Synthesis of 13C6, 3H, and 14C labeled Sch 414319 and 35S labeling of an analog, Sch 225336

Full text of DOI:10.1002/jlcr.1183

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 264–272.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1183
Short Research Article
13
3
14
35
Synthesis of C6, H, and C labeled Sch 414319 and S
labeling of an analog, Sch 225336y
CAROLEE FLADER LAVEY*, DAVID HESK, SHARON HENDERSHOT, DAVID KOHARSKI, SURINDERJIT SALUJA and
PAUL MC NAMARA
Schering-Plough Research Institute, 2015 Galloping Hill Road, K15-4-4545, Kenilworth, NJ 07033, USA
Received 20 September 2006; Accepted 4 October 2006
Keywords: tritium; carbon-13; carbon-14; sulfur-35; Sch 414319
Introduction
Schering-Plough Research Institute1 and is outlined in
Scheme 2. The label was introduced using [13C6]fluor-
obenzene, which was deprotonated using sec-butyl-
lithium and the resulting anion quenched with carbon
dioxide2 to give benzoic acid 4. Aniline 5 was generated
by the treatment of 4 with sodium azide and was
subsequently converted to thiophenol 6 via diazonium
salt formation followed by reaction with potassium
ethyl xanthate3. Disulfide 7 was formed by the oxida-
tion of thiophenol 6 using sodium perborate4. An
impurity was present that co-eluted with the disulfide
during column chromatography}the impure disulfide
was used in the subsequent reaction and the impurity
was removed at a later stage by column chromatogra-
phy. Sulfide 9 was generated by deprotonating chlor-
osulfone 8 with n-butyllithium and quenching the
anion with disulfide 7. The 2-fluoro[13C6]thiophenol
(6) that was generated in this reaction could be
recovered and potentially re-used in future syntheses.
Crude sulfide 9 was oxidized to sulfone 10 using a
urea/hydrogen peroxide complex and trifluoroacetic
anhydride. Free amine 11 was formed by reaction of
the trifluoroacetanilide in 10 with lithium hydroxide
and was subsequently converted to [13C6]Sch 414319
(12) using triflic anhydride. Compound 12 was purified
by column chromatography and recrystallized twice
from toluene. A 1.1% impurity persisted and was
removed ultimately by converting [13C6]Sch 414319 to
its piperazine salt (13).
Sch 414319 (1, Figure 1), a cannabinoid inverse
agonist, can potentially be used for the treatment of
psoriasis, multiple sclerosis, and rheumatoid arthritis.
Three labeled forms of Sch 414319 were synthesized
within our group. Carbon-13 labeled Sch 414319 was
prepared for use as a standard in a mass spectrometry
based bioanalytical assay. Tritiated material and a
carbon-14 labeled compound were synthesized to
assess metabolism. In addition, a sulfur-35 labeled
form of the mesylated analog, Sch 225336 (2, Figure 1),
was prepared to study the binding kinetics of the CB2
receptor. The synthesis of each labeled compound is
described in this paper.
Results and discussion
The synthesis of [3H]Sch 414319 is summarized in
Scheme 1, where Sch 414319 was deprotonated using
n-butyllithium and the resulting anion was quenched
with low specific activity tritiated water (50 Ci/ml). An
analysis of a portion of the compound by 3H-NMR
showed that the tritium was distributed throughout the
three aromatic rings.
The synthesis of [13C6]Sch 414319 was based on the
route developed and optimized by Chemical Research
and Chemical Development (unpublished results) at
The first batch of [14C]Sch 414319 (19) was prepared
using a route analogous to that for [13C6]Sch 414319
(12). The radiochemical yield was 1.7% for this eight-
step synthesis, starting from fluoro[U-14C]benzene.
Subsequently, an improved route to the carbon-14
labeled compound was devised (Scheme 3) that
*Correspondence to: Carolee Flader Lavey, Schering-Plough Research
Institute, 2015 Galloping Hill Road, K15-4-4545, Kenilworth, NJ 07033,
USA. E-mail: carolee.lavey@spcorp.com
yProceedings of the Ninth International Symposium on the Synthesis
and Applications of Isotopically Labelled Compounds, Edinburgh,
16–20 July 2006.
Copyright # 2007 John Wiley & Sons, Ltd.

