Synthesis of d3-cerivastatin for use as internal standard in a LC/MS/MS method developed for quantitation of the drug in human serum

Article abstract of DOI:10.1002/jlcr.1050

d₃-Cerivastatin was synthesized as an internal standard for use in a LC/MS/MS method developed for the simultaneous quantitative determination of the drug in human serum. d₃-Cerivastatin was efficiently prepared on large scale from d

Full text of DOI:10.1002/jlcr.1050

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 311–319.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1050
Research Article
Synthesis of d3-cerivastatin for use as internal standard
in a LC/MS/MS method developed for quantitation of
the drug in human serum
Bang-Chi Chen1,*, Mark S. Bednarz1, Huiping Zhang1, Peng Guo1,
Mohammed Jemal2, Jeffrey A. Robl1, Scott A. Biller1, Joseph E. Sundeen1,
Balu Balasubramanian1 and Joel C. Barrish1
1 Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, NJ 08543-4000, USA
2 Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research
Institute, New Brunswick, NJ 08903-0191, USA
Summary
d3-Cerivastatin was synthesized as an internal standard for use in a LC/MS/MS
method developed for the simultaneous quantitative determination of the drug in
human serum. d3-Cerivastatin was efficiently prepared on large scale from
d3-iodomethane using improved procedures. Copyright # 2006 John Wiley
& Sons, Ltd.
Key Words: cerivastatin; deuterium; Horner olefination
Introduction
Cerivastatin, sodium (E)-(+)-(3R,5S)-7-[4-(4-fluorophenyl)-2,6-diisopropyl-
5-methoxymethyl)-pyrid-3-yl]-3,5-dihydroxyhept-6-enoate, is a new synthetic
inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
effective for the treatment of high serum cholesterol.1–3 A LC method, based
on post-column induced fluorometric detection, has been reported for the
quantitation of cerivastatin in human plasma.4 Methods based on radio-
immunoassay, HMG-CoA reductase inhibition assay and capillary gas
chromatography for the quantitation of cerivastatin in human plasma have
also been reported.5 For a project aimed at the development of a bioanalytical
method based on high-performance liquid chromatography (LC) with
electrospray tandem mass spectrometry (MS/MS) for the simultaneous
*Correspondence to: Bang-Chi Chen, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
E-mail: bangchi.chen@bms.com
Copyright # 2006 John Wiley & Sons, Ltd.
Received 20 October 2005
Revised 14 December 2005
Accepted 16 December 2005

312
B.-C. CHEN ET AL.
quantitative determination of cerivastatin in human serum,6 deuterium labeled
cerivastatin was required. While stable isotope labeled cerivastatin has the
same solubility, extraction and chromatographic behavior as its non-labeled
counterpart, its difference in molecular weight makes it distinguishable in LC/
MS/MS from the non-labeled counterpart. Therefore, stable isotope labeled
cerivastatin is an ideal internal standard in a LC/MS/MS assay used for the
quantitation of the drug in biological matrices.
Results and discussion
The methoxy site of cerivastatin appears to be appropriate for labeling with
deuterium because of the ready availability of d3-iodomethane. While the
previously reported procedures for the synthesis of cerivastatin7,8 could be
adapted, significant modifications were needed and new procedures were
developed for the preparation of the deuterated intermediates and
d3-cerivastatin (15) (Schemes 1 and 2). This new and improved synthesis
is featured by a Horner type olefination using diphenyl phosphine oxide
(11) and the readily available chiral non-racemic aldehyde (12),9 to
avoid the chemical and chromatographic resolutions or the lengthy prepara-
tion of chiral non-racemic side chain needed in the previously reported
methods.7,8
Thus, treatment of ethyl isobutyryl acetate (1) with 4-fluorobenzaldehyde
(2) in isopropanol in the presence of catalytic amounts of acetic acid and
piperidine gave ethyl 2-(4-fluorobenzylidene)-4-methyl-3-oxopentanoate (3) in
95% yield as a mixture of cis/trans-isomers. Condensation of 3 with ethyl
O
F
O
F
piperidine
cat. HOAc
H2N
OEt
O
O
4
+
EtOH
60%
OEt
CHO
i-PrOH
95%
O
2
OEt
1
3
F
F
F
O
O
O
O
O
DDQ
Red-Al
HO
OEt
EtO
OEt
CH2Cl2
95%
EtO
OEt
THF
78%
N
7
N
H
N
6
5
Scheme 1.
