Synthesis and stability of a carbon-14-labeled 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor

Article abstract of DOI:10.1002/jlcr.1810

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) is an effective method of lowering plasma low-density lipoprotein cholesterol levels. Hemi-calcium (3R,5S,E)-7-(4-(4-fluorophenyl)-6-isopropyl-2-(methyl(1-methyl-1H- 1,2,4-triazol-5-yl)amino)pyrimidin-5-yl)-3,5-dihydroxyhept-6-enoate (1) is a cholesterol-lowering statin drug that effectively inhibits HMGR. An important step in the development of this compound was the synthesis of a carbon-14-labeled analog for use in preclinical absorption, distribution, metabolism and excretion studies. The synthesis of a carbon-14-labeled analog of the cholesterol-lowering statin drug 1 is described. The carbon-14-labeled compound [¹⁴C]-1 was prepared in 11 steps from [¹⁴C]- labeled urea. The overall radiochemical yield for the synthesis was 22% and the radiochemical purity of [¹⁴C]-1 was 99.9% immediately after synthesis. It was found that [¹⁴C]-1 with a specific activity of 43.2μCi/mg decomposed at a rate of about 1.9%/month when stored at -78°C under argon. Three samples of [¹⁴C]-1 were prepared to study the chemical stability of the molecule. One sample had a specific activity of 3.8μCi/mg and the other two contained radical inhibitors, L-ascorbic acid (1% by weight, specific activity of 10.5μCi/mg) or BHT (1% by weight, specific activity of 9.8μCi/mg). For these samples the decomposition rates were decreased to 0.5%/month, 0.2%/month and 0.1%/month, respectively. Copyright

Full text of DOI:10.1002/jlcr.1810

Research Article
Received 24 February 2010,
Revised 25 May 2010,
Accepted 9 June 2010
Published online 5 August 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1810
Synthesis and stability of a carbon-14-labeled
3-hydroxy-3-methylglutaryl coenzyme-
A reductase inhibitor
Ã
Richard C. Burrell,a Samuel J. Bonacorsi Jr.,b J. Kent Rinehart,b
Saleem Ahmad,b Khehyong Ngu,b and Balu Balasubramanianb
Inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) is an effective method of lowering plasma low-
density lipoprotein cholesterol levels. Hemi-calcium (3R,5S,E)-7-(4-(4-fluorophenyl)-6-isopropyl-2-(methyl(1-methyl-1H-
1,2,4-triazol-5-yl)amino)pyrimidin-5-yl)-3,5-dihydroxyhept-6-enoate (1) is a cholesterol-lowering statin drug that effectively
inhibits HMGR. An important step in the development of this compound was the synthesis of a carbon-14-labeled analog
for use in preclinical absorption, distribution, metabolism and excretion studies.
The synthesis of a carbon-14-labeled analog of the cholesterol-lowering statin drug 1 is described. The carbon-14-labeled
compound [14C]-1 was prepared in 11 steps from [14C]-labeled urea. The overall radiochemical yield for the synthesis was
22% and the radiochemical purity of [14C]-1 was 99.9% immediately after synthesis. It was found that [14C]-1 with a specific
activity of 43.2 mCi/mg decomposed at a rate of about 1.9%/month when stored at À781C under argon. Three samples of
[
14C]-1 were prepared to study the chemical stability of the molecule. One sample had a specific activity of 3.8 mCi/mg and
the other two contained radical inhibitors, L-ascorbic acid (1% by weight, specific activity of 10.5 mCi/mg) or BHT (1% by
weight, specific activity of 9.8 mCi/mg). For these samples the decomposition rates were decreased to 0.5%/month,
0.2%/month and 0.1%/month, respectively.
Keywords: carbon-14 labeling; radical inhibitors; 3-hydroxy-3-methylglutaryl coenzyme-A reductase; statin drugs; radiochemical
decomposition
support the development of 1, a [14C]-labeled analog needed to
be prepared. The labeling strategy for this molecule not only
Introduction
Coronary heart disease is the leading cause of death in the
United States.1 Studies have shown that elevated plasma levels
of low-density lipoprotein (LDL) cholesterol increases ones risk
of experiencing a coronary event.1,2 3-Hydroxy-3-methylglutaryl
coenzyme-A reductase (HMGR) is the rate-limiting enzyme in the
biosynthesis of LDL cholesterol. Inhibition of HMGR has been
clinically proven to be one of the most effective methods of
lowering plasma LDL cholesterol levels and reducing event rates
associated with cardiovascular disease.2 Statins are a class of
drugs that effectively lower plasma LDL cholesterol levels by
inhibiting HMGR. These drugs are considered the gold-standard
in treating hypercholesterolemia.
took into consideration the availability of labeled starting
materials and degree of synthetic complexity but also the
known biotransformation. A conscious effort was made to make
sure the [14C]-label was not in a position known or suspected to
be metabolically labile. After collaboration with our Biotransfor-
mation colleagues, it was decided that the [14C]-label should be
incorporated into the pyrimidine core of the molecule. This
paper describes the synthesis of [14C]-labeled 1 and its chemical
stability.
