The synthesis of three isotopomers of 2-methyl-2-(4-[3-[1-(4-methylbenzyl)- 5-oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]phenoxy)propionic acid, a potent and selective peroxisome proliferator-activated receptor alpha agonist

Article abstract of DOI:10.1002/jlcr.1395

Although fenofibrate (1a) is commercially available and clinically effective in lowering serum triglycerides, its activity and sub-type selectivity at the PPARα receptors are only moderate; therefore, there exists a need for more potent and sub-type selec

Full text of DOI:10.1002/jlcr.1395

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 693–701.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1395
Research Article
The synthesis of three isotopomers of 2-methyl-2-(4-[3-[1-
(4-methylbenzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazol-3-
yl]propyl]phenoxy)propionic acid, a potent and selective
peroxisome proliferator-activated receptor alpha agonist
FENGJIUN KUO*, DEAN K. CLODFELTER, NAGY A. FARID, WILLIAM J. WHEELER and LENNON H. MCKENDRY
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center Indianapolis, IN 46285, USA
Received 10 January 2007; Revised 4 February 2007; Accepted 5 February 2007
Abstract: Although fenofibrate (1a) is commercially available and clinically effective in lowering serum triglycerides,
its activity and sub-type selectivity at the PPARa receptors are only moderate; therefore, there exists a need for more
potent and sub-type selective PPARa agonists. To that end, discovery efforts have identified 2-methyl-2-(4-[3-[1-(4-
methylbenzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]phenoxy)propionic acid (2), a potent and selective
human PPARa receptor agonist. In support of pre-clinical ADME studies and bioanalysis, three isotopomers of 2
have been synthesized. The results of these efforts are described below. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: PPARa; C-14; tritium; SLIS; dyslipidemia
Introduction
propionic acid (2), a potent and selective human PPARa
receptor agonist (IC50¼ 24 nM versus hPPARa, 6500 nM
versus hPPARg and 3800 nM versus hPPARd), which
has been selected for clinical studies.4 Preliminary
pharmacokinetic studies conducted in Beagle dogs and
Fischer 344 rats (Table 1) indicated that 2 had good
oral bioavailability (79 and 100%) as well as a reason-
ably long half-life (6.1 and 6.8 h, respectively). Further
drug disposition studies in laboratory animals and
humans (a preliminary report of the use of 2-[14C] in
humans has been published)5 required C-14-labeled
material. In addition, the tritiated isotopomer (2-[3H])
was synthesized for use in the determination of protein
binding. A stable labeled isotopomer was synthesized
for use as a stable labeled internal standard (SLIS) for
LC–MS–MS bioanalysis of 2 in biological fluids. The
syntheses of the isotopomers of 2 will be detailed in this
paper.
In the developing world, cardiovascular disease is the
leading cause of death.1 Among the associated risk
factors of this disease are hypercholesterolemia and
hypertriglyceridemia. The fibrates (e.g. fenofibrate, 1a,
Figure 1) have been widely used in the treatment of
dyslipidemia and are the current treatment of choice
for hypertriglyceridemia.2 These agents effectively re-
duce serum triglycerides and effectively increase HDL-
cholesterol in humans because of their agonist activity
at the PPARa (peroxisome proliferator-activated recep-
tors) receptor. The PPAR’s are members of the nuclear
receptor hormone super family of ligand-activated
transcription factors. Three mammalian PPAR isoforms
have been identified (PPARa, PPARd and PPARg) and
each receptor sub-type displays distinct biological
actions.3 Although 1b, the active metabolite of 1a
is a ligand for the PPARa receptor, its activity (IC50
¼
24 000 nM versus hPPARa) and sub-type selectivity are
moderate. Xu et al. recently reported on a SAR which
delivered 2-methyl-2-(4-[3-[1-(4-methylbenzyl)-5-oxo-
4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]phenoxy)-
Discussion
Bromination of 2 with bromine/acetic acid afforded
2-Br in nearly quantitative yield. Treatment of 2-Br
with deuterium gas in the presence of 10% Pd–C/Et3N
yielded 2-D with incorporation of deuterium in the
aromatic ring as well as the aryl methyl moiety. From
the 1H-NMR spectrum, it was found that 2-D was a
*Correspondence to: Fengjiun Kuo, Lilly Research Laboratories, Eli
Lilly and Company, Lilly Corporate Center, DC1543, Indianapolis, IN
46285, USA. E-mail: kuo fengjiun@lilly.com
Copyright # 2007 John Wiley & Sons, Ltd.

694 F. KUO ET AL.
RO2C
O
Cl
HO2C
O
N
N
O
N
H
O
1a; R = i-Pr
1b; R = H
2
Figure 1 Structures of early PPAR-alpha agonists.
