Synthesis of [D4]- and [D7]-4β- hydroxycholesterols for use in a novel drug-drug interaction assay

Article abstract of DOI:10.1002/jlcr.1952

Cytochrome P450 3A (CYP3A) enzymes are involved in the metabolism of over half of today's prescription drugs. As a result, drugs metabolized by CYP3A have a risk of drug-drug interactions (DDIs). Recent studies have shown the potential to use 4β-hydroxych

Full text of DOI:10.1002/jlcr.1952

Research Article
Received 5 July 2011,
Revised 21 September 2011,
Accepted 31 October 2011
Published online 8 December 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1952
Synthesis of [D4]- and
[D7]-4b-hydroxycholesterols for use in a
novel drug–drug interaction assay
a
a
b
*
Wesley A. Turley, Richard C. Burrell, Samuel J. Bonacorsi Jr,
Angela K. Goodenough,c and Joelle M. Onoratod
Cytochrome P450 3A (CYP3A) enzymes are involved in the metabolism of over half of today’s prescription drugs. As a result,
drugs metabolized by CYP3A have a risk of drug–drug interactions (DDIs). Recent studies have shown the potential to use
4b-hydroxycholesterol as an endogenous biomarker of CYP3A activity and predictor of potential DDIs. Bristol–Myers Squibb
has developed a liquid chromatography-electron ionization-tandem mass spectrometry method that accurately measures
4b-hydroxycholesterol levels in clinical plasma samples following treatment with a CYP3A inducer or inhibitor. Stable
isotope labeled (SIL) [D4]- and [D7]-4b-hydroxycholesterols were synthesized to assist in the development of this new
quantification method. The SIL analogs were prepared from the appropriate [D4]- or [D7]-cholesterol starting material
in two steps. The labeled cholesterols were oxidized with bromine and silver acetate in pyridine to give an acetate pro-
tected hydroxy group at C4. Hydrolysis of the acetate protecting group provided [D4]- and [D7]-4b-hydroxycholesterols
in 15%–28% overall yield. 4a-Hydroxycholesterol was also required during method development and was prepared in
four steps from cholesteryl benzoate in 1% overall yield.
Keywords: 4b-hydroxycholesterol; 4a-hydroxycholesterol; deuterium; stable isotope labeling; drug–drug interactions
a broader application of the phenotyping approach. Recent
Introduction
studies have shown the potential to use 4b-hydroxycholesterol
as an endogenous biomarker of CYP3A activity.3,8,9 4b-
Drug–drug interactions (DDIs) occur when two drugs share a
common metabolic pathway.1 These interactions may increase,
decrease, or alter a drug’s desired therapeutic effect. DDIs are
well documented, and many drugs have been removed from
Hydroxycholesterol is produced in vivo by the metabolism of
cholesterol and it has been found to be exclusively mediated
by the enzyme CYP3A.8,10 In addition, circulating levels of 4b-
the market due to their negative impact. The cytochrome P450
hydroxycholesterol have been shown to be fairly stable in
family of enzymes, found mainly in mitochondria, are mem-
plasma, eliminating the need for urine collection and minimizing
brane-bound hemeproteins that are vital in the phase I oxidation
blood sample collections as the same sample could be used
of organic molecules.2 Members of the cytochrome P450 3A
for both exposure and 4b-hydroxycholesterol measurements.
(CYP3A) subfamily, in particular CYP3A4 and to a lesser extent
It has been shown that subjects treated with a CYP3A inducer
CYP3A5, are involved in the metabolism of over half of today’s
had an increase in 4b-hydroxycholesterol over basal levels
prescription drugs.2,3 With CYP3A being so heavily involved in
drug metabolism, the enzyme is a common target in the assess-
ment of DDIs.4 Current exogenous markers of CYP3A activity
involve the administration and measurement of probe drugs,
such as midazolam, erythromycin, alprazolam, alfentanil, nifedipine,
dapsone, omeprazole, and dextromethorphan.5 However, dosing
subjects with a probe drug in addition to the drug of interest is
particularly challenging in certain patient populations including
pediatric and geriatric patients, transplant recipients, and cancer
patients.5,6 Measurement of the urinary 6b-hydroxycortisol-to-
cortisol ratio serves as an endogenous marker of CYP3A activity
eliminating the need to dose with a probe drug, but this method
also suffers from several drawbacks including diurnal variation of
cortisol, high inter-individual variation, and poor correlation with
results obtained from a probe drug.7
aRadiochemical Synthesis Group, Bristol–Myers Squibb Research and Development,
5 Research Parkway, Wallingford, CT 06492, USA
bRadiochemical Synthesis Group, Bristol–Myers Squibb Research and Development,
Route 206 and Province Line Road, Princeton, NJ 08543, USA
cDepartment of Analytical and Discovery Analytical Sciences, Bristol–Myers Squibb
Research and Development, Route 206 and Province Line Road, Princeton, NJ
08543, USA
dDepartment of Analytical and Discovery Analytical Sciences, Bristol–Myers Squibb
Research and Development, 311 Pennington and Rocky Hill Road, Pennington, NJ
08534, USA
*Correspondence to: Wesley A. Turley, Radiochemical Synthesis Group, Bristol–
Myers Squibb Research and Development, 5 Research Parkway, Mall Stop 4BC-340,
Wallingford, CT 06492, USA.
Identification of an appropriate endogenous CYP3A substrate
and development of a suitable quantification method would be
an attractive alternative allowing for simplification and possibly
E-mail: wesley.turley@bms.com
J. Label Compd. Radiopharm 2012, 55 61–65
Copyright © 2011 John Wiley & Sons, Ltd.

