Synthesis of a tritium-labeled photo-affinity probe based on an atypical leukotriene biosynthesis inhibitor

Article abstract of DOI:10.1002/jlcr.1804

A radiolabeled photo-affinity probe containing a diazirine group was designed and synthesized based on an atypical leukotriene (LT) biosynthesis inhibitor to determine whether this type of inhibitor (such as 5) interacts with a novel protein, critical for LT biosynthesis. One of the key transformations was to introduce a tritium label in the thiazole ring. This was eventually achieved by treating the iodinated compound with tritium, Pt/C (sulfided) and triethylamine in anhydrous aprotic solvent, to give the tritiated compound with a specific activity of 303 GBq/mmol. The affinity probe will be used to identify the proteins and protein complexes with which this type of molecules interacts with in inflammatory cells. Copyright 2010 John Wiley & Sons, Ltd. Synthesis of a high specific activity (303 GBq/mmol) tritium-labeled photo-affinity probe based on an atypical leukotriene biosynthesis inhibitor is described.

Full text of DOI:10.1002/jlcr.1804

Research Article
Received 15 December 2009,
Revised 7 May 2010,
Accepted 2 June 2010
Published online 5 August 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1804
Synthesis of a tritium-labeled photo-affinity
probe based on an atypical leukotriene
biosynthesis inhibitor
Nag S. Kumar,a Matthew P. Braun,b Ashok G. Chaudhary,b
Ã
and Robert N. Younga
A radiolabeled photo-affinity probe containing a diazirine group was designed and synthesized based on an atypical
leukotriene (LT) biosynthesis inhibitor to determine whether this type of inhibitor (such as 5) interacts with a novel protein,
critical for LT biosynthesis. One of the key transformations was to introduce a tritium label in the thiazole ring. This was
eventually achieved by treating the iodinated compound with tritium, Pt/C (sulfided) and triethylamine in anhydrous
aprotic solvent, to give the tritiated compound with a specific activity of 303 GBq/mmol. The affinity probe will be used to
identify the proteins and protein complexes with which this type of molecules interacts with in inflammatory cells.
Keywords: photo-affinity labeling; tritiation; leukotriene; 5-lipoxygenase
potent inhibitor of 5-LO and it does not have significant
interactions with FLAP or LTC4 synthase. However, compound 5
Introduction
Leukotrienes (LTs) are lipid mediators that are implicated in
numerous diseases, including inflammation, atherosclerosis, and
respiratory diseases such as asthma.1–9 LTs are produced from
arachidonic acid by the action of 5-lipoxygenase (5-LO) and the
integral membrane protein 5-LO-activating protein (FLAP), which
is an essential partner of 5-LO for LT production in cells. Previously,
it has been shown that LT production can be controlled by either
shows greater inhibitory potency in human whole blood cell
assay than the level of 5-LO activity would predict. This suggests
that 5 might have off-target activity within the same 5-LO
pathway, perhaps with an as yet uncharacterized ancillary
protein.17 Hence, it is of interest to develop an affinity probe
based on 5 to identify this putative new molecular target, which
could serve as a new target for drug discovery and optimization.
inhibiting 5-LO or FLAP, which could in turn lead to new therapies
for treatment of diseases associated with LTs.10–13
Crawley et al. have developed 4-methoxytetrahydropyrans
(1–4) as potent and selective inhibitors of 5-LO from which
compounds
aDepartment of Chemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6
bProcess Research, Merck & Co, Rahway, NJ 07065, USA
1 and 2
were chosen for clinical trials.14,15
Independently, compound 5 that also incorporates 4-methox-
*Correspondence to: Robert N. Young, Department of Chemistry, Simon Fraser
University, Burnaby, BC, Canada V5A 1S6.
ytetrahydropyran moiety was designed and synthesized at
Merck Frosst.16 Compound 5 was found to be a moderately E-mail: roberty@sfu.ca
J. Label Compd. Radiopharm 2011, 54 43–50
Copyright r 2010 John Wiley & Sons, Ltd.