Experimental procedure
Materials and methods
Bristol-Myers Squibb has investigated hemi-calcium (3R,5S,E)-7-
(4-(4-fluorophenyl)-6-isopropyl-2-(methyl(1-methyl-1H-1,2,4-triazol-
5-yl)amino)pyrimidin-5-yl)-3,5-dihydroxyhept-6-enoate (1, Figure 1)
in an effort to develop a new statin drug that was more effective
at lowering plasma LDL cholesterol levels than the statin drugs
currently on the market.3 This new compound was designed to
have minimal statin-induced side effects, namely myotoxicity
and rhabdomyolysis.3 An important step in the early develop-
ment of this compound was the initiation of preclinical
absorption, distribution, metabolism and excretion (ADME)
studies to determine the metabolic fate of the molecule using
in vivo animal models. These preclinical ADME studies typically
require a [14C]-labeled analog of the parent compound. To
All experimental procedures were optimized using unlabeled
material. Reactions were run under an inert atmosphere of
aBristol-Myers Squibb Research and Development, Department of Chemical
Synthesis, 5 Research Parkway, Wallingford, CT 06492, USA
bBristol-Myers Squibb Research and Development, Department of Chemical
Synthesis, Route 206 and Province Line Road, Princeton, NJ 08540, USA
*Correspondence to: Richard C. Burrell, Bristol-Myers Squibb, Department of
Chemical Synthesis, 5 Research Parkway, Mail Stop 4BC-377, Wallingford,
CT 06492, USA.
E-mail: richard.burrell@bms.com
J. Label Compd. Radiopharm 2011, 54 72–79
Copyright r 2010 John Wiley & Sons, Ltd.

R. C. Burrell et al.
14C]-Methyl 4-(4-fluorophenyl)-6-isopropyl-2-oxo-1,2,3,4-
HO
HO
O- 0.5 Ca2+
[
tetrahydropyrimidine-5-carboxylate ([14C]-5)4
O
4-Fluorobenzaldehyde (2, 1.89 g, 15.24 mmol), methylisobutyryl
acetate (3, 2.20 g, 15.24 mmol), [14C]-labeled urea ([14C]-4,
0.42 g, 6.78 mmol, 400 mCi, 59 mCi/mmol), unlabeled urea
(0.55 g, 9.22 mmol) (total urea used (0.97 g, 16.0 mmol, 1.05
eq)), copper (I) chloride (15.1 mg, 0.15 mmol), concentrated
H2SO4 ( 0.145 mL) and anhydrous methanol (15 mL) were added
to a heat-dried flask.4 The mixture was heated to 641C and
stirred for 47 h under argon. Afterwards, the reaction mixture
was cooled to room temperature. After 30 min at room
temperature a white solid formed, which was vacuum filtered
and washed with methanol (2 Â 10 mL). To maximize yield, the
mother liquor was heated to 651C and stirred for an additional
66 h. The mixture was cooled to room temperature and after
30 min a white solid formed. The solid was isolated as described
above and washed with methanol (3 Â 5 mL). The white solids
were combined and vacuum dried for 17 h to give 3.95 g (84%
chemical yield based on urea) of [14C]-5.4 The product was
analyzed by: HPLC, method A, RT 16.6 min, UV detector 254 nm
94.5% pure, b-Ram detector 100% pure; LC/MS m/z [14C-M1H]1
F
N
N
N
N
N
N
1
Figure 1. Structure of the HMGR Inhibitor (1).
argon and stirred magnetically unless otherwise noted. Reac-
tions were monitored by HPLC, LC/MS, TLC and NMR. When
possible, comparisons were made to authentic materials. All
reagents and solvents were ACS grade or better and used
without further purification. Carbon-14-labeled urea (400 mCi,
radiochemical purity 99.8%, specific activity 950 mCi/mg, 59 mCi/
mmol) was obtained from GE Healthcare (formerly Amersham
Biosciences). The intermediates 8 and 13 and all authentic
samples were obtained from Bristol-Myers Squibb’s Process
Research & Development department.
1
295, [12C-M1H]1 293; H NMR (300 MHz, CDCl3) d ppm 1.18 (d,
J = 6.97 Hz, 3 H), 1.19 (d, J = 6.97 Hz, 3 H), 3.62 (s, 3 H), 4.16 (sept,
J = 6.97 Hz, 1 H), 5.29 (brs, 1 H), 5.39 (d, J = 2.07 Hz, 1 H), 6.52 (brs,
1 H), 6.96–7.06 (m, 2 H), 7.21–7.31 (m, 2 H).
[
14C]-Methyl 4-(4-fluorophenyl)-2-hydroxy-6-isopropyl-
Instruments
pyrimidine-5-carboxylate ([14C]-6)4,5
Solvent removal under vacuum was accomplished using a Buchi
R-124 rotary evaporator. Column chromatography was per-
formed using a Biotages Flash Chromatography system. Proton
NMR spectra were recorded on a 300 MHz Bruker Avance
spectrometer or a 400 MHz Bruker Avance spectrometer. Mass
spectra were recorded on a Finnigan LCQ system. TLC analyses
(EMD 60 F254 silica gel-coated plates) were performed as
described and using UV (254 nm) and radiochemical detection
(QC-Scan, Bioscan Model B-QC). Specific activities were deter-
mined by gravimetric analysis using liquid scintillation counting
(Wallac Model 1409).
Dihydropyrimidinone [14C]-5 (3.95 g, 13.50 mmol) was added to
a round-bottomed flask under a nitrogen atmosphere. The
flask was cooled to 01C and concentrated HNO3 (8.93 g,
135.0 mmol) was added dropwise with stirring over 10 min.4,5
The mixture was warmed to room temperature and then stirred
for 1 h. At that time the solution was cooled to 01C and 2 N
NaOH was cautiously added dropwise until the pH was adjusted
to 6. The solution was diluted with water (20 mL) and extracted
with CH2Cl2 (3 Â 20 mL). The organic solutions were combined
and concentrated under reduced pressure to give a slightly
yellow solid. The solid was vacuum dried for 16 h to give 3.97 g
of crude [14C]-6.4–6 The product was analyzed by: HPLC, method
A, RT 15.7 min, UV detector 254 nm 99.2% pure, b-Ram detector
100% pure; LC/MS m/z [14C-M1H]1 293, [12C-M1H]1 291; 1H
NMR (300MHz, CDCl3) d ppm 1.43 (d, J= 6.78 Hz, 6 H), 3.25 (sept,
J= 6.78Hz, 1 H), 3.62 (s, 3 H), 7.09–7.19 (m, 2 H), 7.57–7.67 (m, 2 H).