SYNTHESIS OF 13C6, 3H, AND 14C LABELED SCH 414319 265
Figure 1 Sch 414319 (1); Sch 225336 (2)
Scheme 1
resulted in an 8.1% radiochemical yield of 19 after six
steps. For this synthesis, fluoro[U-14C]benzene was
deprotonated using sec-butyllithium and the resulting
anion was quenched with sulfur dioxide to give sulfinic
acid 14. Sulfinate ester 15 was generated by conver-
sion of the sulfinic acid to the sulfonyl chloride using
thionyl chloride, followed by reaction with ethanol and
pyridine5. Compound 15 was reacted directly with the
anion generated from chlorosulfone 8 to give sulfoxide
16, which was oxidized to sulfone 17 using a urea/
hydrogen peroxide complex and trifluoroacetic anhy-
dride. The last two steps in the synthesis of [14C]Sch
414319 (19) parallel those of the carbon-13 synthesis,
with the conversion of the trifluoroacetanilide in
compound 17 to the free amine (compound 18) using
lithium hydroxide, followed by reaction with triflic
anhydride to give the final product (19).
tritiated water was purchased from GE Healthcare, and
methane [35S]sulfonic acid was purchased from Perkin
Elmer. All anhydrous solvents and reagents not listed
above (including gases) were purchased from commer-
cial suppliers (Acros Organics, Fisher, or Aldrich
Chemical Company) and used as received. Compounds
1, 8, and 20 were obtained from either Chemical
Research or Chemical Development at Schering-Plough
Research Institute.
Liquid scintillation counting: Quantitation of radio-
activity was performed using a Packard 2200CA liquid
scintillation analyzer and Scintiverse BD cocktail.
Thin layer chromatography (TLC): Thin layer chroma-
tography was performed using Whatman LK6DF (silica
gel 60) 5 ꢀ 20 cm, 0.25 mm plates. The plates were
scanned on a Bioscan 1000 linear analyzer using the
solvent systems indicated.
Sch 225336, an analog of Sch 414319, was labeled
with sulfur-35 as shown in Scheme 4 in order to aid in
the study of the binding kinetics of the CB2 receptor.
For this synthesis, methane[35S]sulfonic acid was
reacted with oxalyl chloride to give [35S]mesyl chloride.
Amine 20 was mesylated using this crude reagent6
to give [35S]Sch 225336 (21). The radiochemical yield
was 28.7% from methane[35S]sulfonic acid.
High performance liquid chromatography: [3H] and
[
14C]Sch 414319 and [35S]Sch 225336 were analyzed
by HPLC for radiochemical and chemical purity.
13C6]Sch 414319 was analyzed for chemical purity by
[
HPLC. In addition, reaction progress was frequently
monitored by HPLC. Chemical purity was determined
using a Waters 2487 Dual programmable wavelength
detector and radiochemical purity using a Radiomatic
C150TR radioflow detector with Radiomatic Flo-Scint III
liquid scintillation cocktail. The following systems were
used:
Experimental
Materials: [13C6]Fluorobenzene was purchased from
Cambridge Isotope Labs, fluoro[U-14C]benzene was
purchased from either GE Healthcare or Perkin Elmer,
1. Zorbax SB-C18 column, 4.6 mm ID ꢀ 150 mm,
254 nm, (65:35:0.1) acetonitrile:water: trifluoroacetic
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 264–272
DOI: 10.1002.jlcr

266 C. F. LAVEY ET AL.
Scheme 2
acid at 1 ml/min for 15 min followed by a gradient to
acetonitrile.
ammonium acetate pH 8 at 1 ml/min for 15 min
followed by a gradient to acetonitrile.
2. Zorbax SB-C18 column, 4.6 mm ID ꢀ 150 mm,
254 nm, (60:40:0.1) acetonitrile:water: trifluoroace-
tic acid at 1 ml/min for 15 min followed by a
gradient to acetonitrile.
Synthesis of [3H]Sch 414319 (3)
3. Zorbax Extend C18 column, 3 mm ID ꢀ 150 mm,
254 nm, 40:60 acetonitrile: 0.05 M aqueous triethy-
lammonium acetate pH 9 at 0.5 ml/min for 15 min
followed by a gradient to acetonitrile.
A solution of tetrahydrofuran (1 ml) and Sch 414319
(60 mg, 0.10 mmol) was cooled to ꢁ788C under N2 in a
flame-dried round-bottomed flask. n-Butyllithium
(0.25 ml, 1.6 M, 0.4 mmol) was added dropwise and
the resulting orange solution was stirred at ꢁ788C for
15 min before THO (561.6 mCi, 50 Ci/ml) was added.
