was
removed under reduced pressure. The residue was purified with flash chromatography (general
method A; 40−60% ethyl acetate in hexane) followed by preparative HPLC (general method B)
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to give 32 as a white solid (0.25 g; 50%). Mp 52.8−53.5 °C. H NMR (CDCl3, δ) 7.5–7.48 (m,
2H), 7.39–7.26 (m, 5H), 6.92–6.9 (m, 2H), 6.07 (s, 1H), 5.9 (s, 1H), 3.84 (s, 3H). 13C NMR
(CD3OD, δ) 165.86, 165.83, 153.36, 130.88, 130.18, 128.82, 128.53, 127.41, 127.01, 114.56,
100.15, 97.93, 55.56. HPLC (general method C; tR = 10.3 min; >99.9% purity). HRMSꢀESI
(m/z): [M+H]+ calcd for C16H14FN3O2, 300.1148; found, 300.1145.
COX-1 and COX-2 Inhibition Assays. Wholeꢀblood enzymatic assays were carried out
in two steps, as previously described13, with minor modifications. All incubations were
performed in a lightꢀshielded shaker at 500 rpm and 37 ºC. In the first step, blood (rat, monkey
or human) was collected into heparinized tubes. For the COXꢀ1 inhibition assay, blood (220 µL)
was aliquoted into each well of 96ꢀwell plates, treated with test agent (5 µL) to give various final
concentrations (10 pM–100 µM), and then incubated for 60 min. To stimulate COXꢀ1, Ca2+
ionophore (50 µM; A23187) was then added, and incubated for 30 min. For COXꢀ2 inhibition
assay, aspirin solution (4 mM; 6 µL) was added to blood (214 µL) and incubated for 6 h. Test
agent (5 µL) was added, followed by lipopolysaccharides (10 µg/mL; LPS from Escherichia coli
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O26:B6), and incubated for 18 h. To terminate either the COXꢀ1 or COXꢀ2 reaction, heparinized
diclofenac was added to a final concentration of 107 µM, left for 5 min, and centrifuged for 5
min at 4 ºC. Plasma (100 µL) was obtained from each well for enzymatic assay (Step 2).
In the second step, competitive assays were performed using a thromboxane (Tx) B2 (COXꢀ
1) or prostaglandin (PG) E2 (COXꢀ2) enzymatic immunoassay (EIA) kit (Cayman Chemical,
USA). Concentrations of TxB2 or PGE2 in the plasma samples were determined according to the
kit instructions. Inhibitory potencies (IC50’s) of the test agents to each COX isozyme were
calculated using a 4ꢀparameter logistic fit.31 Assays were performed in triplicate.
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Pharmacological Screening. Inhibitors 5−9, 11−14, 20, 24, and 26−28 were submitted
to the National Institute of Mental Health Psychoactive Drug Screening Program (NIMHꢀPDSP)
for assay of binding affinities to a wide range of human recombinant receptors and binding sites,
and the rat brain benzodiazepine site. Detailed assay protocols are available at the NIMHꢀPDSP
web site (http://pdsp.cwru.edu).
Radiochemistry. Radiochemistry was performed in leadꢀshielded hotꢀcells with
automated radiosynthesis apparatus for personnel protection from radiation.
Production of [11C]Carbon Dioxide. NCA [11C]carbon dioxide (~ 85 GBq) was produced
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with a PETtrace cyclotron (GE Medical Systems; Milwaukee, WI) according to the N(p,α)11C
reaction by irradiation of nitrogen gas32 (initial pressure, 160 psi; volume, 75 mL) containing
oxygen (1%) with a proton beam (16.5 MeV, 45 µA) for 40 min.
Production of [11C]Iodomethane. NCA [11C]iodomethane was produced from NCA
[11C]carbon dioxide in a PETtrace MeI Process Module (GE Medical Systems: Severna Park,
MD). Thus, at the end of proton irradiation, [11C]carbon dioxide was delivered to the apparatus
through stainless tubing (OD 1/8 in, ID 1/16 in) over 3 min, trapped on molecular sieves (13X),
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released for reduction to [11C]methane, and recirculated over iodine at 720 °C.33 The generated
[11C]iodomethane was trapped on Porapak Q held in the recirculation path.
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Radiosynthesis of [11C]5. Precursor 33 (0.7 mg, 2.3 ꢀmol) was dissolved in DMF (80 ꢀL).
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tetraꢀnꢀbutylammonium hydroxide (TBAH) in MeOH (0.5 M, 4.0 ꢀL) was added to this solution
and then injected into the loop of an AutoLoop apparatus (Bioscan; Washington, DC).
[11C]Iodomethane was released 1 min later from the PETtrace module and into the loop, which
was then kept at room temperature for 5 min. [11C]5 was isolated with reversed phase HPLC on
a Luna C18 column (10 × 250 mm, 10 ꢀm; Phenomenex) eluted isocratically with MeCN−10
mM aq HCOONH4 (45:55 v/v) at 6 mL/min (tR = 14 min) (see Supporting Information). After
removal of mobile phase, [11C]5 was formulated for intravenous injection in sterile ethanolꢀ
saline (10:90 v/v), and sterileꢀfiltered (MillexꢀMP 0.22 µm, 25 mm; Merck Millipore;
Burlington, MA). The identity of the [11C]5 was confirmed with reversed phase HPLC on a
Luna C18 column (4.6 mm × 250 mm, 10 ꢀm; Phenomenex) eluted isocratically with MeCN−10
mM aq HCOONH4 (55:45 v/v) at 2 mL/min (tR = 5.4 min) (see Supporting Information), and by
LCꢀMS (ESI) (method 1) of associated carrier [tR = 4.80 min, (m/z) [M+H]+: 312.4]. [11C]5 was
obtained in 20 ± 3% yield from [11C]carbon dioxide in > 99% radiochemical purity and with a
molar activity of 125 ± 62 GBq/µmol (n = 12).
Radiosynthesis of [11C]20. [11C]Iodomethane was trapped in a crimpꢀsealed Vꢀvial
containing 19 (0.7 mg, 1.9 µmol) and solid KOH (5 mg) in DMSO (0.4 mL) and heated at 70 °C
for 3 min. [11C]20 was isolated with reversed phase HPLC on a Luna C18 column (10 × 250
mm, 10 ꢀm; Phenomenex) eluted isocratically with MeCN−100 mM aq HCOONH4 (65: 35 v/v)
at 6 mL/min (tR = 9.5 min) (see Supporting Information). After removal of mobile phase,
[11C]20 was formulated for intravenous injection in sterile ethanolꢀsaline (10:90 v/v) containing
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