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF D3-CERIVASTATIN FOR USE AS INTERNAL STANDARD
313
F
F
F
O
LiAlH4
NaH/CD3I
O
D3CO
OH
D3CO
OEt
THF
86%
THF
HO
OEt
100%
N
9
N
N
7
8
F
F
Ph2POEt
PBr3
D3CO
Br
D3CO
P(O)Ph2
CH2Cl2
94%
PhCH3
71%
N
N
10
11
F
TFA
O
O
O
1. n-BuLi/THF
CH2Cl2
96%
OBu-t
2.
O
O
O
D3CO
H
OBu-t
40%
N
13
O
12
F
F
O
OH
OH
O
O
NaOH/THF
99%
ONa
OH
D3CO
D3CO
N
N
d3-Cerivastatin (15)
14
Scheme 2.
3-amino-4-methyl-2-pentenoate (4)10 in refluxing ethanol provided dihydro-
pyridine diester (5) in 60% yield. Oxidation of 5 with DDQ gave pyridine
diester (6) in 95% yield. The diester 6 was then selectively reduced with Red-Al
to give hydroxy ester (7) in 78% yield.
The d3-methyl group was then introduced by treatment of hydroxy ester (7)
with d3-iodomethane using NaH as the base to give d3-methoxy ester (8) in
quantitative yield. Further reduction of d3-methoxy ester (8) with lithium
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B.-C. CHEN ET AL.
aluminum hydride gave d3-methoxy alcohol (9) in 86% yield. Treatment of 9
with phosphorous tribromide in methylene chloride afforded d3-methoxy
bromide (10) in 91% yield. Reaction of the bromide (10) with ethyl
diphenylphosphinite in refluxing toluene gave d3-methoxy phosphine oxide
(11) in 71% yield. Treatment of 11 with n-BuLi followed by addition of the
chiral non-racemic aldehyde (12) gave d3-methoxy enoate (13) in 40% isolated
yield and >98% E stereochemical purity, which was consistently with the
extraordinarily high E selectivity reported previously for this type of Horner
olefination between a related diphenyl phosphine oxide and 12.9 The enoate
(13) was then directly treated with TFA to give d3-cerivastatin lactone (14) in
96% yield. d3-Cerivastatin sodium salt (15) was finally obtained in 99% yield
by reaction of 14 with 1 equiv of sodium hydroxide. The structure of 15 was
confirmed by comparison of the NMR spectral data with those reported for
the non-labeled Cerivastatin and mass spectrometric studies.3,6
Experimental
Enamine (4)10 and the aldehyde (12)9 were prepared according to literature
procedures. d3-Iodomethane (99+ atom% D) was purchased from Aldrich
Company. 1H-NMR spectra were recorded in CDCl3 or CD3OD on a Bruker
ARX-400, Joel Eclipse 270 or 500 instrument using tetramethylsilane as
internal standard. The reactions were monitored by HPLC using a Shimadzu
LC-10AS system. LC/MS analyses were conducted using a Hewlett-Packard
1090L HPLC system and a Finigan TSQ-7000 triple quadruple mass
spectrometer.11
Ethyl 2-(4-fluorobenzylidene)-4-methyl-3-oxopentanoate ð3Þ
In a 3 l round bottomed flask equipped with a magnetic stirrer was placed ethyl
isobutyrylacetate (1, 211.0 g, 1.33 mol), 4-fluorobenzaldehyde (161.3 g,
1.30 mol), isopropanol (760 ml), pyperidine (7.2 ml) and glacious acetic acid
(4.2 ml). The reaction mixture was stirred at room temperature under nitrogen
for 22 h. The solvent was removed under reduced pressure. The residue was
dissolved with methylene chloride (500 ml) and washed with sat. NaHCO3
(3 ꢀ 300 ml) and dried over anhydrous MgSO4. Removal of MgSO4 and
solvent afforded ethyl 2-(4-fluorobenzylidene)-4-methyl-3-oxopentanoate 3
(319.0 g) in 95% yield as a mixture of cis/trans-isomers which was used in the