To support the ADME studies, a carbon-14-labeled
material was required with the tracer labeled at a
metabolically stable position, preferably at the back-
bone of the molecule. The commercially available
3-(4-methoxyphenyl)-propan-1-ol (6) was mesylated
followed by treatment with KCN-[14C] to give the
corresponding nitrile-[14C] 7. Hydrolysis of the nitrile
under basic conditions gave the carboxylic acid 8. De-
methylation9 of 8 followed by re-alkylation of the
resulting phenol 9 with ethyl bromo-iso-butylate (EBIB)
afforded the carboxylic acid 10 (Scheme 3).4,10
Table 1 Pharmacokinetic properties of 2 in Fischer 344 rats
and Beagle dogs
Compound 2
F344 rat
Beagle dog
Bioavailability (%)
T1/2 (h)
CL (mL/min/kg)
Volume of distribution
100
6.8
8.4
4.9
79
6.1
10.8
5.8
mixture of compounds 2a–c in which there were either
2, 3 or 4 deuteriums (83% incorporation in the aryl
moiety and 38% exchange in the aromatic methyl
group). Indeed, when treated with tritium gas under
the same conditions, the 3H-NMR (Figure 2) of 3
showed three peaks in the methyl region; a singlet at
d 2.21 (single tritium), a doublet at 2.23 (J¼ 16:1 Hz,
two tritia), a triplet at 2.26 (J¼ 15:9 Hz, three tritia),
and a singlet at 6.80 ppm corresponding to the position
ortho to the oxygen. By comparing the 1H-NMR of the
tritiated material, there appeared to be 47% tritiation
on the aromatic ring and 12% on the methyl group. The
specific activity as determined by mass spectrometry
was 22 Ci/mmol and the radiochemical purity (RCP)
was 99.7% (Scheme 1).
The carboxylic acid 10 was converted to an acyl
chloride 11 and condensed with a mesylate salt of
hydrazine 12 to give an intermediate 13, which without
isolation was cyclized under acidic conditions (using
excess of camphorsulfonic acid) to a triazolone 14.
Saponification of 14 gave the desired product 2-[14C]
(Scheme 4).4,10
Conclusion
Methods were developed for labeling of a leading can-
didate 2-methyl-2-(4-[3-[1-(4-methylbenzyl)-5-oxo-4,5-
dihydro-1H-[1,2,4]triazol-3-yl]propyl]phenoxy)propionic
acid (2), a potent and selective human PPARa receptor
agonist. Three isotopomers have been synthesized to
support its use in ADME studies and bioanalysis.
Bargellini described the synthesis of a-alkoxy-iso-
butyric acids by the reaction of chloroform–acetone
with alcohols in the presence of base.6 This procedure
was later examined in further detail by Weizmann et al.
and was found to involve the intermediacy of the
a-lactone of a-hydroxy-iso-butyric acid formed by the
reaction of chloroform–acetone with NaOH.7 Nucleo-
philic attack of the lactone by either alkoxide or
phenoxide formed the a-alkoxy- or phenoxy-iso-butyric
acid. Corey et al. reported that treatment of a mixture of
cresol and acetone with powdered NaOH, followed by
the addition of CHCl3, yielded a-tolyloxy-iso-butyric acid
in 45% yield.8 Reduction of 2 with BH3–THF reduced the
carboxyl group to yield 4. Treatment of 4 with BBr3 in
CH2Cl2 at ꢀ788C yielded phenol 5. Reaction of 5 with
powdered NaOH in acetone was stirred at room
temperature. The reaction was chilled to ꢀ158C and
chloroform was added to yield 2 after purification.
Repeating this sequence with acetone-[UL-13C] and
13CHCl3 yielded 2-[13C4] in 50% yield (Scheme 2).
Experimental
To monitor the deuterium labeling, LC–MS analysis
was run on LCQ Classic Mass Spectrometer with inline
PDA UV detector, using a Metachem Polaris C18-A
column (150 ꢁ 2 mm, 5 m). The NMR spectra were
obtained on a Varian Mercury-400 spectrometer at
400 (1H) and 100 (13C) MHz. Chemical shifts are
reported in parts per million (ppm) downfield from
tetramethylsilane. ES-MS was run on a Waters Micro-
mass ZQ single quadrapole mass spectrometer. Exact
masses were determined on a Micromass High Resolu-
tion Q-TOF II mass spectrometer (Manchester, UK).
Flash chromatography was performed on Biotage
system, or as described by Still et al., using E.M.
Science silica gel 60 (230–400 mesh). RCP was
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 693–701
DOI: 10.1002.jlcr

THE SYNTHESIS OF THREE ISOTOPOMERS 695
δ
δ
δ
Figure 2 Tritium NMR of 3-[3H] in CD3OD. Inset is the expanded region from d 2.1 to 2.3 ppm. Figure available in colour online at
www.interscience.wiley.com
HO2C
O
N
N
O
N
H
2
Br2/HOAc
X
Y
Z
O
Br
HO2C
D2/10% Pd-C
Et3N/THF
HO2C
O
D
N
N
N
N
O
O
N
H
N
H
2a; X = Y = H, Z = D
2b; X = H, Y = Z = D
2c; X = Y = Z = D
2-Br
D2/10% Pd-C
Et3N/THF
X
Y
Z
O
T
HO2C
N
N
O
N
H
3a; X = Y = H, Z = T
3b; X = H, Y = Z = T
3c; X = Y = Z = T
Scheme 1 Scheme available in colour online at www.interscience.wiley.com
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 693–701
DOI: 10.1002.jlcr