4. Zorbax Extend C18 column, 4.6 mm ID ꢀ 150 mm,
254 nm, 50:50 acetonitrile: 0.05 M aqueous triethyl-
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 264–272
DOI: 10.1002.jlcr

SYNTHESIS OF 13C6, 3H, AND 14C LABELED SCH 414319 267
Scheme 3
The reaction was stirred at ꢁ788C for 1 h, then
quenched by the addition of water (2 ml) and methylene
chloride (5 ml) and warmed to RT. The aqueous layer
was acidified and extracted with methylene chloride
(3 ꢀ 5 ml). The combined organic layers were washed
with water (2 ml), dried (sodium sulfate) and evapo-
rated, and the resulting crude [3H]Sch 414319 (76.6
mCi, RCP 29.6%) was dissolved in EtOH (10 ml) for
storage. Compound 3 was purified by RP-HPLC (Zorbax
Extend C18, 10 mm ID ꢀ 250 mm, 254 nm, 50:50
acetonitrile: 0.05 M aqueous triethylammonium acet-
ate pH 8 at 5 ml/min); 17 mCi of [3H]Sch 414319 at a
specific activity of 1.05 Ci/mmol was obtained. The
radiochemical purity as determined by radio-HPLC
(system 4) and radio-TLC plate scanning (5:95 2 M
methanolic ammonia:methylene chloride) was 98.7
and 98.6%, respectively. 3H-NMR of [3H]Sch 414319
(CDCl3): d 8.59 (t, H60), 8.38 (d, H30), 8.26 (t, H6), 7.98
(d, H200), 7.84 (d, H40), 7.13 (t, H3).
(25 ml) and [13C6]fluorobenzene (1.0 g, 9.8 mmol). The
clear, colorless solution was cooled to ꢁ788C and sec-
butyllithium (8.6 ml, 1.14 M, 9.8 mmol) was added
dropwise over 30 min. The cloudy yellow solution was
stirred at ꢁ788C for 2.5 h, then transferred via a glass
syringe to an open 250 ml flask (previously dried)
containing dry ice and ether (100 ml). The cloudy,
white mixture was warmed to 08C and quenched with
aqueous hydrochloric acid (30 ml, 1 N). The layers were
separated and the ether layer was washed with water
(30 ml), then extracted with sodium hydroxide
(3 ꢀ 30 ml, 0.2 M). The combined aqueous extracts were
washed with ether (20 ml), then acidified with aqueous
hydrochloric acid (1 M) and extracted with ether
(5 ꢀ 40 ml). The combined organic extracts were
washed with water (2 ꢀ 30 ml) and brine (30 ml), dried
(sodium sulfate) and evaporated to give 4 as an off-
white solid (1.27 g, 89%). 1H NMR (d6-DMSO): d 13.21
(s, 1 H), 8.07 (m, 0.5 H), 7.83 (m, 0.5 H), 7.66 (m, 0.5 H),
7.51 (m, 0.5 H), 7.43 (m, 0.5 H), 7.09 (m, 0.5 H).
Synthesis of [13C6]Sch 414319 piperazine salt (13)
2-Fluoro[13C6]aniline (5). A solution of 2-fluoro[13C6]-
benzoic acid (4, 1.27 g, 8.7 mmol) in sulfuric acid
(4.4 ml, concentrated) was heated to 508C in a 25 ml
2-Fluoro[13C6]benzoic acid (4). Into a dry, 100 ml,
round-bottomed flask were added tetrahydrofuran
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 264–272
DOI: 10.1002.jlcr

268 C. F. LAVEY ET AL.
Scheme 4
round-bottomed flask equipped with a reflux conden-
ser. Sodium azide (0.62 g, 9.6 mmol) was added in
seven portions over 1 h – bubbling ensued and the
reaction turned yellow. The solution was heated at
508C for 6 h, then cooled to RT, diluted with water
(5 ml), cooled to 08C, and made basic using aqueous
sodium hydroxide (12 M). The aqueous solution was
extracted with methylene chloride (3 ꢀ 30 ml). The
combined organic layers were washed with brine
(20 ml), dried (sodium sulfate) and evaporated at room
temperature to give the crude product as a brown
liquid. The residue was purified by column chromato-
graphy (methylene chloride) to give 5 as a brown/
orange oil (0.82 g, 81%). 1H NMR (CDCl3): d 7.18 (m,
0.5 H), 7.14 (m, 0.5 H), 6.97 (m, 0.5 H), 6.89 (m, 0.5 H),
6.78 (m, 0.5 H), 6.74 (m, 0.5 H), 6.58 (m, 0.5 H), 6.49
(m, 0.5 H), 3.71 (bs, 2 H).
water (30 ml) and methylene chloride (50 ml). The
layers were separated and the aqueous layer was
extracted with methylene chloride (2 ꢀ 50 ml). The
combined organic layers were dried (sodium sulfate)
and evaporated to give a mixture of 6 and disulfide
7 (0.92 g) as an orange oil. The material was directly
oxidized to the disulfide.