N. S. Kumar et al.
In order to explore the biomolecular target(s) of compound 5, 0.066 mmol), and dppf (148 mg, 0.267 mmol) was dissolved in
a radiolabeled photo-affinity probe would be useful. From NMP (8 ml) followed by the addition of Et3N (0.45 ml, 3.23 mmol).
structure activity relationship, we noted that the oxime moiety The mixture was purged with N2 for 15 min and then a solution
could be modified without having significant impact on the of thiol 13 (ꢀ2.08 mmol) in NMP (3 ml) was added. The reaction
biological activity.16 We envisioned that 6, a closely analogous mixture was heated at 601C for 4 h and then partitioned
diarizine relative of 5, should retain the activity and this could be between EtOAc (150 ml) and brine (50 ml). The organic phase
used to identify these new targets. The radioactive photo- was washed with brine (50 ml), dried over anhydrous Na2SO4,
affinity ligand, such as compound 7, could be incubated with and concentrated. The crude product was purified by flash
inflammatory cells and photolysed to give covalent conjugation chromatography (EtOAc-hexanes, 1:3-1:1) to give 8 as a white
with target proteins. The tritium label could then be followed to solid (379 mg, 57%) as 1:9 inseparable mixture of isomers.
characterize the complexes. Hence, we report herein the
Major isomer: 1H NMR (DMSO): d 12.95 (1H, s, NOH), 7.78 (1H,
synthesis of the protio-compound 6 and the tritio-compound s, H-4), 7.70 (2H, d, J2 ,3 = 8.3 Hz, H-300), 7.57 (2H, d,
00 00
00
0
00 00
7 in which the oxime group is replaced with a photo-labile J2 ,3 = 8.3 Hz, H-2 ), 3.62–3.60 (4H, m, H-2 ), 2.98 (3H, s, OCH3),
diazirine group. In view of the fact that compounds 6 and 7 1.99–1.90 (4H, m, H-30); 13C NMR (DMSO): d 162.3 (C-2), 147.1
have a trifluoromethyl group, instead of a methyl group as in (C-5), 144.5 (1C, JC,F = 31.5 Hz, C-500), 142.2 (C-4), 134.8 (C-400),
the parent oxime 5, we also synthesized the trifluoromethyl 132.7 (C-300), 130.7 (C-200), 127.8 (C-100), 121.7 (1C, JC,F = 273.8 Hz,
derivative 8 to verify whether there is any impact in the C-600), 73.2 (C-40), 63.4 (C-20), 50.2 (OCH3), 36.6 (C-30).
biological activity when a methyl group is modified.
HRMS calcd for (C17H17F3N2O3S21H)1 419.0710, found
419.0705.
5-(4-Methoxytetrahydropyran-4-yl)-2-(4-(3-
(trifluoromethyl)-diazirin-3-yl)phenylthio)thiazole (6)
Diazirine 12 (335 mg, 1.07 mmol), Pd2dba3 (45 mg, 0.049 mmol),
and dppf (112 mg, 0.202 mmol) was dissolved in NMP (6 ml)
followed by the addition of Et3N (0.45 ml, 3.23 mmol) in
dark. The mixture was purged with N2 for 15 min and then a
solution of freshly prepared thiol 13 (ꢀ1.52 mmol) in NMP
(2 ml) was added. The reaction mixture was heated at 601C
for 5 h and then partitioned between EtOAc (150 ml) and
brine (50 ml). The organic phase was washed with brine (50 ml),
dried over anhydrous Na2SO4, and concentrated. The
crude product was purified by flash chromatography (EtOAc-
hexanes, 1:3-1:2) to give 6 as pale yellow syrup (128 mg, 29%).
Experimental
General methods
1H and 13C NMR spectra were recorded at frequencies of 500 and
125 MHz, respectively. All assignments were confirmed with the
aid of two-dimensional 1H, 1H (gCOSY) and 1H, 13C (gHMQC)
experiments using standard Varian pulse programs. Processing of
the spectra was performed with MestRec software. The high-
resolution mass spectra were recorded in positive-ion mode with
an ESI ion source on an Agilent Time-of-Flight LC/MS mass
spectrometer. Analytical thin-layer chromatography (TLC) was
performed on aluminum plates precoated with silica gel 60F-254
as the adsorbent. The developed plates were air-dried, exposed
to UV light and/or dipped in KMnO4 solution and heated. Column
chromatography was performed with Silica gel 60 (230–400
mesh). All procedures with diazirine functionality were carried out
in flask/column covered by aluminum foil to exclude light.
1H NMR (DMSO): d 7.78 (1H, s, H-4), 7.70 (2H, d, J2 ,3 = 8.6 Hz,
00 00
00
0
H-300), 7.38 (2H, d, J2 ,3 = 8.8 Hz, H-2 ), 3.63–3.60 (4H, m, H-2 ),
2.97 (3H, s, OCH3), 1.99–1.91 (4H, m, H-30); 13C NMR (DMSO): d
162.0 (C-2), 147.3 (C-5), 142.2 (C-4), 135.1 (C-400), 133.4 (C-300),
129.1 (C-100), 128.5 (C-200), 122.4 (1C, JC,F = 274.9 Hz, C-600), 73.2
(C-40), 63.4 (C-20), 50.2 (OCH3), 36.6 (C-30), 28.7 (1C, JC,F = 40.1 Hz,
C-500). HRMS calcd for (C17H16F3N3O2S21H)1 416.0714, found
416.0719.