HPLC
HPLC analyses were performed on a Varian instrument equipped
with two PrepStar pumps (Model SD-218, 25 mL pump heads), a
ProStar PDA detector (model 330, for analytical UV detection), a
ProStar UV-1 detector (model 320, for semi-preparative UV
detection) and an IN/US b-ram detector (model 3B, for radio-
chemical purity measurements). The following analytical meth-
od (method A) was used for all in process and final product
purity analyses.
[
14C]-Methyl 2-chloro-4-(4-fluorophenyl)-6-isopropylpyrimi-
dine-5-carboxylate ([14C]-7)4,5
Crude [14C]-6 (3.97 g, 13.66 mmol) and POCl3 (14.01 mL, 23.05 g,
150.3 mmol) were added to a round-bottomed flask.4 The
solution was heated to 1001C and stirred for 3 h under nitrogen.
At that time the mixture was cooled to 01C and slowly quenched
with a dropwise addition of water (100 mL). When the addition
was complete the mixture was warmed to room temperature
and stirred for 5 min. The aqueous solution was extracted with
CH2Cl2 (4 Â 25 mL). The organic solutions were combined and
concentrated under reduced pressure. The resulting solid
residue was dissolved in tert-butyl methyl ether (TBME, 40 mL)
and concentrated. The concentrate was vacuum dried for 15 h to
give 3.90 g of yellow/brown solid. The crude material was
purified by column chromatography (silica, 90:10 hexanes/ethyl
HPLC Method A:
Column: YMC-Pack Pro C18, 3.0 mm (4.6 Â 150 mm).
Mobile Phase A: Water with 0.05% trifluoroacetic acid.
Mobile Phase B: Acetonitrile with 0.05% trifluoroacetic acid.
Conditions: 0% B, 0–5 min; 0–100% B, 5–20 min; 100% B,
20–25 min; 100–0% B 25–30 min.
Flow rate: 1 mL/min, Injection size: 20 mL.
Detection: UV at 254 nm and radiochemical (b-ram).
All Semi-preparative HPLC purifications were conducted on
the Varian instrument described above using the methods
described in the experimental section.
J. Label Compd. Radiopharm 2011, 54 72–79
Copyright r 2010 John Wiley & Sons, Ltd.
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R. C. Burrell et al.
acetate) to give 3.40 g (82% for the two steps) of [14C]-7 as a slowly quenched with the careful addition of methanol (5 mL)
white solid.4 The product was analyzed by: TLC Rf = 0.54 (UV) and then 2 N NaOH (50 mL). The mixture was warmed to room
using 70:30 hexanes/ethyl acetate; HPLC, method A, RT 21.3 min, temperature and stirred for 30 min. The solution was diluted
UV detector 254 nm 93.0% pure, b-Ram detector 96.4% pure; LC/ with water (200 mL) and extracted with CH2Cl2 (4 Â 50 mL).
MS m/z [14C-M1H]1 311, [12C-M1H]1 309; 1H NMR (300 MHz, The organic solutions were combined and concentrated
CDCl3) d ppm 1.33 (d, J = 6.78 Hz, 6 H), 3.13 (sept, J = 6.78 Hz, under reduced pressure. Toluene (50 mL) was added and then
1 H), 3.75 (s, 3 H), 7.11–7.21 (m, 2 H), 7.62–7.72 (m, 2 H).
removed under reduced pressure. The concentrate was vacuum
dried for 14 h to give 3.18 g of [14C]-11 as a yellow solid. The
crude product was analyzed by: HPLC, method A, RT 17.4 min,
UV detector 254 nm 84.7% pure, b-Ram detector 87.0% pure;
LC/MS m/z [14C-M1H]1 359, [12C-M1H]1 357; 1H NMR (300 MHz,
CDCl3) d ppm 1.22 (d, J = 6.78 Hz, 6 H), 3.41 (sept, J = 6.78 Hz,
1 H), 3.59 (s, 3 H), 3.67 (s, 3 H), 4.60 (s, 2 H), 7.08–7.15 (m, 2 H),
7.67–7.75 (m, 2 H), 7.87 (s, 1 H).
[
14C]-Methyl 4-(4-fluorophenyl)-6-isopropyl-2-(1-methyl-1H-
1,2,4-triazol-5-ylamino)pyrimidine-5-carboxylate ([14C]-9)5,7
The chloride [14C]-7 (3.40 g, 11.03 mmol), amino triazole 85,7,8
(1.30 g, 13.25 mmol) and DMF (44 mL) were added to a round-
bottomed flask under argon.5,7 The solution was cooled to 01C
and sodium tert-butoxide (2.12 g, 22.06 mmol) was added
portion-wise over 20 min. After the addition was complete, the
dark yellow solution was warmed to room temperature and
stirred for 1 h 45 min. At that time the solution was quenched
with water (250 mL) and the mixture was extracted with CH2Cl2
(4 Â 50 mL). The combined organic solutions were concentrated
under reduced pressure. The oil that remained was diluted with
toluene (50 mL) and concentrated. This process was repeated
two more times. The concentrate was vacuum dried for 15 h to
give 3.69 g of [14C]-9 as a yellow solid.5,7 The crude product was
analyzed by: HPLC, method A, RT 16.8 min, UV detector 254 nm
94.1% pure, b-Ram detector 97.7% pure; LC/MS m/z [14C-M1H]1
373, [12C-M1H]1 371; 1H NMR (300 MHz, CDCl3) d ppm 1.24
(d, J = 6.78 Hz, 6 H), 3.15 (sept, J = 6.78 Hz, 1 H), 3.68 (s, 3 H), 3.83
(s, 3 H), 7.07–7.18 (m, 2 H), 7.52–7.63 (m, 2 H), 7.84 (s, 1 H).