696 F. KUO ET AL.
BH3 (THF)
THF
O
HO2C
O
N
N
N
HO
N
O
O
N
N
H
H
4
2
BBr3/CH2Cl2
*
*
O
HO
HO2C
acetone-[UL-13C]
N
N
N
N
*
*
O
O
13CHCl3/NaOH
N
H
N
H
2-13C4
5
* = 13C
Scheme 2
O
O
O
1. Ms2O, DMAP
30% H2O2
OH
*
*
CN
CO2H
Et3N
2. K *CN
1N NaOH
6
7
8
Pyr HCl
EtO2C
O
HO
EBIB
*
*
CO2H
CO2H
NaOEt/EtOH
10
9
Scheme 3
O
EtO2C
O
EtO2C
O
O
H2N
+
pyridine/EtOAc
N
NH2
*
N
NH2
COR
N
H
*
R = OH, 10
R = Cl, 11
O
(COCl)2/EtOAc
13
MsOH
12
camphor-
sulfonic acid
EtOAc
EtO2C
O
HO2C
O
aq. NaOH
N
N
N
N
O
O
N
H
N
*
*
H
2-[14C]
14
Scheme 4
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 693–701
DOI: 10.1002.jlcr

THE SYNTHESIS OF THREE ISOTOPOMERS 697
assessed by autoradiography. As a further check of the
RCP, the sample was subjected to radio-HPLC.
1H-[1,2,4]triazol-3-yl]propyl]phenoxy)-2-methylpropio-
nic acid (0.0313 g, 0.064 mmol), 10% Pd–C (0.032 g)
and THF were added (3 mL). A deuterium-filled balloon
was added and the reaction mixture was cooled in a dry
ice/acetone bath. The flask was evacuated and re-filled
with deuterium (this was repeated twice). The cooling
bath was removed and the mixture was stirred over an
atmosphere of deuterium. The progress of the reaction
was followed by HPLC (vide infra) and after stirring for
2.75 h, Et3N (45 mL, 0.323 mmol, 5 eq) was added and
the mixture was stirred overnight. The mixture was
filtered through Celite. The filter cake was washed with
THF (3 ꢁ 1 mL) and the filtrate was concentrated in
vacuo. The residue was re-dissolved in CH3OH and
concentrated (1 mL, repeated four times) to remove any
exchangeable deuterium. The residue was partitioned
between Et2O and water (1:1); concentration of the
Et2O extract and crystallization from Et2O yielded
2-methyl-2-(4-[3-[1-(4-methylbenzyl)-5-oxo-4,5-dihy-
dro-1H-[1,2,4]triazol-3-yl]propyl]phenoxy)propionic-
[2-phenoxy-methylphenyl-2H] (0.023 g, 87%) as a white
solid which was 98% pure by HPLC. ES-MS: ½M þ Hꢂþ,
m=z ¼ 411, 412, 413, 414; 1H-NMR (acetone/d6)
d 1.41 (s, 6H, O–C(CH3)2), 1.79 (dt, 2H, J¼ 7:7 Hz,
CH2CH2CH2), 2.15 (s, 1.86H, ArCH3, diminished in
size and broadened because of the H–D coupling), 2.35
(t, 2H, J¼ 7:45 Hz; triazole–CH2), 2.45 (t, 2H,
J¼ 7:42 Hz, ROPh–CH2), 4.67 (s, 2H, ArCH2), 6.68 (d,
1H, J¼ 8:9 Hz, 50-ArOR), 6.94 (m, 1.17 H, 2060-ArOR),
6.99 (d, 2H, J¼ 8:02 Hz, 30, 50-Ar–triazole), 7.07 (d, 2H,
J¼ 8:02 Hz, 20, 60-Ar–triazole) and 10.56 (bs, 1H, NH).
Integration of the NMR showed about 83% incorpora-
tion in the aromatic ring and 38% exchange in the
aromatic methyl group.
Synthesis of 2-(2-bromo-4-[3-[1-(4-methylbenzyl)-5-
oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]phe-
noxy)-2-methylpropionic acid, 2-Br
An acetic acid solution (6 mL) of 2-methyl-2-(4-[3-[1-(4-
methylbenzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazol-
3-yl]propyl]phenoxy)propionic acid (2, 0.527 g, 1.286
mmol) was stirred and treated dropwise with bromine
(100 mL, 1.940 mmol, 1.5 eq). The reaction was followed
by HPLC (vide infra); the RT ð2Þ ¼ 7:65 and RT
ð2-BrÞ ¼ 8:12. After 30 min, the reaction was approxi-
mately 50% complete. An additional 33 mL of bromine
was added and stirring was continued for 15 min.
HPLC showed the reaction to be 77% complete.