00 00
5-Bromo-2-(methylthio)thiazole (16)
To a stirred solution of 2-(methylthio)thiazole 9 (5.0g, 38.2mmol)
in DMF (100ml) was added NBS (8.15 g, 45.8mmol). The mixture
was stirred in dark for 16h and then partitioned between EtOAc
(300ml) and H2O (150ml). The organic phase was washed with
brine (150ml), dried over anhydrous Na2SO4, and concentrated.
The crude product was purified by flash chromatography (EtOAc-
5-(4-Methoxytetrahydropyran-4-yl)-2-(4-(2,2,2-trifluoro-
1-(hydroxyimino)ethyl)phenylthio)thiazole (8)
1
hexanes, 1:8) to give 16 as pale yellow oil (6.3g, 79%). H NMR
CDCl3): d 7.54 (1H, s, H-4), 2.75 (3H, s, SCH3); 13C NMR (CDCl3): d
167.7 (C-2), 144.0 (C-4), 106.7 (C-5), 16.7 (SCH3). HRMS calcd for
(C4H4BrNS21H)1 209.9046, found 209.9041.
To a stirred solution of 14 (505 mg, 2.08 mmol) in CHCl3 (15 ml)
at 01C was added MCPBA (470 mg, 77%, 2.09 mmol) and the
mixture was stirred for 90 min. Ca(OH)2 (247 mg, 3.33 mmol) was
added and the mixture was stirred at rt for 20 min, filtered, and
concentrated to give essentially pure sulfoxide. The residue was
dissolved in TFAA (8 ml) and refluxed for 30 min and concen-
trated. The mixture was diluted with MeOH–Et3N (1:1, 20 ml) and
evaporated to dryness. The residue was dissolved in CHCl3
(150 ml) and washed with sat. NH4Cl, dried over anhydrous
Na2SO4, and concentrated to provide thiol 13. In a separate
flask, oxime 11 (500 mg, 1.59 mmol), Pd2dba3 (60 mg,
4-Bromo-5-(4-hydroxytetrahydropyran-4-yl)-2-
(methylthio)thiazole (17)
A solution of compound 16 (3.0 g, 14.3 mmol) in THF (8 ml) was
added to a stirred solution of freshly prepared LDA (n-BuLi
(11.4 ml, 2.5 M in hexanes, 28.5 mmol) and diisopropylamine
(4.0 ml, 28.5 mmol) in THF (20 ml)) at ꢁ781C under N2. After
www.jlcr.org
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N. S. Kumar et al.
stirring for 20 min at rt, the reaction mixture was cooled to HRMS calcd for (C17H15BrF3N3O2S21H)1 493.9819, found
ꢁ781C, followed by addition of tetrahydropyran-4-one 10 493.9818.
(2.65 ml, 28.6 mmol) dropwise. The mixture was stirred at rt for
16 h and then partitioned between EtOAc (200 ml) and 1 M HCl
(50 ml). The organic phase was washed with H2O (50 ml), brine
(50 ml), dried over anhydrous Na2SO4, and concentrated. The
crude product was purified by flash chromatography (EtOAc-
hexanes, 1:2-1:1) to give 17 as brown syrup (1.92 g, 43%). H
NMR (CDCl3): d 3.87–3.84 (4H, m, H-2ax0, H-2eq0), 2.93 (1H, s, OH),
5-Bromo-2-(4-(3-(trifluoromethyl)-diazirin-3-yl)phe-
nylthio)thiazole (21)
Diazirine 12 (600 mg, 1.92 mmol), Pd2dba3 (90 mg, 0.09 mmol),
and dppf (230 mg, 0.41 mmol) was dissolved in NMP (12 ml)
followed by the addition of Et3N (0.70 ml, 5.0 mmol) in dark. The
mixture was purged with N2 for 15 min and then a solution of
2-mercaptothiazole 20 (320 mg, 2.73 mmol) in NMP (3 ml) was
added. The reaction mixture was heated at 601C for 24 h and
then partitioned between EtOAc (150 ml) and brine (50 ml). The
organic phase was washed with brine (50 ml), dried over
anhydrous Na2SO4, and concentrated. Polar impurities were
removed by flash chromatography (EtOAc-hexanes, 1:9) and the
residue was dissolved in DMF (50 ml). NBS (400 mg, 2.24 mmol)
was added and the reaction mixture was stirred at rt in dark for
18 h. The mixture was diluted with EtOAc (150 ml) and washed
with H2O (50 ml), brine (50 ml), dried over anhydrous Na2SO4,
and concentrated. The crude product was purified by flash
chromatography (EtOAc-hexanes, 1:10) to give compound 21 as
pale yellow oil (378 mg, 51%).