General procedure for the preparation and titration
of the bleach solution
A commercial bleach solution that was advertized to contain 5%
aqueous NaOCl was added to a round-bottomed flask. The pH of
the solution was adjusted to 9.1–9.2 (actual pH was 9.15) by the
addition of solid NaHCO3. The buffered bleach solution
(approximately 1.5 g, the exact amount was recorded) was
added to an Erlenmeyer flask along with water (50 mL), KI (2.0 g)
and 6 N acetic acid (10 mL). The black solution was titrated to a
near end point with 0.1004 N sodium thiosulfate. A 1% starch
solution (3 mL) was added and the titration was continued until
a clear end point was reached. The exact amount of sodium
thiosulfate added was recorded. This data was used to calculate
the percentage of NaOCl in the stock solution. This process was
repeated a second time and the results were averaged (the
average concentration was 5.06%). This stock solution was used
in the next step.
[
14C]-Methyl 4-(4-fluorophenyl)-6-isopropyl-2-(methyl(1-
methyl-1H-1,2,4-triazol-5-yl)amino)pyrimidine-5-carboxylate
([14C]-10)5
Crude [14C]-9 (3.69 g, assume 9.97 mmol), dimethylcarbonate
(18.5 mL, 5 mL/g of [14C]-9) and 1,4-diazabicyclo[2.2.2]octane
(1.12 g, 9.97 mmol) were added to a round-bottomed flask.5 The
mixture was heated to 901C and stirred for 6 h under argon. At
that time the solution was cooled to room temperature and
diluted with water (75 mL). The aqueous solution was extracted
with CH2Cl2 (3 Â 25 mL). The organic solutions were combined,
washed with 10% H3PO4 (35 mL), brine (25 mL) and then
concentrated under reduced pressure. The material that
remained was diluted with toluene (50 mL) and concentrated
to remove residual water. The concentrate was vacuum dried for
12 h to give 3.67 g of [14C]-10 as a dark yellow viscous oil. The
crude product was analyzed by: HPLC, method A, RT 19.3 min,
UV detector 254 nm 81.9% pure, b-Ram detector 89.3% pure;
LC/MS m/z [14C-M1H]1 387, [12C-M1H]1 385; 1H NMR (300 MHz,
CDCl3) d ppm 1.19 (d, J = 6.59 Hz, 6 H), 3.14 (sept, J = 6.59 Hz,
1 H), 3.61 (s, 3 H), 3.67 (s, 3 H), 3.68 (s, 3 H), 7.06–7.15 (m, 2 H),
7.52–7.63 (m, 2 H), 7.88 (s, 1 H).
[
14C]-4-(4-Fluorophenyl)-6-isopropyl-2-(-(methyl(1-methyl-
1H-1,2,4-triazol-5-yl)amino)pyrimidine-5-carboxaldehyde
([14C]-12)5,7
Crude [14C]-11 (3.18 g, assume 8.92 mmol), TEMPO (69.7 mg,
0.45 mmol), KBr (106 mg, 0.89 mmol) and CH2Cl2 (25 mL) were
added to a round-bottomed flask. The solution was cooled to
01C and the aqueous bleach solution (pH 9.15, 5.06% NaOCl,
14.43 g, 9.81 mmol) was added dropwise over 30 min.5,7 The
internal temperature was kept below 51C during the addition.
The mixture was stirred for 1 h under argon at 01C. At that time
the reaction was quenched with 1 N sodium thiosulfate (15 mL)
and then warmed to room temperature. Next, 0.5 N NaOH
(20 mL) was added and the solution was stirred for 10 min.
Finally, water (100 mL) was added and the mixture was extracted
with CH2Cl2 (3 Â 40 mL). The organic solutions were combined
and concentrated under reduced pressure. The concentrate was
diluted with TBME (40 mL) and concentrated under reduced
pressure. The material that remained was vacuum dried for 68 h
to give 2.98 g of a dark yellow solid. The crude material was
purified by column chromatography (silica, 1:1 hexanes/ethyl
acetate) to give 2.56 g (65% for the four steps) of [14C]-12 as a
slightly yellow solid. The product was analyzed by: TLC Rf = 0.22
(UV) using 1:1 hexanes/ethyl acetate; HPLC, method A, RT
19.2 min, UV detector 254 nm 90.3% pure, b-Ram detector 97.5%
pure; LC/MS m/z [14C-M1H]1 357, [12C-M1H]1 355; 1H NMR
(300 MHz, CDCl3) d ppm 1.18 (d, J = 6.59 Hz, 6 H), 3.66 (s, 3 H),
3.71 (s, 3 H), 3.96 (sept, J = 6.59 Hz, 1 H), 7.13–7.22 (m, 2 H),
7.47–7.57 (m, 2 H), 7.92 (s, 1 H), 9.90 (s, 1 H).