Additional bromine (33 mL) was added twice more for a
total of 199 mL (3.86 mmol, 3 eq). Three hours after the
final addition of bromine (total reaction time was ca.
6 h), HPLC showed a single peak corresponding to 2-Br.
The reaction mixture was chilled in an ice bath and
treated with Na2SO3 (0.42 g, 3.31 mmol) in 15 mL of
water whereupon a white gum was deposited. The
mixture was filtered and the residue was washed with
water (3 ꢁ 5 mL) and then hexanes (5 mL). The semi-
solid residue was dissolved in Et2O (5 mL) and stirred
at 58C for 1 h. A white crystalline precipitate formed
which was collected by filtration, washed with
fresh Et2O (3 ꢁ 3 mL) and dried in vacuo to yield 2-(2-
bromo-4-[3-[1-(4-methylbenzyl)-5-oxo-4,5-dihydro-1H-
[1,2,4]triazol-3-yl]propyl]phenoxy)-2-methylpropionic
acid (2-Br, 0.612 g, 97%). ES-MS: ½M þ Hꢂþ,
m=z ¼ 488=490; ½M þ Naꢂþ, m=z ¼ 512=514. In MS–
MS, the base peak was 105, corresponding to CH3–
PhCHþ2 which confirms the location of the site of the
bromination. 1H-NMR (acetone/d6) d 1.59 (s, 6H,
O–C(CH3)2), 1.94 (dt, 2H, J ¼ 7:4 and 8 Hz,
CH2CH2CH2), 2.29 (s, 3H, ArCH3), 2.50 (dd, 2H, J ¼
7:2 and 7.4 Hz, triazole–CH2), 2.61 (dd, 2H, J ¼ 7:4 and
7.7 Hz, ROPh–CH2), 4.79 (s, 2H, ArCH2), 6.94 (d, 1H,
J¼ 8:3 Hz, 50-ArOR), 7.10 (dd, 1H, J ¼ 2 and 7.5 Hz,
60-ArOR), 7.13 (d, 2H, J¼ 7:7 Hz, 30, 50-Ar–triazole),
7.21 (d, 2H, J¼ 7 Hz, 20, 60-Ar–triazole), 7.43 (d, 1H,
J¼ 2 Hz, 30-ArOR) and 10.44 (bs, 1H, NH).
HPLC
method:
Zorbax
SB-phenyl
column
(4.6 ꢁ 250 mm) with gradient elution at 2 mL/min
(Solvent A ¼ CH3CN with 0.1% TFA; Solvent B=0.1%
aqueous TFA) and UV detection at 230 nm. Gradient:
100% of solvent B to 100% of solvent A in 10 min; hold
at 100% A for 5 min, then gradient to 100% B in 1 min.
Synthesis of 2-methyl-2-(4-[3-[1-(4-methylbenzyl)-5-
oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]phe-
noxy)propionic-[2-phenoxy-3H] acid, 3a–c
HRMS (QTOF): Calculated for
C
23H26BrN3O4:
A mixture of 2-(2-bromo-4-[3-[1-(4-methylbenzyl)-5-
oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]-phe-
noxy)-2-methylpropionic acid (0.0054 g, 0.011 mmol)
and 10% Pd–C (0.0087 g) in DMF (1.5 mL) containing
i-Pr2NEt (0.05 mL) was stirred under tritium gas (5 Ci)
for 2 h. The reaction mixture was filtered and the
filtrate was concentrated in vacuo. The residue was re-
dissolved in EtOH and subsequently concentrated to
remove the labile tritium (this was repeated twice).
487.1107. Found: 487.1099.
Synthesis of 2-methyl-2-(4-[3-[1-(4-methylbenzyl)-5-
oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]phe-
noxy)propionic-[2-phenoxy-2H] acid, 2a–c
A three-necked flask was purged with dry nitrogen; 2-
(2-bromo-4-[3-[1-(4-methylbenzyl)-5-oxo-4,5-dihydro-
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 693–701
DOI: 10.1002.jlcr

698 F. KUO ET AL.
The residue was purified by reversed-phase HPLC on an
Ultrasphere ODS column and gradient elution similar to
the one used above. The pure 2-methyl-2-(4-[3-[1-(4-
methylbenzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]
propyl]-phenoxy)propionic-[2-phenoxy-methylphenyl-
3H] was concentrated in vacuo and re-dissolved in
EtOH at 1 mCi/mL.
3H-NMR (CD3OD): d 2.21 (s); 2.23 (d, J¼ 16:1 Hz) 2.26
(t, J¼ 15:9 Hz), 6.80 (s) (Figure 2). By comparing the
1H-NMR of the tritiated material, there appeared to be
47% tritiation on the aromatic ring and 12% on the
methyl group. The specific activity as determined by
mass spectrometry was 22 Ci/mmol and the RCP was
99.7%, showing a single peak by HPLC on a Spherisorb
Phenyl column (4.6 ꢁ 250 mm) with gradient elution at
1 mL/min; 0% B to 100% B over 2 min (Solvent A ¼ 1%
TFA in water; Solvent B ¼ 1% TFA in CH3CN). UV dete-
ction was at 230 nm. FAB-MS: ½M þ Hꢂþ, m=z ¼ 410,
412, 414.
in CH2Cl2, 7.0 mmol) was added dropwise over 15 min.