1
0
0
2.66 (3H, s, SCH3), 2.43 (2H, ddd, J2ax ,3eq = 7.4 Hz,
0
0
0
0
0
J2ax ,3ax = 10.0 Hz, J3ax ,3eq = 13.9 Hz, H-3ax ), 1.87–1.84 (2H, m,
H-3eq0); 13C NMR (CDCl3): d 164.9 (C-2), 139.7 (C-5), 118.4 (C-4),
69.6 (C-40), 63.2 (C-20), 37.0 (C-30), 16.5 (SCH3). HRMS calcd for
(C9H12BrNO2S21H)1 309.9571, found 309.9566.
4-Bromo-5-(4-methoxytetrahydropyran-4-yl)-
2-(methylthio)thiazole (18)
NaH (100 mg, 60% in oil, 2.5 mmol) was added to a stirred
solution of compound 17 (460 mg, 1.5 mmol) in DMF (10 ml) at
01C under N2. After stirring for 10 min, MeI (115 ml, 1.8 mmol) was
added and the mixture was stirred at rt for 2 h. The reaction
mixture was partitioned between EtOAc (100 ml) and H2O
(50 ml). The organic phase was washed with brine (50 ml), dried
over anhydrous Na2SO4, and concentrated. The crude product
was purified by flash chromatography (EtOAc-hexanes, 1:3) to
give 18 as pale yellow solid (420 mg, 88%, mp 85–871C). 1H NMR
CDCl3: d 3.81–3.79 (4H, m, H-20), 3.17 (3H, s, OCH3), 2.69 (3H, s,
SCH3), 2.22–2.12 (4H, m, H-30); 13C NMR (CDCl3): d 165.7 (C-2),
135.4 (C-5), 120.9 (C-4), 73.7 (C-40), 63.3 (C-20), 50.3 (OCH3), 35.2
(C-30), 16.3 (SCH3). HRMS calcd for (C10H14BrNO2S21H)1
323.9727, found 323.9724.
1H NMR (CDCl3): d 7.63 (1H, s, H-4), 7.57 (2H, d, J2 ,3 = 8.4 Hz,
00 00
H-300), 7.20 (2H, d, J2 ,3 = 8.6 Hz, H-2 ); 13C NMR (CDCl3): d 162.8
00
00 00
(C-2), 143.9 (C-4), 132.9 (C-40), 131.7 (C-30), 129.2 (C-10), 126.6
(C-20), 120.8 (1C, JC,F = 274.8 Hz, C-600), 109.2 (C-5), 27.2
(1C, JC,F = 40.7 Hz, C-500). HRMS calcd for (C11H5BrF3N3S21H)1
379.9138, found 379.9137.
Alternative method for the synthesis of 4-Bromo-5-
(4-methoxytetrahydropyran-4-yl)-2-(4-(3-(trifluoromethyl)-
diazirin-3-yl)phenylthio)thiazole (15)
4-Bromo-5-(4-methoxytetrahydropyran-4-yl)-2-(4-(3-
(trifluoromethyl)-diazirin-3-yl)phenylthio)thiazole (15)
A solution of compound 21 (635 mg, 1.67 mmol) in THF (4 ml)
was added to a stirred solution of freshly prepared LDA (n-BuLi
(1.6 ml, 2.5 M in hexanes, 4.0 mmol) and diisopropylamine
(0.56 ml, 4.0 mmol) in THF (6 ml)) at ꢁ781C under N2. After
stirring for 15 min at rt, the reaction mixture was cooled
to ꢁ781C, followed by addition of tetrahydropyran-4-one
10 (0.4 ml, 4.32 mmol) dropwise. The mixture was stirred at rt
for 16 h and then partitioned between EtOAc (150 ml) and 1 M
HCl (50 ml). The organic phase was washed with H2O (50 ml),
brine (50 ml), dried over anhydrous Na2SO4, and concentrated.
Most impurities were removed by flash chromatography (EtOAc-
hexanes, 1:3-1:2) and the residue was dissolved in DMF (15 ml).