Di-2-fluoro[13C6]benzenedisulfide (7). A mixture of
2-fluoro[13C6]thiophenol (6) and di-2-fluoro[13C6]ben-
zenedisulfide (7) (0.92 g total) was dissolved in metha-
nol (17 ml) and water (6.9 ml). Sodium perborate
(1.37 g, 13.72 mmol) was added and the yellow solution
became thick yellow. After 20 min, the reaction pro-
gress was checked by HPLC (system 1). When all
starting thiophenol was gone, water (5 ml) was added
and the aqueous solution was extracted with methy-
lene chloride (3 ꢀ 30 ml). The combined organic layers
were dried (sodium sulfate) and evaporated to give the
crude product as a brown oil (0.7 g). The material was
purified by column chromatography (hexanes) to give
purified 7 (0.158 g) and a mixture of 7 and a byproduct
(0.192 g).z
2-Fluoro[13C6]thiophenol (6). A cloudy orange mixture
of 2-fluoro[13C6]aniline (5, 0.82 g, 7 mmol) in sulfuric
acid:water (1:6, 7.4 ml) was cooled to 08C. Sodium
nitrite (0.58 g, 8.4 mmol) in water (3.4 ml) was added
dropwise and the diazonium salt, a clearer orange
solution, was stirred at 08C for 2 h. In a separate
flask, a mixture of potassium ethyl xanthate7 (2.02 g,
12.6 mmol), sodium tetraborate decahydrate (7.74 g,
20.3 mmol), and water (31 ml) was heated to 728C. After
the yellow mixture became clear, the diazonium salt
was added dropwise. The reaction immediately turned
cloudy yellow and an orange oil formed. The mixture
was heated at 728C for 1 h, stirred at RT for 1 h, then
allowed to stand overnight. Water (30 ml) was added
and the aqueous solution was extracted with methy-
lene chloride (3 ꢀ 50 ml). The combined organic layers
were dried (sodium sulfate) and evaporated to give
the crude intermediate xanthate (1.75 g) as an orange
oil. This compound was hydrolyzed by refluxing in a
solution of potassium hydroxide (0.90 g, 16.1 mmol) in
ethanol (7.8 ml) for 5 h. After the reaction was cooled to
RT, it was evaporated to remove the ethanol. The dark
orange solid that resulted was partitioned between
N-(1-{4-[4-Chloro-2-(2-fluoro[13C6]phenylsulfanyl)benze-
nesulfonyl]phenyl}ethyl)-2,2,2-trifluoroacetamide (9).
A
solution of N-{1-[4-(4-chlorobenzenesulfonyl)-phenyl]
ethyl}-2,2,2-trifluoroacetamide (8, 0.21 g, 0.54 mmol)
and 1,10-phenanthroline (trace amount) in tetrahydro-
furan (2.3 ml) was cooled to ꢁ45 to ꢁ508C. n-Butyl-
lithium (0.99 ml, 1.08M, 1.07mmol) was added
dropwise until the solution turned dark red. The
reaction was stirred at ꢁ45 to ꢁ508C for 45min, then
added dropwise via Teflon cannula to a solution of the
z The byproduct is a less polar impurity that forms during the
oxidation reaction. Both the purified di-2-fluoro[13C6]benze-
nedisulfide (7) and the mixture of di-2-fluoro[13C6]benzene-
disulfide and the byproduct are used independently in the
following two reactions, then the purified products from those
reactions are combined and subjected together to the last
reactions in the series.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 264–272
DOI: 10.1002.jlcr

SYNTHESIS OF 13C6, 3H, AND 14C LABELED SCH 414319 269
purified disulfide 7 (0.158 g, 0.59mmol) in tetrahydro-
furan (1.6 ml) at ꢁ20 to ꢁ258C. The orange solution was
stirred for 15min, then checked by HPLC (system 1).
When the reaction was complete, it was warmed to 08C
and acetic acid (0.0936 ml, 1.62mmol) was added.
After 10min, water (20 ml) and ethyl acetate (30 ml)
were added and the pH was raised to 10 using aqueous
sodium hydroxide (25%). The layers were separated,
water (20 ml) was added and the pH was again raised
to 10. The layers were separated and the organic layer
was washed with aqueous sodium chloride (20%, 10ml),
dried (sodium sulfate) and evaporated to give crude
9 (0.461 g, chemical purity 64%) as an orange oil.}
The material was used without purification in the
following reaction. [The same reaction was carried out
using the mixture of di-2-fluoro[13C6]benzenedisulfide
and a byproduct to give the crude product (0.592 g) as a
yellow oil.]
turned cloudy, then clear light yellow. The reaction was
stirred for 19 h, then checked by HPLC (system 2). When
all starting material was gone, aqueous sodium chloride
(10%, 5 ml) and methyl tert-butyl ether (10 ml) were
added. The layers were separated and the organic layer
was washed again with aqueous sodium chloride (10%,
5 ml), then dried (sodium sulfate) and evaporated. The
residue was coevaporated with acetonitrile (3 ꢀ 10 ml) to
give 11 (0.206 g, 86%) as a light yellow oil that was dried
under vacuum overnight.