subsequent step without further purification. 1H NMR spectral data of 3 were
consistent with those reported previously.12
Diethyl 4-(4-fluorophenyl)-2,6-diisopropyl-1,4-dihydropyridine-3,5-dicarboxy-
late ð5Þ
In a 2 l round bottomed flask equipped with a magnetic stirrer and a con-
denser was placed ethyl 2-(4-fluorobenzylidene)-4-methyl-3-oxopentanoate
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF D3-CERIVASTATIN FOR USE AS INTERNAL STANDARD
315
(3, 237.8 g, 0.90 mol), enamine (4, 141.5 g, 0.90 mol) and absolute ethanol
(750 ml). The reaction mixture was heated to reflux and stirred under nitrogen
for 16.5 h. The solvent was distilled until the pot temperature reached to
1708C. The reaction mixture was then stirred at 170–1738C for 4 h and cooled
to 608C. Hexane (950 ml) was added. The resulting slurry was stirred at room
temperature for 0.5 h, filtered, washed with hexane (3 ꢀ 200 ml) and suction
dried at room temperature for 17 h to give 5 (217.8 g) in 60.3% yield. 1H NMR
(CDCl3) d 7.15–7.26 (m, 2H), 6.84–6.88 (m, 2H), 6.12 (s, 1H), 4.98 (s, 1H),
4.12–4.22 (m, 2H), 4.04–4.11 (m, 4H), 1.15–1.25 (m, 18H).
Diethyl 4-(4-fluorophenyl)-2,6-diisopropylpyridine-3,5-dicarboxylate ð6Þ
In a 3 l round bottomed flask equipped with a mechanical stirrer and a
condenser was placed dihydropyridine (5, 217.7 g, 0.54 mol), methylene
chloride (1.7 l) and DDQ (123.1 g, 0.54 mol). The reaction was stirred at
room temperature for 1 h. The reaction mixture was filtered through of a pad
of silica gel (310 g) and the pad washed with methylene chloride (3 ꢀ 350 ml).
The filtrate was concentrated under reduced pressure. The resulting solid was
1
dried under vacuum to give pyridine 6 (206.5 g) in 95% yield. H NMR
(CDCl3) d 7.15–7.19 (m, 2H), 6.93–6.99 (m, 2H), 3.92 (q, J ¼ 7:3 Hz, 4H),
2.97–3.05 (m, 2H), 1.21 (d, J ¼ 6:8 Hz, 12H), 0.88 (t, J ¼ 7:3 Hz, 6H).
Ethyl 4-(4-fluorophenyl)-5-(hydroxymethyl)-2,6-diisopropylnicotinate ð7Þ
In a 5 l three-necked round-bottomed flask equipped with a mechanical stirrer,
pressure-equalized addition funnel and rubber septum was placed pyridine
ester (6, 94.29 g, 0.23 mol) and tetrahydrofuran (1 l). The reaction mixture was
stirred to give a solution under nitrogen and cooled to ꢁ118C. Red-Al (215 ml,
65 wt% in toluene, 0.69 mol) was added over 18 min while keeping the reaction
temperature below ꢁ88C. After 22.5 h, an additional Red-Al (40 ml, 65 wt% in
toluene, 0.13 mol) was added and the reaction was stirred at rt for another
22 h. The reaction mixture was cooled to ꢁ108C and water (1.1 l) was carefully
added to destroy any excess Red-Al. The reaction mixture was transferred into
a separation funnel and the phases separated. The aqueous phase was
extracted with ethyl acetate (2 ꢀ 1 l). The organic layers were combined and
dried over anhydrous MgSO4. Removal of drying agent and solvent afforded a
crude product which was purified by flash chromatography using 4:1
heptane:ethyl acetate as the eluent to give hydroxy ester 7 (65.69 g) in 78%
yield. 1H NMR (CDCl3) d 7.00–7.30 (m, 4H), 4.41 (s, 2H), 3.94 (q, J ¼ 7:0 Hz,
2H), 3.37–3.52 (m, 1H), 2.95–3.10 (m, 1H), 1.29 (d, J ¼ 6:8 Hz, 6H), 1.24
(d, J ¼ 6:8 Hz, 6H), 0.94 (t, J ¼ 7:2 Hz, 3H).