[
14C]-4-(4-Fluorophenyl)-6-isopropyl-2-(methyl(1-methyl-1H-
1,2,4-triazol-5-yl)amino)pyrimidin-5-yl)methanol ([14C]-11)5,7
Crude [14C]-10 (3.67 g, assume 9.55 mmol) and anhydrous
toluene (30 mL) were added to a round-bottomed flask. The
mixture was cooled to À781C and DIBAL (1 M in toluene,
28.6 mL, 28.6 mmol) was added dropwise over 20 min.5,7 The
internal temperature was kept below À601C during the
addition. When the addition was complete the solution was
warmed to 01C and stirred for 1 h. At that time the reaction was
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R. C. Burrell et al.
minimize impurity formation.10 At that time the solution was
diluted with methanol (10 mL) and concentrated under reduced
pressure. The material that remained was dissolved in 65:35
water/acetonitrile with 17.5 mmol/L of ammonium acetate
(5 mL) and the desired product was purified by semi-preparative
HPLC (0.5 mL injections on a YMC Pack Pro C18 column s-5 mm,
250 Â 20 mm. Solvent: A = 65:35 water/acetonitrile with
17.5 mmol/L of ammonium acetate; B = 90:10 acetonitrile/water
with 17.5 mmol/L of ammonium acetate. Conditions: 0% B,
0–13 min, 6 mL/min; 0–100% B, 13–15 min, 6–15 mL/min; 100%
B, 15–23 min, 15 mL/min; 100–0% B, 23–25 min, 15 mL/min; 0%
B, 25–30 min, 15 mL/min; 0% B, 30–31 min, 15–6 mL/min, 0% B,
31–33 min, 6 mL/min. Wavelength: 230 nm). The fractions that
contained the desired product were combined and partially
concentrated. The pH of the aqueous solution was lowered to
5.2 by the addition of saturated aqueous NH4Cl (approximately
200 mL were added, the pH of the saturated NH4Cl solution was
adjusted to 3.3 with 1 N HCl prior to the addition). Care was
taken to insure the pH of the solution containing the product
did not go below 5. When the pH was lowered below 5 large
amounts of the lactone ([14C]-17) impurity were formed.5,7 The
aqueous solution was extracted with CH2Cl2 (8 Â 50 mL). The
organic solutions were combined and concentrated to a volume
of about 10 mL. The CH2Cl2 solution was filtered to remove
traces of solid ammonium chloride. The filter was washed with
CH2Cl2 (3 Â 5 mL). The organic solutions containing the acid
product were combined (total CH2Cl2 volume 25 mL) and
treated with NH3 (2 M solution in MeOH, 2.5 mL).5 The reaction
flask was capped and allowed to stand at room temperature for
1.5 h. At that time the suspension was concentrated using a
stream of nitrogen to give a white solid. The solid was vacuum
dried for 5 h to yield 0.104 g (61% for the two steps and salt
formation) of [14C]-15 with a specific activity of 48.6 mCi/mg
(24.4 mCi/mmol, total activity 5.1 mCi, 64% radiochemical yield).
The ammonium salt was analyzed by: HPLC, method A, RT
16.1 min, UV detector 254 nm 99.7% pure, b-Ram detector 99.9%
[14C]-tert-Butyl 2-((4R,6S)-6-((E)-2-(4-(4-fluorophenyl)-6-isopro-
pyl-2-(methyl(1-methyl-1H-1,2,4-triazol-5-yl)amino)pyrimidin-
5-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate ([14C]-14)3,5,7
The aldehyde [14C]-12 (2.56 g, 7.22mmol), sulfone 137,9 (3.59 g,
7.94mmol) and THF (30 mL) were added to a round-bottomed
flask. The solution was cooled to À781C and lithium bis
(trimethylsilyl)amide (1 M in THF, 9.39mL, 9.39mmol) was added
dropwise over 15min.5,7 The internal temperature was kept
below À701C during the addition. The reaction was stirred for
30min at À781C under argon. At that time the solution was
quenched with 7.5% aqueous NH4Cl (20 mL). The mixture was
warmed to room temperature and diluted with water (200mL).
The aqueous solution was extracted with CH2Cl2 (4 Â 50mL). The
organic solutions were combined, washed with 5% NaHCO3
(25 mL) and concentrated under reduced pressure. TBME (40 mL)
was added and the solution was concentrated to give a yellow oil.
Analysis by HPLC showed the E/Z ratio to be greater than 95:5 in
favor of the desired E isomer. The crude material was purified by
column chromatography (silica, 1:1 hexanes/ethyl acetate) to give
a white solid. The white solid was vacuum dried for 21h to yield
3.50g (84%) of [14C]-14 with a specific activity of 39.8mCi/mg
(23.1 mCi/mmol, total activity 139.2 mCi, 35% radiochemical yield
from urea).5,7 The product was analyzed by: TLC Rf = 0.22 (UV)
using 1:1 hexanes/ethyl acetate; HPLC, method A, RT 23.0min, UV
detector 254 nm 90.9% pure, b-Ram detector 93.4% pure; LC/MS
m/z [14C-M1H]1 583, [12C-M1H]1 581; 1H NMR (300 MHz, CDCl3)
d ppm 1.07–1.19 (m, 7 H), 1.38 (s, 3 H), 1.42–1.53 (m, 13 H),
2.22–2.34 (m, 1 H), 2.36–2.48 (m, 1 H), 3.22–3.37 (m, 1 H), 3.60
(s, 3 H), 3.68 (s, 3 H), 4.20–4.32 (m, 1 H), 4.32–4.44 (m, 1 H), 5.40
(dd, J = 16.20, 5.65Hz, 1 H), 6.47 (dd, J = 16.20, 1.13 Hz, 1 H),
6.99–7.09 (m, 2 H), 7.50–7.61 (m, 2 H), 7.89 (s, 1 H).