(S)-N-[1-[4-[[4-Chloro-2-[(2-fluoro[13C6]phenyl)-sulfonyl]-
phenyl]sulfonyl]phenyl]-ethyl]trifluoromethanesulfona-
mide (12). A mixture of 11 (0.206 g, 0.448 mmol) in
acetonitrile (5.1 ml) was heated in a 508C oil bath to
put the compound into solution, then cooled to ꢁ20 to
ꢁ258C. N-methylmorpholine (0.054 ml, 0.493 mmol)
was added followed by the dropwise addition of triflic
anhydride (0.0753 ml, 0.448 mmol) over 30 min. After
10 min, the reaction progress was checked by HPLC
(system 2). If starting material remained, an appro-
priate amount of N-methylmorpholine and triflic
anhydride was added and the reaction was again
checked after 10 min. When the reaction was com-
plete, it was diluted with water (10 ml) and ethyl
acetate (10 ml) and the pH was lowered to 4.5 with
aqueous hydrochloric acid (1 N). Sodium chloride
(0.03 g) was added and the aqueous layer was
extracted with ethyl acetate (2 ꢀ 30 ml). The combined
organic layers were dried (sodium sulfate) and evapo-
rated to give crude [13C6]Sch 414319 (0.6 g) as a yellow
oil, which was purified by column chromatography
(2:8 ethyl acetate:hexanes) to give 12 (0.191 g, 72%) as
a white foam. The compound was recrystallized twice
from toluene, then converted to the piperazine salt
(13) to remove an impurity that was present in 1.1%.
(S)-N-[1-[4-[[4-Chloro-2-[(2-fluoro[13C6]phenyl)sulfoxyl]-
phenyl]sulfonyl]phenyl]-ethyl]-2,2,2-trifluoroacetamide
(10). To a solution of crude 9 (0.461 g, chemical purity
64%, 0.56 mmol) in ethyl acetate (1.8 ml) was added
urea/hydrogen peroxide (0.238 g, 2.52 mmol). The
reaction, under argon, was placed in a RT water bath
and acetonitrile (0.22 ml) was added. Trifluoroacetic
anhydride (0.32 ml, 2.24 mmol) was added dropwise to
the yellow mixture over 1 h}the reaction became clear
yellow. The reaction was stirred for 2.5 h at RT then
checked by HPLC (system 1). When all of 9 was gone,
the reaction was diluted with ethyl acetate (5 ml) and
cooled to 10–158C. Sodium sulfite (0.213 g, 1.68 mmol)
in water (0.9 ml) was added dropwise}the reaction
turned cloudy, then clear yellow. The pH was raised to
8 with aqueous sodium hydroxide (25%, 0.6 ml) and the
aqueous solution was extracted with ethyl acetate
(4 ꢀ 15 ml). The combined organic layers were washed
with brine (15 ml), dried (sodium sulfate) and evapo-
rated to give the crude product as an orange oil, which
was purified by column chromatography (2:8 ethyl
acetate:hexanes) to give the product (10) as a white
solid. This material was combined with the purified
product generated from the disulfide+byproduct start-
ing material to give 10 (0.29 g) as a white solid.
Piperazine salt of [13C6]Sch 414319 (13). A mixture of
piperazine (0.215 g, 0.25 mmol) in acetone (0.2 ml) was
heated in a 508C oil bath until it was clear and
colorless. Compound 12 (0.148 g, 0.25 mmol) in acet-
one (1 ml) was added and the mixture was heated for
15 min at 508C, then slowly cooled to ꢁ108C over 1.5 h.
The acetone was removed by passing a stream of
nitrogen over the flask until 0.3 ml remained. Toluene
(3 ml) was added and the reaction became cloudy. The
flask was stored at ꢁ208C overnight to induce crystal-
lization, then the solvent was filtered away to give 13
(0.161 g, 95%) as a white solid with a chemical purity of
97.5%. FAB Mass Spectrometry of unlabeled Sch
414319 1: m/z 586.266 (M), 490, 452, 437, 426, 341,
333, 317, 277, and 269. FAB mass spectrometry of 13:
m/z 592.229 (M), 490, 458, 443, 426, 341, 339, 323,
277, and 275. 1H NMR of 13 (d6-DMSO): 8.40 (d, 1 H),
(S)-4-[[4-Chloro-2-[(2-fluoro[13C6]phenyl)sulfonyl]phe-
nyl]sulfonyl]-alpha-methylbenzenemethanamine (11). A
mixture of lithium hydroxide (0.0375 g, 1.56 mmol) in
water (1.4 ml) was stirred for 5 min until it turned clear. A
solution of 10 (0.29 g, 0.52 mmol) in acetonitrile (0.7 ml)
and tetrahydrofuran (1 ml) was added}the reaction
} The basic aqueous layers can be acidified and extracted with
methylene chloride to recover 2-fluoro[13C6]thiophenol (6).