The resulting mixture was stirred at 58C for 0.5 h and
then quenched by the dropwise addition of methanol
(5 mL). After 5 min, the quenched mixture was poured
into a mixture of ice and water and stirred for 0.5 h. The
resulting white precipitate was collected by filtration,
washed with water and then hexanes (3 ꢁ 25 mL).
HPLC (vide supra) showed the material to be pure
(RT¼ 7:18 min). Air drying afforded 5-[3-(4-hydroxy-
phenyl)-propyl]-2-(4-methylbenzyl)-2,4-dihydro-[1,2,4]
triazol-3-one as a white solid (1.70 g, 86.7%): 1H-NMR
(acetone/d6)
d 1.90 (dt, 2H, J ¼ 7:4 and 7.7 Hz,
CH2CH2CH2), 2.29 (s, 3H, ArCH3), 2.47 (dd, 2H, J ¼
7:0 and 8.1 Hz, triazole–CH2), 2.56 (dd, 2H, J ¼ 7:7 and
7.8 Hz, ArCH2), 4.81 (s, 2H, CH3PhCH2), 6.74 (d, 2H,
J¼ 8:4 Hz, 30, 50-HOAr), 7.01 (d, 2H, J¼ 8:3 Hz, 20,60-
HOAr), 7.13 (d, 2H, J¼ 7:3 Hz, 30, 50-CH3Ar), 7.21 (d,
2H, J¼ 8:5 Hz, 20, 60-CH3Ar), 8.14 (s, 1H, OH) and
1
10.47 ppm (bs, H, NH); ES-MS: ½M þ Hꢂþ, m=z ¼ 324,
½M þ Naꢂþ, m=z ¼ 346.
Synthesis of 5-[3-[4-(2-hydroxy-1,1-dimethylethoxy)
phenyl]2-(4-methylbenzyl)-2,4-dihydro[1,2,4]triazol-
3-one, 4
HRMS (QTOF): Calculated for C19H21N3O2: 323.1634.
Found: 323.162.
Synthesis of 2-methyl-2-(4-[3-[1-(4-methylbenzyl)-5-
oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]propyl]phenoxy)
propionic-[2-methyl-2-propionyl-UL-13C] acid, 2-13C
A THF solution (25 mL) solution of 2-methyl-2-(4-[3-[1-
(4-methylbenzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazol-
3-yl]propyl]phenoxy)propionic acid (2.50 g, 6.11 mmol)
was cooled in an ice bath under nitrogen and treated
dropwise (over 10 min) with borane–THF complex
(30 mL, 1 M in THF, 30 mmol). The resulting clear
solution was stirred in the cold for 1.5 h and then
allowed to warm to room temperature. After 90 h, the
Sodium hydroxide (0.866 g, 21.64 mmol, 11.5 eq) was
ground in a mortar and pestle and added to a flask
containing 5-[3-(4-hydroxyphenyl)-propyl]-2-(4-methyl-
benzyl)-2,4-dihydro-[1,2,4]triazol-3-one (0.605 g, 1.88
mmol). Acetone-[UL-13C] (4.0 g, 65.5 mmol, 35 eq) was
added and the resulting mixture was stirred at room
temperature under a balloon of nitrogen for 20 min. A
gummy precipitate was formed; the mixture was cooled
to ca. ꢀ158C and CHCl3–[13C] (1.08 g, 8.97 mmol,
4.8 eq) was added dropwise over 4 min. After stirring
at ꢀ158C for 2 h, the reaction mixture was allowed to
warm to room temperature and stirring was continued
overnight. The excess acetone–[UL-13C] was distilled
off; the residue was cooled in an ice bath and diluted
with water (5 mL) and EtOAc (10 mL). The mixture was
acidified to pH 1 with HCl (2 mL, 5 N) and the EtOAc
layer was separated and filtered through anhydrous
MgSO4. The filtrate was concentrated in vacuo. HPLC of
the residue showed the desired compound (36.5%) as
well as several smaller peaks due to impurities. The
residue was dissolved in CH3OH and divided into three
1.5 mL aliquots. Each was purified by preparative
HPLC (TAI Gel-ODS column) eluting with CH3CN/H2O
(each containing 0.01% TFA, 47:53) at 8 mL/min. The
combined eluate containing the desired product was
concentrated in vacuo and extracted with EtOAc
(4 ꢁ 25 mL). The combined extracts were dried (anhy-
THF solution was slowly decanted into
a stirred
mixture of ice/water and then extracted with Et2O
(100 mL). The aqueous layer was re-extracted with
Et2O (3 ꢁ 50 mL). The combined Et2O extracts were
washed with water (2 ꢁ 100 mL) and then NaHCO3
(2 ꢁ 50 mL). The Et2O extract was dried (anhydrous
MgSO4) and concentrated in vacuo to yield 2.65 g of a
yellow viscous oil. The oil was triturated with pentane,
methyl tert. butyl ether to yield 5-[3-[4-(2-hydroxy-1,
1-dimethylethoxy)-phenyl]2-(4-methylbenzyl)-2,4-dihy-
dro[1,2,4]triazol-3-one as a white gummy solid (2.40 g,
99.3%). HPLC (vide supra) showed that this material
was slightly impure but free of starting material.