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B.-C. CHEN ET AL.
Ethyl 4-(4-fluorophenyl)-2,6-diisopropyl-5-(d3-methoxymethyl)nicotinate ð8Þ
In a 2 l three-necked round bottomed flask equipped with a magnetic stirrer
and rubber septum was placed hydroxy ester (7, 34.84 g, 97 mmol) and
tetrahydrofuran (1 l). The reaction mixture was stirred to give a solution under
nitrogen and cooled to ꢁ538C. d3-Iodomethane (25.06 g, 173 mmol) was added
followed by sodium hydride (5.84 g, 60 wt%). The reaction mixture was then
allowed to warm to rt. After stirring over weekend, the reaction mixture was
carefully quenched by water (500 ml) and concentrated under reduced pressure
to a volume of ꢂ700 ml. This mixture was extracted with methyl t-butyl ether
(2 ꢀ 400 ml). The combined extract was dried over anhydrous MgSO4.
Removal of drying agent and solvent afforded d3-methoxy ester 8 (38.19 g)
in quantitative yield. 1H NMR (CDCl3) d 7.00–7.30 (m, 4H), 4.07 (s, 2H), 3.96
(q, J ¼ 7:2 Hz, 2H), 3.30–3.45 (m, 1H), 2.96–3.08 (m, 1H), 1.30 (d, J ¼ 6:8 Hz,
6H), 1.26 (d, J ¼ 6:8 Hz, 6H), 0.93 (t, J ¼ 7:2 Hz, 3H).
(4-(4-Fluorophenyl)-2,6-diisopropyl-5-(d3-methoxymethyl)pyridin-3-yl)
methanol ð9Þ
In a 2 l three-necked round bottomed flask equipped with a magnetic stirrer,
reflux condenser and rubber septum was placed lithium aluminum hydride
(6.02 g, 95%, 151 mmol) and dry tetrahydrofuran (240 ml). The suspension
was stirred under argon and was heated to 608C. A solution of d3-methoxy
ester (8, 38.02 g, 101 mmol) in dry tetrahydrofuran (350 ml) was added. The
reaction was heated to reflux and stirred for 50 min. The pinkish gray
suspension was allowed to cool to 358C and was quenched carefully by
aqueous KOH (40 ml, 17 w/w%). Water (800 ml) was then added and the
mixture was stirred for 10 min. The mixture was transferred into a separation
funnel and extracted with methyl t-butyl ether (2 ꢀ 400 nml). The combined
extract was washed with saturated aqueous brine (500 ml). Removal of
solvent afforded a crude product which was purified by flash chromatography
using 9:1 heptane:ethyl acetate as the eluent to give d3-methoxy alcohol 9
1
(27.97 g) in 86% yield. H NMR (CDCl3) d 7.05–7.26 (m, 4H), 4.33 (s, 2H),
4.00 (s, 2H), 3.29–3.46 (m, 2H), 1.41 (s, 1H), 1.31 (d, J ¼ 6:7 Hz, 6H), 1.28
(d, J ¼ 6:7 Hz, 6H).