The mixture was cooled to 01C and then NaH (49 mg, 60% in oil,
1.22 mmol). After stirring for 10 min, MeI (60 ml, 0.96 mmol) was
added and the mixture was stirred at rt for 90 min. The reaction
mixture was partitioned between EtOAc (50 ml) and H2O (20 ml).
The organic phase was washed with brine (20 ml), dried over
anhydrous Na2SO4, and concentrated. The crude product was
purified by flash chromatography (EtOAc-hexanes, 1:4) to give
15 as colorless syrup (207 mg, 25%).
To a stirred solution of 18 (450 mg, 1.39 mmol) in CHCl3 (15 ml)
at 01C was added MCPBA (340 mg, 77%, 1.51 mmol) and the
mixture was stirred for 90 min. Ca(OH)2 (190 mg, 2.56 mmol)
was added and the mixture was stirred at rt for 20 min, filtered,
and concentrated to give essentially pure sulfoxide. The
residue was dissolved in TFAA (8 ml) and refluxed for 45 min
and concentrated. The mixture was diluted with MeOH–Et3N
(4:1, 10 ml) and evaporated to dryness under high vacuum to
give crude thiol 19 as the triethylammonium salt. In a separate
flask, diazirine 12 (450 mg, 1.45 mmol), Pd2dba3 (80 mg,
0.087 mmol), and dppf (196 mg, 0.354 mmol) was dissolved in
NMP (8 ml) followed by the addition of Et3N (0.60 ml,
4.31 mmol) in dark. The mixture was purged with N2 for
15 min and then heated to 601C.
A solution of 19
(ꢀ1.39 mmol) in NMP (4 ml) was added over a 30-min period.
The reaction mixture was stirred at 601C for 16 h and then
partitioned between EtOAc (150 ml) and brine (50 ml). The
organic phase was washed with brine (50 ml), dried over
anhydrous Na2SO4, and concentrated. The crude product was
purified by flash chromatography (EtOAc-hexanes, 1:7-1:5)
4-Iodo-5-(4-methoxytetrahydropyran-4-yl)-2-
(methylthio)thiazole (22)
1
to give 15 as colorless syrup (115 mg, 22%). H NMR (CD3OD):
00
00 00
00 00
d 7.73 (2H, d, J2 ,3 = 8.2 Hz, H-3 ), 7.36 (2H, d, J2 ,3 = 8.6 Hz,
H-200), 3.74–3.72 (4H, m, H-20), 3.12 (3H, s, OCH3), 2.19–2.03 (4H, nBuLi (0.4 ml, 2.5 M in hexanes, 1 mmol) was added to a stirred
m, H-30); 13C NMR (CD3OD): d 163.8 (C-2), 139.4 (C-5), 133.9 solution of compound 18 (220 mg, 0.68 mmol) in Et2O (10 ml)
(C-300), 133.4 (C-400), 130.6 (C-100), 128.0 (C-200), 122.2 at ꢁ781C under Ar. After stirring for 30 min, a solution of
(1C, JC,F = 281.4 Hz, C-600), 121.6 (C-4), 73.9 (C-40), 63.0 (C-20), 1,2-diiodoethane (280 mg, 0.99 mmol) in Et2O (3 ml) was added
49.7 (OCH3), 34.9 (C-30), 28.7 (1C, JC,F = 40.4 Hz, C-500). dropwise. The mixture was stirred at ꢁ781C for 30 min and then
J. Label Compd. Radiopharm 2011, 54 43–50
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N. S. Kumar et al.
at rt for 2 h. The reaction was quenched with sat. NH4Cl (5 ml) Hydrogenation of compound 24
and partitioned between EtOAc (100 ml) and H2O (50 ml). The
Pt/C sulfided (10 mg, 5% on C) was added to a stirred solution of
compound 24 (5 mg, 9 mmol) in EtOH (2 ml). The suspension was
briefly purged with hydrogen gas and then a balloon filled with
hydrogen gas was attached. The suspension was stirred at room
temperature for 18 h. The solid material was filtered and washed
with EtOH (1 ml). The filtrate was concentrated and the residue
was purified by flash chromatography (EtOAc-hexanes, 1:2) to
give 6 as colorless syrup (3 mg, 78%).
organic phase was washed with brine (50 ml), dried over
anhydrous Na2SO4, and concentrated. The crude product was
purified by flash chromatography (EtOAc-hexanes, 1:3) to give
22 as a white solid (194 mg, 77%, mp 109–1111C). 1H NMR
CDCl3: d 3.81–3.79 (4H, m, H-20), 3.14 (3H, s, OCH3), 2.68 (3H, s,
0
0
0
0
SCH3), 2.24 (2H, ddd, J2eq ,3eq = 2.6 Hz, J2ax ,3eq = 4.9 Hz,
J3ax ,3eq = 14.2 Hz, H-3eq ), 2.08–2.04 (2H, m, H-3ax ); 13C NMR
(CDCl3): d 167.1 (C-2), 138.9 (C-5), 90.9 (C-4), 73.6 (C-40), 63.3 (C-
20), 50.2 (OCH3), 35.5 (C-30), 16.5 (SCH3). HRMS calcd for
(C10H14INO2S21H)1 371.9588, found 371.9584.