Synthesis of 5-[3-(4-hydroxyphenyl)propyl]-2-(4-methyl-
benzyl)-2,4-dihydro-[1,2,4]triazol-3-one, 5
A methylene chloride solution (25 mL) of 5-[3-[4-(2-
hydroxy-1,1-dimethylethoxy)-phenyl]2-(4-methylben-
zyl)-2,4-dihydro[1,2,4]triazol-3-one as a white gummy
solid (2.40 g, 6.07 mmol) was cooled in an ice bath
under nitrogen and stirred; boron tribromide (7 mL, 1 M
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002.jlcr

THE SYNTHESIS OF THREE ISOTOPOMERS 699
drous MgSO4) and concentrated in vacuo. The residue
was crystallized from Et2O to yield 2-methyl-2-(4-[3-[1-
(4-methylbenzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]tria-
zol-3-yl]propyl]-phenoxy)propionic-[2-methyl-2-pro-
pionyl-UL-13C] acid (0.387 g, 50%) as a white solid
which was >97.5% pure by HPLC (RT ¼ 7:69, vide
infra). ES-MS: ½M þ Hꢂþ, m=z ¼ 414 (judged to be
>95% 13C4 and 5% 13C3); 1H-NMR (acetone/d6) d
1.41 (ddd, 6H, J13C2H ¼ 128:7, 4.1, 3.9 Hz,
13C(13CH3)2), 1.92 (dt, 2H, J ¼ 7:7 and 7.4 Hz,
CH2CH2CH2), 2.16 (s, 3H, ArCH3), 2.48 (t, 2H,
J¼ 7:5 Hz, triazole–CH2), 2.59 (t, 2H, J¼ 7:5 Hz,
ArCH2), 4.78 (s, 2H, CH3PhCH2), 6.82 (d, 2H,
J¼ 8:3 Hz, 30, 50-HOAr), 7.08 (d, 2H, J¼ 8:3 Hz, 20,60-
HOAr), 7.13 (d, 2H, J¼ 7:7 Hz, 30, 50-CH3Ar), 7.21 (d,
2H, J¼ 7:7 Hz, 20, 60-CH3Ar),10.38 (bs, 1H, NH) and
11.2 ppm (bs, 1H, CO2H); 13C-NMR (acetone/d6) d
24.7 (d, 13CH3), 78.54 (m, 13C(13CH3)2) and 174.71
(d, 13CO2H).
water (2 ꢁ 10 mL), and concentrated in vacuo with
absolute EtOH (20 mL). The residue was further dried
in vacuo overnight to give the desired acid 8 as beige
solid (440 mg, 2.26 mmol, 97.8% chemical purity by
HPLC). The crude product 8 was used in the next step
without further purification.
HPLC conditions: (Zorbax SB-Phenyl, 4.6 ꢁ 250 mm).
UV at 220 nm, temperature at rt, flow rate at 1 mL/min.
Solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in
ACN. Gradient: A=B ¼ 70:30 to A=B ¼ 50:50 in 15 min,
then maintain at 50:50 for 10 min. Product is at
Rt¼ 11:27 min.
1H-NMR of mesylate (CDCl3): 7.10 (d, 2H), 6.84 (d,
2H), 4.10 (t, 2H), 3.79 (s, 3H), 3.00 (s, 3H), 2.67 (t, 2H),
2.05 (tt, 2H). MS: ½M þ NH4ꢂþ¼262:2. 1H-NMR of
unlabeled acid 8 (CDCl3): 7.08 (d, 2H), 6.82 (d, 2H),
3.89 (s, 3H), 2.60 (t, 2H), 2.35 (t, 2H), 1.94 (tt, 2H). MS:
½M þ NH4ꢂþ ¼ 212:2.
HRMS (QTOF): Calculated for C1193C4H27N3O4:
413.4393. Found: 413.2136.
Synthesis of 4-[4-(1-ethoxycarbonyl-1-methylethoxy)
phenyl]-butyric acid-[14C], 10
Synthesis of 4-(4-methoxyphenyl)butyric acid-[14C], 8
The crude acid 8 (440 mg, 2.26 mmol) was mixed with
pyridinium HCl salt (1.2 g) in absolute EtOH (15 mL) to
give a colorless, clear solution. The solution was
concentrated in vacuo to remove most of the ethanol
to give a residue as a wet solid containing residual
ethanol. The flask containing the wet solid was heated
in an oil bath (about 1008C) with stirring to melt the
solid to give a colorless clear solution. The resulting
solution was then heated at 190–2008C (oil tempera-
ture) overnight (about 21 h) under an argon atmo-
sphere. After cooling to room temperature, the crude
product was stirred with MTBE (20 mL) and 1 N HCl
(20 mL). The layers were separated. The aqueous layer
was extracted with MTBE (2 ꢁ 10 mL), and the com-
bined MTBE extracts were washed once with 1 N HCl
(20 mL) and with water (2 ꢁ 20 mL). The solution was
concentrated in vacuo. The resulting residue was taken
into absolute EtOH (20 mL) and re-concentrated in
vacuo. The residue was further dried in vacuo overnight
to give a crude product 9 as a beige solid (392 mg,
2.15 mmol). Without further purification, the crude
compound 9 (390 mg) was dissolved in anhydrous
EtOH (12 mL) containing anhydrous EtOAc (0.2 mL) to
give a clear solution. To the solution was added a
solution of 21% sodium ethoxide in EtOH (2 mL), and
the resulting solution was heated to reflux for 70 min.
To the refluxing solution was added ethyl bromo-iso-
butyrate (1 mL, 3 eq) via a syringe, and 0.6 mL more of it
was added after refluxing for about 3.75 h. The result-
ing solution was then refluxed overnight (about 15 h).
The solution was cooled in an ice bath, acidified with
conc. H3PO4 (1 mL) in water (10 mL), and concentrated
To a colorless, clear solution of mesylate (734 mg,
3 mmol, prepared from corresponding alcohol 6) in
anhydrous DMF (10 mL) was added KCN–[14C]
(100 mCi from ARC, 55 mCi/mmol), KCN (100 mg),
and the resulting heterogeneous solution was heated
in an oil bath (about 908C). The oil temperature
continued to go up to about 958C. To avoid overheating,
the flask was removed from the oil bath, and was
replaced when the oil temperature dropped to 808C.
The solution was stirred at that temperature overnight
(about 27 h), then cooled to room temperature, and
stirred overnight. To work up the reaction, the solution
was taken into a separatory funnel containing water
(75 mL) and ethyl acetate (50 mL). The layers were
separated. The organic layer was washed with water
(50 mL), and concentrated with ethanol (20 mL) under
reduced pressure to give a crude product 7 (about
550 mg as a light yellowish liquid). Without further
purification, the crude product was taken into ethanol
(10 mL). To the solution was added 1 N NaOH (10 mL),
followed by 30% H2O2 (0.5 mL) at room temperature
with stirring. The solution was stirred at 40–508C (oil
bath) for 4.5 h, then refluxed overnight (about 21.5 h).
After cooling to room temperature, the solution was
diluted with water, and then concentrated in vacuo to
remove most of ethanol. The remaining aqueous
solution was extracted with ethyl ether (2 ꢁ 10 mL) to
remove any neutral species, adjusted to about pH 2,
and extracted with ethyl acetate (2 ꢁ 10 mL). The
combined ethyl acetate extracts were washed with
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 693–701
DOI: 10.1002.jlcr

700 F. KUO ET AL.
in vacuo to remove most of ethanol. To the remaining
aqueous solution containing an oily product was added
Na2CO3 solution (3.3 g in 30 mL of water). Some white
salt precipitated. Water was added to dissolve the salt,
and the resulting clear aqueous solution was extracted
with ethyl ether (3 ꢁ 20 mL) to remove any neutral
species. The aqueous layer was adjusted to about pH
2–3 with concentrated H3PO4 (about 3.6 mL) in the
presence of ethyl acetate (30 mL). The layers were
separated, and the aqueous layer was extracted once
with ethyl acetate (20 mL). The combined ethyl acetate
solution was washed with water (2 ꢁ 30 mL), and
concentrated in vacuo with ethanol (30 mL). The
residue was dissolved in a minimum amount of CH2Cl2,
and purified by Biotage (SiO2, 100:10:1=CH2Cl2:EtOA-
c:HOAc). The desired fractions were pooled, concen-
trated in vacuo with ethanol (10 mL) and toluene
(10 mL) in sequence. The desired product 10 was
obtained as viscous oil initially; further drying in vacuo
gave a white crystalline solid (511 mg, 1.72 mmol,
80%). For cold 9: 1H-NMR (acetone-d6): 7.04 (d, 2H),
6.75 (d, 2H), 2.56 (t, 2H), 2.28 (t, 2H), 1.83 (tt, 2H). MS:
½M þ NH4ꢂþ ¼ 198:2. For cold 10: 1H-NMR (CDCl3): 7.02
(d, 2H), 6.77 (d, 2H), 4.23 (q, 2H), 2.60(t, 2H), 2.37 (t,
2H), 1.93 (tt, 2H), 1.56 (s, 6H), 1.27 (t, 3H). MS
ðM þ NH4Þþ ¼ 312:3.