[
14C]-Hemi-calcium (3R,5S,E)-7-(4-(4-fluorophenyl)-6-isopro-
pyl-2-(methyl(1-methyl-1H-1,2,4-triazol-5-yl)amino)pyrimi-
din-5-yl)-3,5-dihydroxyhept-6-enoate ([14C]-1)5
The acetonide [14C]-14 (0.20 g, 0.34 mmol, 8.0 mCi, 39.8 mCi/mg), pure; the material co-eluted with an authentic sample of 15; the
acetonitrile (2 mL) and 0.02 N HCl (1 mL, 0.02 mmol) were added 1H NMR was also identical to an authentic sample.
to a round-bottomed flask.3,5,7 The solution was heated to 401C
The ammonium salt [14C]-15 (48.6 mg, 0.097 mmol, 48.6 mCi/
and stirred for 6 h. Analysis by HPLC showed complete mg, 2.4 mCi) and water (1 mL) were added to a round-bottomed
conversion to the free diol (method A, b-Ram detector 88.5% flask. An aqueous solution of CaCl2-2H2O (7.4 mg, 0.0504 mmol,
pure). Care was taken to stop the reaction as quickly as possible 0.52 eq, in 0.3 mL of water) was added to the reaction mixture.5
after completion to minimize impurity formation.10 At that time The suspension was stirred for 16 h at room temperature. At that
the solution was diluted with methanol (10 mL) and concen- time the reaction mixture was diluted with MeOH (2 Â 10 mL)
trated under reduced pressure. The off-white solid that and concentrated to give a white solid. The solid was vacuum
remained was vacuum dried for 16 h. The solid was dissolved dried for 17 h to yield 55.3 mg (100%) of [14C]-1 with a specific
in acetonitrile (5 mL) and the desired product was purified by activity of 43.2 mCi/mg (21.8 mCi/mmol, total activity 2.4 mCi,
semi-preparative HPLC (0.75 mL injections on a YMC Pack Pro 100% radiochemical yield).11 The calcium salt was analyzed by:
C18 column s-5 mm, 250 Â 20 mm. Solvent: A = water with 0.05% HPLC, method A, RT 16.1 min, UV detector 254 nm 99.1% pure,
TFA; B = acetonitrile with 0.05% TFA. Conditions: 75% B, b-Ram detector 99.9% pure; the material coeluted with an
0–10 min; 75–100% B, 10–12 min; 100% B, 12–19 min; 100–75% authentic sample of 1; 1H NMR (400 MHz, DMSO-D6) d ppm 1.09
B, 19–20 min. Flow 10 mL/min. Wavelength: 230 nm). The (d, J = 6.80 Hz, 3 H), 1.10 (d, J = 6.80 Hz, 3 H), 1.27–1.36 (m, 1 H),
fractions that contained the desired product were combined 1.46–1.54 (m, 1 H), 2.02–2.10 (m, 1 H), 2.15–2.22 (m, 1 H),
and concentrated to give 0.148 g of a white solid. The diol 3.27–3.37 (m, 1 H), 3.45 (s, 3 H), 3.61 (s, 3 H), 3.70–3.79 (m, 1 H),
product was analyzed by: HPLC, method A, RT 19.4 min, UV 4.12–4.20 (m, 1 H), 5.44 (dd, J= 15.99, 5.67 Hz, 1 H), 6.43 (d,
detector 254 nm 99.3% pure, b-Ram detector 99.4% pure. J=15.99Hz, 1 H), 7.21–7.28 (m, 2 H), 7.59–7.65 (m, 2 H), 7.90 (s, 1 H);
1
The diol (0.148 g, 0.27 mmol), acetonitrile (2 mL) and 1 N NaOH the H NMR spectrum was identical to an authentic sample.
(0.5 mL, 0.5 mmol) were added to a round-bottomed flask.3,5,7
The solution was stirred for 4 h at room temperature. Analysis
by HPLC showed complete hydrolysis of the ester (method A,
b-Ram detector 99.2% pure). Care was taken to stop the reaction
as quickly as possible after the reaction was complete to
Stability studies
Small samples of [14C]-15 (48.6 mCi/mg) and [14C]-1 (43.2 mCi/
mg) were saved to study the chemical stability of high specific
J. Label Compd. Radiopharm 2011, 54 72–79
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

R. C. Burrell et al.
activity material. A sample of [14C]-1 with lower specific activity samples of [14C]-1 containing the radical inhibitors L-ascorbic acid
was prepared by dissolving [14C]-1 (34.0 mCi/mg, 10.1 mg) and and BHT showed decomposition rates of about 0.2%/month and
unlabeled 1 (91.4 mg) in EtOH (190 proof, 10 mL). The clear 0.1%/month, respectively. As many as five decomposition
solution was concentrated and vacuum dried for 65 h to give a products were observed. The major decomposition product
white solid. The specific activity of this material was determined (4.8% for the sample of [14C]-1 with a specific activity of 43.2mCi/
to be 3.8 mCi/mg. A sample with lower specific activity contain- mg) was isolated by semi-preparative HPLC (YMC Pack Pro C18
ing L-ascorbic acid (1% by weight) was prepared by dissolving column s-5 mm, 250 Â 20mm. Solvent: A = 65:35 water/acetoni-
[
14C]-1 (43.2 mCi/mg, 16.4 mg), unlabeled 1 (50.4 mg) and trile with 17.5mmol/L of ammonium acetate. Conditions: 10% A,
L-ascorbic acid (1 mg/mL solution in EtOH (190 proof), 0.65 mL, 0–15min, 6 mL/min. Wavelength: 230 nm). This material was
0.65 mg) in EtOH (190 proof, 5 mL). The clear solution was determined to be the allylic ketone [14C]-16 (method A, RT
concentrated and vacuum dried for 15 h to give a white solid. 16.8min) after comparison of its NMR spectrum with an authentic
The specific activity of this material was determined to be sample. The next largest impurity (0.7% for the sample of [14C]-1
10.5 mCi/mg. This material was analyzed by: HPLC, method A, RT with a specific activity of 43.2mCi/mg) was determined to be the
16.1 min, UV detector 254 nm 99.5% pure, b-Ram detector 99.9% lactone [14C]-17 (method A, RT 17.3min) by coinjection with an
pure. Finally, a sample with lower specific activity containing authentic sample. The structures of the other minor impurities
BHT (1% by weight) was prepared by dissolving [14C]-1 were not determined.