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002.jlcr

270 C. F. LAVEY ET AL.
8.35 (m, 1 H), 8.14 (dd, 1 H), 8.10 (m, 1 H), 7.79
(m, 1 H), 7.74 (d, 1 H), 7.53 (d, 1 H), 7.51 (m, 1 H),
7.36 (m, 1 H), 4.42 (q, 1 H), 2.82 (s, 8 H), 1.20 (d, 3 H).
(0.6 ml) and ether (2.4 ml). The mixture was sonicated,
then cooled to 08C under argon. A solution of ethanol
(0.088 ml, 1.50 mmol) in pyridine (0.24 ml) was added
dropwise. The cloudy, light yellow reaction was warmed
to room temperature and stirred for 2 h. Distilled water
(2 ml) and ether (7 ml) were added. The layers were
separated and the organic layer was washed with
hydrochloric acid (2 ml, 1 N) and brine (2 ml). The
aqueous layers were washed with methylene chloride
(2 ml) and this was added to the ether solution. The
combined organic layers were dried (sodium sulfate)
and evaporated to afford a yellow oil (75.28 mCi) that
had a radiochemical purity (RCP) of 59% by radio-TLC
(1:9 ethyl acetate:hexanes). The crude material was
purified using a Waters silica gel ‘sep-pak’ (5 g, 1:99
ethyl acetate:hexanes) to give [14C]-sulfinate ester 15
as a yellow oil (39.34 mCi, RCP 99%, 44% yield from
Synthesis of [14C]Sch 414319 (19)
[
14C]-o-Fluorobenzene sulfinic acid (14). Into a dry,
25 ml, round-bottomed flask was added tetrahydrofur-
an (3 ml) under argon. Three freeze/thaw cycles were
performed, and then
[
14C]-fluorobenzene (90 mCi,
64.75 mCi/mmol, 1.39 mmol) was vacuum transferred
from a break-seal ampoule into the flask over 1 hr. After
the flask was removed from the vacuum transfer
system, it was cooled to ꢁ788C under argon. sec-
Butyllithium (0.72 M, 1.93 ml) was added dropwise
over 15 min and the resulting clear, light yellow
solution was stirred at ꢁ788C for 2.5 h. Into a dry,
100 ml, round-bottomed flask was added tetrahydro-
furan (30 ml) under argon. The solvent was cooled to
ꢁ50 to ꢁ608C and then sulfur dioxide was bubbled into
the solvent for 10 min while the flask was minimally
open to the air. The anion was added quickly via a
teflon cannula to the sulfur dioxide/tetrahydrofuran
solution. The flask originally containing the anion was
rinsed with tetrahydrofuran (2 ꢀ 1 ml) and this was
added to the reaction flask via a teflon cannula. The
reaction was warmed slowly to 08C under argon over
1.3 h (if the reaction warmed to ꢁ408C too quickly, then
[
14C]-fluorobenzene).
(S)-N-[1-[4-[[4-Chloro-2-[([14C]-2-fluorophenyl)sulfoxyl]-
phenyl]sulfonyl]phenyl]-ethyl]-2,2,2-trifluoroacetamide
(16). To a dry 25 ml, round-bottomed flask were added
(S)-N-[1-[4-[[4-chlorophenyl]sulfonyl]
phenyl]-ethyl]-
2,2,2-trifluoroacetamide (9, 0.262 g, 0.669 mmol),
1,10-phenanthroline (1 mg), tetrahydrofuran (2.7 ml),
and N,N,N’,N’-tetramethylethylenediamine (0.101 ml,
0.669 mmol). The solution was cooled to ꢁ55 to ꢁ608C
under argon. n-Butyllithium (1.28 ml, 1 M, 1.40 mmol)
was added dropwise (the indicator was used to
determine when 1 eq. of base had been added, even
though the base had been previously titrated). The
resulting dark brown solution was stirred under argon
for 45 min at ꢁ55 to ꢁ608C and then added via a teflon
cannula to a solution of ethyl [14C]-o-fluorobenzene
sulfinate ester (15, 39.34 mCi, 64.75 mCi/mmol,
0.608 mmol) in tetrahydrofuran (1.9 ml) at ꢁ45 to
ꢁ508C. The flask originally containing the anion was
rinsed with tetrahydrofuran (2 ꢀ 1 ml) and this was
added to the reaction flask via a teflon cannula. After
15 min, the reaction conversion was 55% by HPLC
(system 1). The reaction was warmed to 08C and acetic
acid (0.104 ml, conc.) was added. The cloudy yellow
mixture was warmed to room temperature and stirred
for 10 min. Water (4 ml) and ethyl acetate (4 ml) were
added and the pH was raised to 11 with sodium
hydroxide (0.04 ml, 25%). The layers were separated
and the organic layer was extracted with pH 11 water
(3 ml). The layers were separated and the combined
aqueous layers were extracted with ethyl acetate (3 ml).