0
0
0
0
Deuteriation of compound 24
Pt/C sulfided (9 mg, 5% on C) was added to a stirred solution of
compound 24 (3 mg, 5.5 mmol) in EtOAc (1.5 ml) and Et3N (5 ml,
35 mmol). The suspension was briefly purged with deuterium gas
and then a balloon filled with deuterium gas was attached. The
suspension was stirred at room temperature for 48 h. The solid
material was filtered and the filtrate was concentrated. H NMR
and MS of the crude material show that there was 50%
deuterium incorporation.
4-Iodo-5-(4-methoxytetrahydropyran-4-yl)-2-(4-(3-
(trifluoromethyl)-diazirin-3-yl)phenylthio)thiazole (24)
To a stirred solution of 22 (302 mg, 0.81 mmol) in CHCl3 (10 ml)
at 01C was added MCPBA (195 mg, 77%, 0.88 mmol) and the
mixture was stirred for 90 min. Ca(OH)2 (142 mg, 1.92 mmol)
was added and the mixture was stirred at rt for 20 min, filtered,
and concentrated to give essentially pure sulfoxide. The
residue was dissolved in TFAA (8 ml) and refluxed for 45 min
and concentrated. The mixture was diluted with MeOH-Et3N
(4:1, 10 ml) and evaporated to dryness under high vacuum to
give crude thiol 23 as the triethylammonium salt. In a separate
flask, diazirine 12 (350 mg, 0.98 mmol), Pd2dba3 (80 mg,
87 mmol), and dppf (196 mg, 0.35 mmol) was dissolved in
NMP (8 ml) followed by the addition of Et3N (0.40 ml, 2.8 mmol)
in dark. The mixture was purged with N2 for 15 min and then
heated to 601C. A solution of 23 (ꢀ0.81 mmol) in NMP (4 ml)
was added over a 20-min period. The reaction mixture was
stirred at 601C for 16 h and then partitioned between EtOAc
(150 ml) and brine (50 ml). The organic phase was washed with
brine (50 ml), dried over anhydrous Na2SO4, and concentrated.
The crude product was purified by flash chromatography
(EtOAc-hexanes, 1:7-1:5) to give 24 as pale yellow syrup
1
Tritiation of compound 24
Following the deuteriation procedure with tritium gas (at ca.
200 mm Hg and extending the reaction period to 4 days) gave
the tritiated compound 7. The crude product was purified by
reverse-phase HPLC (Luna Polar RP, CH3CN/H2O 30:70 isocratic)
to give probe 7 (0.222 GBq) with specific activity of 303 GBq/
mmol representing a 28% radiochemical yield. Identity of 7 was
confirmed by LC/MS and HPLC co-elution with unlabeled
reference.
Results and discussion
Retrosynthetic analysis indicated that thioether A could be
synthesized by coupling thiol B with aryl iodide C (Scheme 1).
The key intermediate B could, in turn, be prepared from
commercially available 2-(methylthio)thiazole (9) and tetrahy-
dropyran-4-one (10). This route was chosen in order to provide
flexibility in synthesizing compounds that have different
functional groups attached to the phenyl ring.
The oxime (11) and the diazirine (12) were synthesized from
1,4-diiodobenzene using the procedure of Topin et al. (Scheme 2).18
The key intermediate 13, corresponding to B, was prepared from
(118 mg, 27%). 1H NMR (CD3OD): d 7.72 (2H, d, J2 ,3 = 8.3 Hz,
00 00
00
0
H-300), 7.35 (2H, d, J2 ,3 = 8.7 Hz, H-2 ), 3.76–3.73 (4H, m, H-2 ),
3.10 (3H, s, OCH3), 2.26–2.21 (2H, m, H-3eq0), 2.03–1.97 (2H, m,
H-3ax0); 13C NMR (CD3OD): d 164.9 (C-2), 143.0 (C-5), 133.8 (C-400),
133.7 (C-300), 130.5 (C-100), 127.9 (C-200), 122.2 (1C, JC,F = 281.5 Hz,
C-600), 91.9 (C-4), 73.7 (C-40), 63.0 (C-20), 49.5 (OCH3), 35.2 (C-30),
28.2 (1C, JC,F = 40.3Hz, C-500). HRMS calcd for (C17H15F3IN3O2S21H)
00 00
1
541.9680, found 541.9683.