(10 mL), followed by camphorsulfonic acid (2.32 g). The
resulting solution was refluxed overnight (about 16 h),
then cooled and kept at room temperature for another
24 h. The solution was then stirred with 1 N HCl
(10 mL). The organic layer was separated, washed with
1 N HCl (2 ꢁ 25 mL), sat. Na2CO3 (3 ꢁ 30 mL), water
(2 ꢁ 20 mL), and concentrated with ethanol (20 mL) in
vacuo to give a crude product as viscous oil (0.75 g).
The crude product was purified by Biotage (SiO2,
80:20:1 ¼ CH2Cl2:EtOAc:HOAc) to give the desired
product 14 as a viscous oil (0.65 g, 1.48 mmol, 86%).
For cold 14: 1H NMR (CD3OD): 7.14 (AB q, 4 H), 7.02 (d,
2H), 6.74 (d, 2H), 4.81 (s, 2H), 4.20 (q, 2H), 2.57 (t, 2H),
2.46 (t, 2H), 2.30 (s, 3H), 1.90 (dd, 2H), 1.52 (s, 6H),
1.27 (q, 3H). MS ½M þ Hꢂþ ¼ 438:3.
Synthesis of 2-[4-[3-[2,5-dihydro-1-[(4-methylphenyl)-
methyl]-5-oxo-1H-1,2,4-triazol-3-yl]propyl]phenoxy]-
2-methylpropanoic acid-[14C], 2-[14C]
The compound 14 (310 mg, 0.7 mmol) was taken into
toluene (10 mL), concentrated in vacuo, and re-dis-
solved in toluene (3 mL). To the solution was added 1 N
NaOH (1.4 mL) and water (1.7 mL), and the resulting
heterogeneous solution was stirred at room tempera-
ture overnight (about 17 h). A small amount of 14
remained as monitored by TLC (SiO2, CH2Cl2:
EtOAc:HOAc ¼ 80:25:1). About 0.5 mL of 1 N NaOH
was added to the solution, and the reaction was stirred
for another 3.5 h. The solution was then diluted with
water (15 mL), and the aqueous layer was extracted
with toluene (3 ꢁ 15 mL) to remove any neutral species.
The aqueous solution was then adjusted to about pH 2
with conc. HCl, and extracted with ethyl acetate
(20 mL). The ethyl acetate extract was washed with
water (3 ꢁ 20 mL), and concentrated in vacuo with
ethanol (20 mL) to give a viscous residue. The residue
was re-dissolved in ethyl acetate (10 mL), and any
insoluble particles were filtered off. The filtrate was
concentrated and dried in vacuo again to give viscous
oil (220 mg). The viscous oil was dissolved in 1 mL of
ethyl acetate, seeded with cold 2, and stirred at room
temperature until some white solid formed. The solu-
tion was stored in refrigerator over the weekend. After
stirring in an ice bath for about 40 min, the white solid
was filtered, rinsed with cold ethyl acetate (3 mL), and
dried in vacuo at room temperature. The desired
Synthesis of ethyl 2-[4-[3-[2,5-dihydro-1-[(4-methyl-
phenyl)-methyl]-5-oxo-1H-1,2,4-triazol-3-yl]propyl]-
phenoxy]-2-methylpropanoate-[14C], 14
To a clear solution of the acid 10 (510 mg, 1.72 mmol)
in anhydrous EtOAc (4.5 mL) was added three drops of
anhydrous DMF, followed by adding of oxalyl chloride
(175 mL, 1.15 eq) dropwise during a period of 36 min
while the solution was cooled in an ice bath. The
resulting solution was stirred for another 30 min in a
water bath to generate the corresponding acyl chloride
11. Meanwhile, in another flask, compound 12 (0.5 g,
1 eq) was stirred with anhydrous pyridine (0.6 mL) in
anhydrous EtOAc (10 mL) as a heterogeneous solution
for about 60 min. The above acyl chloride solution of 11
was concentrated in vacuo to remove the solvent and
the excess oxalyl chloride. The residue was re-dissolved
in anhydrous EtOAc (4.5 mL). Some solid was insolu-
ble. The supernatant was taken out via syringe and
added dropwise to the heterogeneous solution contain-
ing 12 during a period of 30 min while the reaction was
cooled in an ice bath. After the addition, the resulting
solution was stirred at room temperature for about 3 h.
Most of the starting material (acyl chloride of 10) was
consumed, and a new compound, presumably the
intermediate 13, was generated as detected by HPLC.
To the reaction solution was added anhydrous EtOAc
product 2-[14C] was obtained as
a white solid
(137 mg, 0.334 mmol) at 64.3 mCi/mg with 99.3% RCP
by HPLC. For cold 2: 1H-NMR (CD3OD): 7.15 (AB q,
4H), 7.03 (d, 2H), 6.80 (d, 2H), 4.80 (s, 2H), 2.57 (t, 2H),
2.46 (t, 2H), 2.30 (s, 3H), 1.90 (dd, 2H), 1.52 (s, 6H).
MS: ½M þ Hꢂþ ¼ 410:2.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 693–701
DOI: 10.1002.jlcr

THE SYNTHESIS OF THREE ISOTOPOMERS 701
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