The combined organic layers were washed with water
(2 ml), brine (2 ml), dried (sodium sulfate) and
evaporated to give [14C]-sulfoxide 16 as a yellow/
orange oil (37.34 mCi, RCP 60%). The material was
used without purification in the following reaction.
the deprotonated fluorobenzene decomposed to
a
benzyne intermediate). The resulting cloudy white
mixture was warmed to room temperature while open
to the air. The stir bar was removed and the reaction
was evaporated to give a white solid. Hydrochloric acid
(5 ml, 1 N) was added and the suspension was evapo-
rated and then coevaporated with acetonitrile (4 ꢀ 5 ml)
to remove residual water. The resulting white solid was
extracted with methylene chloride (2 ꢀ 5 ml) and ethyl
acetate (2 ꢀ 5 ml). The combined organic extracts were
dried (sodium sulfate) and evaporated in a 50 ml
round-bottomed flask to give a light yellow oil that
was dried in a vacuum desiccator overnight. Crude 14
(0.271 g, assume quantitative yield) was used without
purification in the following reaction.
Ethyl [14C]-o-fluorobenzene sulfinate ester (15). Into a
50 ml, round-bottomed flask containing crude [14C]-o-
fluorobenzene sulfinic acid (14, assume 1.39 mmol)
was added toluene (4.5 ml). The mixture was sonicated
and then stirred under argon while thionyl chloride
(0.51 ml, 6.95 mmol) was added dropwise. The reaction
was stirred at room temperature for 1.5 h, then
evaporated. The residue was coevaporated with toluene
(3 ꢀ 2 ml) to remove residual thionyl chloride. To the
cloudy yellow oil with white solid were added toluene
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 264–272
DOI: 10.1002.jlcr

SYNTHESIS OF 13C6, 3H, AND 14C LABELED SCH 414319 271
(S)-N-[1-[4-[[4-Chloro-2-[([14C]-2-fluorophenyl)sulfonyl]-
phenyl]sulfonyl]phenyl]-ethyl]-2,2,2-trifluoroacetamide
(17). Into a 25 ml, round-bottomed flask were added
mmol, 0.281mmol) was added acetonitrile (3.2 ml). The
solution was cooled to ꢁ20 to ꢁ258C under argon.
N-methylmorpholine (0.034ml, 0.309mmol) was added
followed by the dropwise addition of triflic anhydride
(0.0473ml, 0.281mmol). After 10 min, the reaction
progress was checked by HPLC (system 2). If the re-
action was incomplete, the appropriate equivalents of
N-methylmorpholine and triflic anhydride (correspond-
ing to the amount of starting material still present) were
added to the reaction and the reaction progress was
again checked by HPLC. This addition was repeated one
more time. Water (3 ml) and ethyl acetate (5 ml) were
added. Hydrochloric acid (0.03 ml, 1N) was added to
lower the pH to 4–4.5. The layers were separated and the
aqueous layer was extracted with ethyl acetate (2 ꢀ 3 ml).
The combined organic layers were washed with brine
(2 ml), dried (sodium sulfate) and evaporated to give
crude trifluoromethanesulfonamide 19 as a yellow oil
(16.9 mCi). The crude material was first purified using a
Waters silica gel ‘sep-pak’ (5 g, 1:2 ethyl acetate:hexanes)
to give compound 19 as a yellow oil (9.81mCi, RCP
88.9%), which was further purified using NP-HPLC
[
14C]-sulfoxide 16 (37.34 mCi, 64.75 mCi/mmol, 0.577
mmol), ethyl acetate (2 ml), urea/hydrogen peroxide
(0.244 g, 2.60 mmol), and acetonitrile (0.23 ml) under
argon. The cloudy yellow mixture was placed in a room
temperature water bath. Trifluoroacetic anhydride
(0.326 ml, 2.31 mmol) was added dropwise over
30 min; the reaction turned clear yellow. After 2.5 h,
the reaction was complete by HPLC (system 1). Ethyl
acetate (4 ml) was added and the reaction was cooled to
10–158C. Sodium sulfite (0.218 g, 1.73 mmol) in water
(1.2 ml) was added dropwise; the reaction turned
cloudy, then clear yellow. Sodium hydroxide (0.4 ml,
25%) was added to raise the pH to 5–9. The layers were
separated and the aqueous layer was extracted with
ethyl acetate (3 ꢀ 3 ml). The combined organic layers
were washed with brine (2 ml), dried (sodium sulfate)
and evaporated to give crude [14C]-sulfone 17 as a
yellow oil (33.93 mCi, RCP 59%). The crude product
was purified by silica gel chromatography (2:8 ! 4:6
ethyl acetate:hexanes) to give [14C]-sulfone 17 as a
white solid (20.5 mCi, RCP 96.9%, 55% yield from [14C]-
sulfinate ester 15, HPLC system 2).