3-(Bromomethyl)-4-(4-fluorophenyl)-2,6-diisopropyl-5-(d3-methoxymethyl)
pyridine ð10Þ
In a 2 l round bottomed flask equipped with a magnetic stirrer, a pressure-
equalized addition funnel and rubber septum was placed d3-methoxy alcohol
(9, 27.80 g, 83 mmol) and methylene chloride (750 ml). The mixture was stirred
to give a solution at room temperature. Phosphorous tribromide (16 ml,
170 mmol) was added over 3 min while keeping the reaction temperature below
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SYNTHESIS OF D3-CERIVASTATIN FOR USE AS INTERNAL STANDARD
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288C. After stirring for 1 h, the reaction was carefully quenched by saturated
aqueous sodium bicarbonate (500 ml). Additional methylene chloride (500 ml)
was added. The organic phase was separated and dried over anhydrous
MgSO4. Removal of the drying agent and solvent afforded d3-methoxy
1
bromide 10 (31.17 g) in 94% yield. H NMR (CDCl3) d 7.24–7.29 (m, 2H),
7.11–7.18 (m, 2H), 4.20 (s, 2H), 3.97 (s, 2H), 3.34–3.39 (m, 2H), 1.32
(d, J ¼ 6:7 Hz, 6H), 1.29 (d, J ¼ 6:7 Hz, 6H).
3-((Diphenylphosphoryl)methyl)-4-(4-fluorophenyl)-2,6-diisopropyl-5-
(d3-methoxymethyl)pyridine ð11Þ
In a 1 l three necked round bottomed flask equipped with a magnetic stirrer, a
reflux condenser and rubber septum was placed d3-methoxy bromide
(10, 19.43 g, 49 mmol), toluene (200 ml) and ethyl diphenylphosphinite
(10.5 ml, 49 mmol). The solution was heated to reflux (ꢂ1168C) and stirred
under nitrogen for 4 h and allowed to cool to room temperature overnight.
The solvent was removed to give a pasty off white solid which was dissolved in
refluxing heptane (500 ml). This turbid solution was polish filtered while hot.
The filtrate was stirred and allowed to cool to rt. The resulting heavy slurry
was diluted with heptane (100 ml). After cooling to ꢂ158C, the slurry was
filtered, washed with heptane and air dried to afford d3-methoxy phosphine
1
oxide 11 (17.90 g) in 71% yield. H NMR (CDCl3) d 7.43–7.47 (m, 2H),
7.29–7.33 (m, 8H), 6.80–6.84 (m, 2H), 6.73–6.76 (m, 2H), 3.87 (s, 2H), 3.62
(d, J ¼ 13:5 Hz, 2H), 3.19–3.31 (m, 2H), 1.28 (d, J ¼ 6:7 Hz, 6H), 1.12
(d, J ¼ 6:1 Hz, 2H).
tert-Butyl 2-((4R,6S)-6-((E)-2-(4-(4-fluorophenyl)-2,6-diisopropyl-5-(d3-metho-
xymethyl)pyridin-3-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate ð13Þ
In a 500 ml oven dried round-bottomed flask equipped with a magnetic stirrer
and rubber septum was placed d3-methoxy phosphine oxide (11, 17.90 g,
34.5 mmol) and dry tetrahydrofuran (180 ml). The reaction mixture was stirred
to give a solution under nitrogen and cooled to ꢁ98C. n-BuLi (14 ml, 2.5 M in
hexane, 35 mmol) was added over 3 min while keeping the reaction
temperature below 38C. The resulting red–orange solution was stirred at
3–58C for 50 min.
In another oven dried 1 l round-bottomed flask equipped with a magnetic
stirrer and rubber septum was placed activated 4 A molecular sieves (28.5 g),
aldehyde (12, 11.27 g, 61 mmol) and dry tetrahydrofuran (100 ml). After
stirring at ꢂ08C for 20 min, the aldehyde/THF solution was added via a
cannulation into the above prepared phosphine oxide anion solution over
4 min while keeping the pot temperature below 58C. After addition, the
reaction mixture was stirred at 08C for 10 min and rt for 1 h. The reaction was
then quenched by saturated aqueous brine (200 ml) and diluted with methyl
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B.-C. CHEN ET AL.