(43.2 mCi/mg, 14.8 mg), unlabeled 1 (50.6 mg) and BHT (1 mg/
mL solution in EtOH (190 proof), 0.65 mL, 0.65 mg) in EtOH (190
proof, 5 mL). The clear solution was concentrated and vacuum
Results and discussion
dried for 5 h to give a white solid. The specific activity of this
material was determined to be 9.8 mCi/mg. This material was
analyzed by: HPLC, method A, RT 16.1 min, UV detector 254 nm
99.5% pure, b-Ram detector 99.9% pure.
All of the samples prepared above were stored at À781C under
argon and analyzed by HPLC (method A) about once a month
over the next 14 weeks. The samples of [14C]-15 (48.6 mCi/mg)
and [14C]-1 (43.2 mCi/mg) with high specific activities showed
decomposition rates of about 1.0%/month and 1.9%/month,
respectively. The sample of [14C]-1 (3.8mCi/mg) with low specific
activity showed a decomposition rate of about 0.5%/month. The
Our labeling strategy for compound 1 was to incorporate the
[14C]-label into the pyrimidine core. The synthetic plan that was
developed took advantage of some existing synthetic metho-
dology that was being used by the Bristol-Myers Squibb PR&D
department.5 The key step in the construction of the pyrimidine
core utilized a Biginelli three-component coupling reaction.12
This provided the option of using any one of the three starting
materials to incorporate the [14C]-label (Scheme 1). Carbon-14-
labeled urea was chosen as the label source because it was the
cheapest and most readily available of the three potential
starting materials.
F
F
O
OMe
OMe
+
O
O
14C
O
CuCl, H2SO4
MeOH, reflux
HNO3
3
HN14 NH
C
O
H
F
+
O
H2N
NH2
2
14C]-4
[14C]-5
[
O
OMe
F
O
N
OMe
N
POCl3
tBuONa, DMF
1C4
1C4
NH2
N
N
N
N
OH
Cl
14C]-7
N
[
8
[
14C]-6
F
O
OMe
F
O
N
N
OMe
N
Dimethylcarbonate
DABCO
N 14
N
H
1C4
N
C
N
N
N
N
N
N
[14C]-10
[
14C]-9
Scheme 1.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 72–79

R. C. Burrell et al.
The Biginelli reaction was conducted using 4-fluorobenzalde- coupled via a Julia olefination to give [14C]-14 in an 84%
hyde (2), methylisobutyryl acetate (3) and [14C]-labeled urea yield.3,5,7 Deprotection of the acetonide ([14C]-14) with HCl,
([14C]-4, 400 mCi) as shown in Scheme 1. This gave the desired hydrolysis of the tert-butyl ester with NaOH and then formation
dihydropyrimidinone ([14C]-5) in an 84% yield.4 Most impor- of the ammonium salt of the carboxylic acid gave [14C]-15 in a
tantly this intermediate had all of the functionality around the 61% yield.3,5,7 The ammonium salt [14C]-15 was treated with
core in the appropriate positions to complete the synthesis. The CaCl2 to produce the desired calcium salt [14C]-1 in 100% yield.5
dihydropyrimidinone ([14C]-5) was oxidized with nitric acid to This material had a radiochemical purity of 99.9% and a specific
produce the pyrimidinol [14C]-6.4–6 Treatment of this inter- activity of 43.2 mCi/mg.
mediate with POCl3 produced the corresponding chloropyrimi-
Radiolabeled [14C]-1 was used successfully in preclinical
dine ([14C]-7) in an 82% yield for the two steps.4 The ADME studies to determine the metabolic fate of the molecule.
chloropyrimidine ([14C]-7) was coupled with amino triazole In the course of this work it was discovered that the ammonium
85,7,8 to give the secondary amine [14C]-9.5,7 The secondary salt [14C]-15 (48.6 mCi/mg) and the calcium salt [14C]-1, (specific
amine [14C]-9 was methylated with dimethylcarbonate to yield activity above 30 mCi/mg) had poor chemical stability. These
the tertiary amine [14C]-10.5
materials decomposed at rates of about 1.0%/month and 1.9%/
Next, the olefinic top portion of the molecule was installed month respectively to two major components and several minor
and the synthesis was completed as shown in Scheme 2. The components when stored at À781C under argon (Table 1).
methyl ester [14C]-10 was reduced with DIBAL to produce the A semi-preparative HPLC method was developed to purify
primary alcohol [14C]-11.5,7 A TEMPO oxidation of [14C]-11 gave impure [14C]-1. However this purification was difficult in practice
the corresponding aldehyde [14C]-12 in a 65% overall yield for since the impurities eluted close to the desired product.
the four steps.5,7 The aldehyde [14C]-12 and sulfone 137,9 were Fortunately the bulk supply of the material was stored as the
F
OH
N
F
O
N
N
OMe
N
DIBAL
TEMPO, Bleach
KBr, CH2Cl2
1C4
N
Toluene
N
N
1C4
N
N
N
N
N
[
14C]-10
[
14C]-11
O
N
O
O
O
F
O
H
N
F
LiHMDS, THF
O
O
N14
N
C
1C4
N
O
O
O
S
O
N
N
N
N
N
N
13
N
N
N
[
N N
14C]-14
[
14C]-12
HO
HO
O-NH4
+
HO
HO
O- 0.5 Ca2+
O
O
F
F
CaCl2
water
1. 0.02 M HCl, ACN
2. NaOH, water, ACN
3. NH3 in MeOH
N14
N
N 14
N
C
C
N
N
N
N
N
N
[
N
[
N
14C]-15
14C]-1
Scheme 2.