(Phenomenex Prodigy 5 m silica (30), 10 mm ID
x
250mm, 8 ml/min, 254 nm, 18:82 ethyl acetate:hex-
anes, column cleaned with ethyl acetate between injec-
tions) to give [14C]Sch 414319 (19) as a white solid
(7.3 mCi, RCP 99.2%).
S-4-[[4-Chloro-2-[([14C]-2-fluorophenyl)sulfonyl]phenyl]-
sulfonyl]-alpha-methylbenzenemethanamine (18). Into
a 15ml, round-bottomed flask was added compound 17
(20.5 mCi, 64.75 mCi/mmol, 0.317 mmol), tetrahydro-
furan (2ml), and acetonitrile (0.7 ml). Lithium hydroxide
(22.8 mg, 0.951 mmol) in water (0.9 ml) was added
dropwise; the reaction became clear and then cloudy
yellow. The reaction was stirred at room temperature
overnight, then diluted with t-butylmethyl ether (4ml)
and washed with sodium chloride (2ꢀ 2 ml, 10%). The
combined aqueous extracts were washed with t-butyl-
methyl ether (2ml). The combined organic extracts were
dried (sodium sulfate) and filtered into a 25ml round-
bottomed flask. It was estimated that there was
20.2 mCi of crude [14C]-amine 18. (The radioactivity of
the product was determined by counting the radio-
activity in the aqueous washes and subtracting this
number from that of the starting material.) The solvent
was removed by passing nitrogen over the solution, and
the [14C]-amine was used immediately in the following
reaction. For the purposes of calculating the amounts of
the reactants needed for the next reaction, it was
assumed that the product was 90% pure.
Synthesis of [35S]Sch 225336 (21)
[
35S]Mesyl chloride. To a 10 ml Reactiware Vial was
added solution of methane
35S]sulfonic acid
a
[
(10.0 mCi, 1438 Ci/mmol) in water (97 ml). Ethanol
(800 ml) and potassium hydroxide (5 ml, 1 N) were added
and the solvent was removed by passing nitrogen over
the solution. Ethanol (800 ml) was added again and then
removed by passing nitrogen over the solution. Anhy-
drous methylene chloride (1500 ml) and oxalyl chloride
(200 ml, 2.1 mmol) were added and the clear, colorless
solution was stirred under argon (gas evolves). After
1 h, 10% dimethylformamide in methylene chloride
(30 ml) was added and more gas evolved. The reaction
was stirred at room temperature under argon for 18 h.
Methylene chloride (1.5 ml) was added and the reac-
tion was cooled to 08C. The organic solution was
washed with ice cold aqueous sodium bicarbonate
(2 ꢀ 2 ml, 1%), room temperature sodium bicarbonate
(2 ml, 1%), and room temperature sodium hydrogen-
sulfite (1 ml, 2%). The organic solution was dried
(sodium sulfate) for 1 h, then filtered through a plug
of sodium sulfate into a flame-dried 15 ml pear-
shaped flask. The methylene chloride solution was
distilled under atmospheric pressure at 608C until the
volume was reduced to approximately 100 ml. This
(S)-N-[1-[4-[[4-Chloro-2-[([14C]-2-fluorophenyl)-sulfonyl]-
phenyl]sulfonyl]phenyl]-ethyl]trifluoromethanesulfona-
mide (19). Into
a
25ml round-bottomed flask
containing crude [14C]-amine 18 (18.2 mCi, 64.75mCi/
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 264–272
DOI: 10.1002.jlcr

272 C. F. LAVEY ET AL.
[
35S]mesyl chloride solution was used immediately in
the following reaction.
Acknowledgements
The authors would like to thank the Schering-Plough Re-
search Institute Molecular Spectroscopy group for analy-
sis of [13C6]Sch 414319and DrT. M. Chan from this group
for tritium NMR work. Thanks are also due to SPRI Chem-
ical Development and Discovery Researchers for inter-
mediates and unlabeled Sch 414319 and Sch 225336.
[
35S]Sch 225336 (21). To a dry 0.3 ml Reactiware vial
were added compound 20 (10 mg, 21.7 mmol), methy-
lene chloride (20 ml), and triethylamine (5 ml, 35.9 mmol).
The solution of
[
35S]mesyl chloride in methylene
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 264–272
DOI: 10.1002.jlcr