N
S
N
SH
X
O
O
S
X
S
+
I
CF3
O
O
CF3
B
C
A
O
N
S
9
+
S
O
10
Scheme 1. Retrosynthetic analysis of compound A.
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J. Label Compd. Radiopharm 2011, 54 43–50

N. S. Kumar et al.
commercially available 2-(methylthio)thiazole (9) (Scheme 3).19 thioether 18. The methyl group on the sulfide was cleaved using
Palladium catalyzed cross-coupling of thiol 13 with either oxime a mild, one-pot, three-step procedure via Pummerer rearrange-
11 or diazirine 12 gave the target compounds 8 and 6, ment of the corresponding sulfoxide to give thiol 19 as the
respectively (Scheme 4).
To save the expensive isotopically enriched reagent and to with diazirine 12 gave the desired compound 15.
reduce the number of steps involving radioactive intermediates, Alternatively, compound 15 was synthesized from commer-
triethylammonium salt.21 Palladium-catalyzed cross-coupling of 19
we envisioned that the tritium could be introduced as the final cially available 2-mercaptothiazole (20), as shown in Scheme 6.
synthetic step by tritium reduction of brominated compound Diazirine 12 was first coupled with 2-mercaptothiazole (20) and
15. Several attempts were made to directly introduce a bromine on treatment with NBS gave compound 21. The brominated
atom at C-4 of the thiazole moiety of compound 6. However, in compound 21 underwent halogen dance reaction when
each case, there was considerable decomposition under the subjected to LDA and quenching the lithiated intermediate
standard condition for lithiation/bromination or with NBS and with tetrahydropyran-4-one (10) gave an alcohol, which was
the desired product could not be isolated. Hence, we introduced subsequently methylated to give compound 15.
bromine at an earlier step, as shown in Scheme 5.
Attempts were made to dehalohydrogenate compound 15
2-(Methylthio)thiazole (9) was treated with NBS to give compound using H2. Various conditions and catalysts were tried; however,
16 with bromine at C-5. When compound 16 was subjected to we could not isolate the desired product in which the bromide is
LDA, it underwent the ‘halogen dance’ (halogen migration) exchanged with hydrogen. With Pd as catalyst, only decom-
reaction where the bromide migrates to C-4.20 Quenching the posed starting material was isolated, whereas with Pt, there was
reaction with water gave compound with a bromide at C-4 no reaction as indicated by TLC. Presumably, the rate at which Pt
position. The reason for halogen dance reaction can be reacts is too slow, whereas Pd is reacting faster with thioether
explained in terms of the acidity of position 4 and 5 of thiazole, than with bromide, which in turn, cleaves the thioether and
which correlates with the stability of the lithiated species poisons the catalyst. Hence, we envisioned that an iodinated
formed. The 5-position is more acidic than the 4-position, hence, compound might be more appropriate, as it will undergo
the ultimate lithiation is more favored at position 5.20 Treating hydrogenation reaction faster than bromide and perhaps the
the lithiated intermediate with tetrahydropyran-4-one (10) gave iodide would react faster than thioether with Pd as catalyst.
alcohol 17, which was subsequently methylated to give In addition, the iodo-hydrogen exchange might be fast enough
to be carried out with Pt as catalyst.
Numerous attempts were made to iodinate compound 15
OH
N
N
directly by metallation; however, no desired product was obtained.
The lithium–iodine exchange was successful with intermediate 18
and treating the resulting lithiated intermediate with 1,2-iodoethane
gave compound 22 (Scheme 7). The methyl group on the sulfide
was cleaved to give intermediate 23, which was subsequently
coupled with diazirine 12 to furnish iodinated compound 24.
The hydrogenation of compound 24 proceeded smoothly
with H2 and 5% Pt/C (sulfided) in ethanol to give compound 6 in
N
CF3
CF3
I
I
Ref. 18
Ref. 18
two steps
three steps
I
I
11
12
Scheme 2. Synthesis of oxime 11 and diazirine 12.
1) i. MCPBA / CHCl3
N
N
ii. Ca(OH)2
N
S
SH
Ref. 19
O
O
2) TFAA 40°C
3) i. Et3N / MeOH
ii. NH4Cl
S
S
S
S
two steps
O
O
9
14
13
Scheme 3. Synthesis of thiol 13.