J. Label Compd. Radiopharm 2011, 54 72–79
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

R. C. Burrell et al.
Table 1. Summary of stability data (14 weeks)
14C]-15
[
[
14C]-1
[
14C]-1
[
14C]-1
[
14C]-1
48.6 mCi/mg 43.2 mCi/mg 3.8 mCi/mg 10.5 mCi/mg Ascorbic acid 9.8 mCi/mg BHT
Rate of decomposition (%/month)
[
[
1.0
2.1
0.4
1.9
4.8
0.7
0.5
0.7
0.4
0.2
0.3
0.2
0.1
0.2
0.1
14C]-16 formed (%)
14C]-17 formed (%)
O
HO
O-R
fully protected intermediate [14C]-14, which was chemically
stable. The requests for highly pure [14C]-1 were met by
preparing fresh batches of material as needed from [14C]-14 and
using them as quickly as possible after preparation. This strategy
was fine for the preclinical ADME studies, but would not work
for the preparation of clinical supplies needed to fund the
human ADME study. For these studies the radiolabeled material
needed to be relatively stable for several weeks to several
months to allow for drug product manufacturing and dosing.
Confirming the identity of the two major decomposition
products was the first step taken to understand how and why
OH
O
O
O
F
F
N 14
N
N 14
N
+
R = 0.5 Ca2+ or NH4
C
C
N
N
N
N
N
N
N
N
14C]-16
[
14C]-17
[
[
14C]-1 was decomposing. The larger of the two impurities as
Figure 2. Structures of the two major decomposition products.
measured by HPLC peak integration was isolated by semi-
preparative HPLC. This compound was determined to be allylic
significantly lower than the sample with specific activity
3.8 mCi/mg with no radical inhibitor. These results show that
there is an advantage to adding radical inhibitors to [14C]-1, a
compounds susceptible to radical promoted decomposition.
These results also show that BHT is slightly better than L-ascorbic
acid at stabilizing this [14C]-labeled compound. However, both
provided potential solutions to the stability issue that needed to
be resolved before the human ADME study could be conducted.
1
ketone [14C]-16 by comparison of its H NMR spectrum to an
authentic sample (Figure 2). The smaller impurity was deter-
mined to be lactone [14C]-17 by coinjection with an authentic
sample. Based on the structure of the largest impurity ([14C]-16),
the decomposition pathway is believed to be
a radical
promoted, oxidative decomposition process that is initiated by
b particle emission from carbon-14. This phenomenon has been
documented for other [14C]-labeled compounds.13–15 It is worth
noting that under similar conditions, this decomposition was
not observed with unlabeled 1.
Conclusion
The synthesis of [14C]-1 was completed in 11 steps from [14C]-
labeled urea in a 22% overall yield. Unfortunately [14C]-1 with a
specific activity of 43.2 mCi/mg decomposed at a rate of 1.9%/
month when stored at À781C. As the decomposition pathway
was believed to be radical initiated, samples were prepared that
contained the radical inhibitors L-ascorbic acid or BHT. By adding
L-ascorbic acid the decomposition rate was reduced to about
0.2%/month and by adding BHT the decomposition rate was
reduced to about 0.1%/month. The use of radical inhibitors to
stabilize compounds labeled with radioactive isotopes has been
shown to be effective numerous times in the literature. Most of
these examples focus on the use of radical inhibitors in the
solution phase. It is not always practical or feasible to store
radiolabeled compounds as solutions, especially when the
radiolabeled compounds are intended for use in human clinical
studies. The radical inhibitors L-ascorbic acid and BHT have not
yet been shown to be effective at stabilizing a large number of
[14C]-labeled compounds being stored as solids. However, it is
believed that this approach could be applicable to other [14C]-
labeled compounds that undergo rapid degradation due to
radical-promoted decomposition.
Three samples were prepared to study the stability of [14C]-1.
Sample one, with a specific activity of 3.8 mCi/mg, was prepared
by diluting [14C]-1 (34.0 mCi/mg) with unlabeled 1. This sample
was prepared because [14C]-1 with lower specific activity should
have better stability. Samples two and three were prepared to
study the effects of radical inhibitors on sample stability.14,15
Sample two (10.5 mCi/mg) was prepared by diluting [14C]-1
(43.2 mCi/mg) with unlabeled 1 and L-ascorbic acid (1% by
weight).15,16 The hope was that radical inhibitors would reduce
or even inhibit the decomposition of [14C]-1. Sample three
(9.8 mCi/mg) was prepared by diluting [14C]-1 (43.2 mCi/mg) with
unlabeled 1 and 2,6-di-tert-butyl-4-methylphenol (BHT, 1% by
weight).15,16 This sample was prepared to compare the
effectiveness of the different radical inhibitors. The radical
inhibitors L-ascorbic acid and BHT were specifically chosen
because they are commonly found in foods, supplements and
consumer products, making them acceptable additives to a
drug substance administered to human subjects.
All samples were stored at À781C under argon and analyzed
periodically by HPLC over the next 14 weeks. The sample of
[
14C]-1 with a specific activity of 3.8 mCi/mg decomposed at a
rate of about 0.5%/month. This was better than the high specific
activity material but the stability still did not meet the
requirements necessary for a human ADME study. The samples
Acknowledgements
of [14C]-1 with L-ascorbic acid (1% by weight, specific activity We thank the BMS Process Research and Development
of 10.5 mCi/mg) and BHT (1% by weight, specific activity of Department for intermediates, procedures and helpful discus-
9.8 mCi/mg) decomposed at rates of about 0.2%/month and sions. We also thank the BMS Biotransformation and Medicinal
0.1%/month, respectively. These decomposition rates were Chemistry Departments for helpful discussions.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 72–79

R. C. Burrell et al.
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Copyright r 2010 John Wiley & Sons, Ltd.
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