t-butyl ether (200 ml). The mixture was filtered. The filtrate was transferred
into a separation funnel and the phases separated. The aqueous phase was
extracted with methyl t-butyl ether (200 ml). The combined organic was dried
over anhydrous MgSO4. Removal of drying agent and solvent afforded a
crude product which was slurried in a mixture of 4:1 heptane:ethyl acetate
(95 ml) at ꢂ428C. The resulting solids were removed by filtration. The filtrate
was concentrated and purified by flash chromatography using 4:1 hepta-
ne:ethyl acetate as the eluent to give d3-methoxy enoate 13 (14.96 g) in 40%
1
yield. H NMR (CDCl3) d 6.98–7.12 (m, 4H), 6.17 (d, J ¼ 16:1 Hz, 1H), 5.19
(dd, J ¼ 6:1, 16.1 Hz, 1H), 4.14–4.23 (m, 2H), 4.00–4.06 (m, 2H), 3.26–3.35
(m, 2H), 2.19–2.39 (m, 2H), 0.8–1.45 (m, 29H).
(4R,6S,E)-6-(2-(4-(4-fluorophenyl)-2,6-diisopropyl-5-(d3-methoxymethyl)-
pyridin-3-yl)vinyl)-4-hydroxy-tetrahydropyran-2-one ð14Þ
In a 500 ml round-bottomed flask equipped with a magnetic stirrer was placed
d3-methoxy enoate (13, 6.88 g, 14.2 mmol) and methylene chloride (300 ml).
The reaction mixture was stirred under nitrogen to give a pale yellow solution
and trifluoroacetic acid (28 ml) was then added. After stirring at room
temperature for 16.25 h, the reaction mixture was concentrated under reduced
pressure. The residue was redissolved in methylene chloride (200 ml), washed
with pH 7.5 sodium phosphate buffer (3 ꢀ 200 ml) and dried over anhydrous
MgSO4. Removal of drying agent and solvent afforded a crude product which
was purified by flash chromatography using 4:1 to 2:1 heptane:ethyl acetate as
1
the eluent to afford d3-methoxy lactone 14 (4.43 g) in 81% yield. H NMR
(CDCl3) d 7.05–7.14 (m, 4H), 6.39 (d, J ¼ 16:2 Hz, 1H), 5.27 (dd, J ¼ 6:4,
16.2 Hz, 1H), 5.02–5.08 (m, 1H), 4.12–4.18 (m, 1H), 4.03–4.09 (m, 2H,
3.20–3.40 (m, 2H), 2.50–2.68 (m, 2H), 1.96 (s, 1H), 1.63–1.71 (m, 1H),
1.45–1.54 (m, 1H), 1.32 (d, J ¼ 6:8 Hz, 6H), 1.23 (d, J ¼ 6:4 Hz, 6H).
Sodium (3R,5S,E)-7-(4-(4-fluorophenyl)-2,6-diisopropyl-5-(d3-methoxymethyl)
pyridin-3-yl)-3,5-dihydroxyhept-6-enoate (d3-cerivastatin, ð15Þ
In a 200 ml round-bottomed flask equipped with a magnetic stirrer and rubber
septum was placed d3-methoxy lactone (14, 1.00 g, 2.25 mmol) and freshly
distilled inhibitor-free tetrahydrofuran (8 ml). The reaction mixture was stirred
to give a pale yellow solution. Then a diluted solution of sodium hydroxide
(2.28 ml, 0.996 N) in distilled water (6 ml) was added. After stirring at room
temperature for 1.25 h, the reaction was concentrated under reduced pressure
to remove the bulk of the tetrahydrofuran. The residue was lyophilized to
afford d3-cerivastatin 15 (1.08 g) in 99% yield. 1H NMR (CD3OD) d 7.15–7.26
(m, 4H), 6.36 (d, J ¼ 16:2 Hz, 1H), 5.40 (dd, J ¼ 6:5, 16.2 Hz, 1H), 4.93
(m, 3H), 4.20–4.17 (m, 1H), 4.12–4.18 (m, 2H), 3.81–3.89 (m, 1H), 3.35–3.53
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SYNTHESIS OF D3-CERIVASTATIN FOR USE AS INTERNAL STANDARD
319
(m, 2H), 2.10–2.38 (m, 2H), 1.54–1.63 (m, 1H), 1.35 (d, J ¼ 6:4 Hz, 6H), 1.30
(d, J ¼ 6:6 Hz, 6H).
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