N
N
S
S
OH
N
SH
OH
N
Pd2dba3 / dppf
Et3N / NMP
O
O
S
+
I
CF3
60°C
57%
O
O
CF3
13
11
8
N
N
S
S
N
SH
Pd2dba3 / dppf
Et3N / NMP
O
O
N
S
N
+
I
N
CF3
60°C
29%
O
O
CF3
13
12
6
Scheme 4. Palladium catalyzed cross-coupling of thiol 13 with oxime 11 and diazirine 12.
J. Label Compd. Radiopharm 2011, 54 43–50
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

N. S. Kumar et al.
Br
N
S
N
S
N
S
1) LDA / THF
2) O
NBS / DMF
79%
S
S
S
O
Br
O
OH
17
9
10
16
43%
NaH / MeI
DMF
0°C - rt
88%
1) i. MCPBA / CHCl3
ii. Ca(OH)2
Br
Br
N
N
S
S
SH
O
O
2) TFAA 40°C
S
3) Et3N / MeOH
O
O
19
18
N
N
Pd2dba3 / dppf
Et3N / NMP
I
CF3
60°C
12
22% (two steps)
Br
N
S
O
S
N
N
O
CF3
15
Scheme 5. Synthesis of compound 15.
1)
Pd2dba3 / dppf
Et3N / NMP
N
N
S
S
SH
+
12
S
N
60°C
Br
N
2) NBS / DMF
CF3
20
21
51% (two steps)
1) i. LDA / THF
ii. O
O
10
2) NaH / MeI /DMF
25% (two steps)
Br
N
S
O
S
N
N
O
CF3
15
Scheme 6. Alternative method for the synthesis of compound 15.
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J. Label Compd. Radiopharm 2011, 54 43–50

N. S. Kumar et al.
Br
I
N
S
N
S
1) n-BuLi / Et2O -78°C
S
S
O
O
I
2)
I
O
O
77%
18
22
1) i. MCPBA / CHCl3
ii. Ca(OH)2
2) TFAA 40°C
3) Et3N / MeOH
I
I
N
N
Pd2dba3 / dppf
Et3N / NMP
S
SH
S
O
O
S
N
12
60°C
N
O
O
27% (two steps
from 22)
CF3
24
23
Scheme 7. Synthesis of iodinated compound 24.
H
N
H
N
S
S
O
O
S
N
N
S
N
N
O
O
CF3
CF3
6
6
H2 / Pt/C (sulfided)
D2 / Pt/C (sulfided)
EtOH
78%
EtOH
70%
24
D2 / Pt/C (sulfided)
EtOAc / Et3N
77%,
T2 / Pt/C (sulfided)
EtOAc / Et3N
D
N
T
N
S
S
O
O
S
S
N
N
N
N
O
O
CF3
CF3
7 (303 GBq/mmol)
25
(50% deuterated)
Scheme 8. Hydrogenation/deuteriation/tritiation of compound 24.
78% yield (Scheme 8). The hydrogenation reaction also worked Similar reaction with tritium gas afforded the desired tritiated
with Pd as catalyst, however, there was formation of side compound 7, which was confirmed by LC/MS and HPLC
products as indicated by TLC. Unfortunately, when the reaction co-elution with cold standard 6. The specific activity of
was repeated using deuterium gas, there was no incorporation compound 7 was determined to be 303 GBq/mmol (28%
of deuterium as indicated by MS and 1H NMR. The proton radiochemical yield).
introduced is most likely coming from the protic solvent. When
the same reaction was carried out in aprotic solvent, 50% ligand (6) and tritiated photo-affinity probe (7) with
In summary, we have prepared oxime (8), photo-affinity
a
deuterated product (25) was isolated. The Pt/C catalyst and/or trifluoromethyldiazirine unit. These compounds are currently
the deuterium gas might contain trace amount of H2O, which being tested against 5-LO and in human whole blood cell assay.
could account for the 50% of the undesired protonated product Compound 7 will be used to photo-affinity label protein and
6 although no improvement in incorporation was obtained protein complexes with which this type of compound interact
even after rigorously drying of the solvents and reagents. with in inflammatory cells.
J. Label Compd. Radiopharm 2011, 54 43–50
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

N. S. Kumar et al.
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Acknowledgements
We thank the British Columbia Government Leading Edge
Endowment Fund, Merck Frosst, and Simon Fraser University for
financial support.
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J. Label Compd. Radiopharm 2011, 54 43–50