W. A. Turley et al.
and subjects treated with a CYP3A inhibitor had a decrease in to ensure chromatographic resolution of 4a-hydroxycholesterol 2
4b-hydroxycholesterol levels using an isotope-dilution Gas and 4b-hydroxycholesterol for accurate quantification. Our initial
chromatography–mass spectrometry method.9,11,12 The develop- plan was to synthesize [D4]-4b-hydroxycholesterol [D4]-1, [D7]-4b-
ment of an liquid chromatography–mass spectrometry assay hydroxycholesterol [D7]-1 and unlabeled 4a-hydroxycholesterol 2
was considered more amenable to the routine measurement following the procedure described in the literature by Shibuya.13
of 4b-hydroxycholesterol levels in clinical samples for predicting In this route, cholesteryl acetate was converted in several steps to
potential DDIs.
a mixture containing largely 4b-hydroxycholesterol 1 and a lesser
A liquid chromatography-electron ionization-tandem mass amount of 4a-hydroxycholesterol 2 (Scheme 1). This synthetic
spectrometry (LC-ESI-MS/MS) method was developed for accu- plan appeared advantageous for several reasons. Several
rately measuring 4b-hydroxycholesterol levels in clinical plasma deuterium labeled cholesterol analogs are commercially avail-
samples following treatment with a CYP3A inducer or inhibitor.6 able, which would make sourcing necessary starting materials
SIL [D4]-4b-hydroxycholesterol [D4-1], [D7]-4b-hydroxycholesterol relatively easy. More importantly, one synthetic route could be
[D7-1], and unlabeled 4a-hydroxycholesterol 2 were required for used to synthesize both 4b-hydroxycholesterols [D4]-1 and
both method development and clinical sample analyses (Figure 1). [D7]-1 and 4a-hydroxycholesterol 2.
The SIL [D4]-4b-hydroxycholesterol [D4]-1 analog was utilized
In Shibuya’s procedure, cholesteryl acetate 3 undergoes allylic
as the internal standard in the assay for quantification. Because oxidation using selenium dioxide and formic acid to provide a
of the endogenous presence of 4b-hydroxycholesterol, [D7]-4b- mixture of allylic alcohols (4 and 5) (Scheme 1). The crude alcohols
hydroxycholesterol [D7]-1 was employed as the calibrant in (4 and 5) were then treated with lithium aluminum hydride to give
the standard curve and quality control (QC) samples prepared in a 3:1 mixture of 4b- and 4a-hydroxycholesterol isomers 1 and 2 in
human plasma. The 4a-hydroxycholesterol 2 isomer is isobaric with a 57% yield. We attempted to reproduce these results in hopes of
4b-hydroxycholesterol but is produced from the auto-oxidation isolating pure samples of 4b- and 4a-hydroxycholesterol isomers 1
of cholesterol and is not an indicator of CYP3A activity; therefore, and 2. In our hands, the oxidation step to produce the allylic
it was necessary to ensure chromatographic resolution of the alcohols (4 and 5) provided two major products and several minor
isomers for the accurate quantification of 4b-hydroxycholesterol. products according to thin layer chromatography (TLC). The two
4a-hydroxycholesterol 2 and [D4]-4b-hydroxycholesterol [D4-1] are major products were isolated by column chromatography and
not commercially available and although [D7]-4b-hydroxycholes- based on the literature, were assumed to be the 4b- and 4a-
1
terol [D7-1] is, it is relatively expensive in the amounts required for hydroxycholesterol acetate isomers 4 and 5. H NMR analysis of
the method development and clinical sample analyses. As a result, the isolated products revealed that the 4b-hydroxycholesterol
samples of all three 4-hydroxycholesterol analogs needed to acetate 5 and re-arrangement product 6 with the acetate pro-
be synthesized. It is important to note that the success of this analy- tecting group on C4 were actually isolated (Scheme 2) and not
tical approach was strongly influenced by the chemical and the desired 4a-hydroxycholesterol acetate 5. All of our attempts
isotopic purities of [D4]-4b-hydroxycholesterol [D4-1] and [D7]-4b- to reproduce Shibuya’s results resulted in either no alpha isomer
hydroxycholesterol [D7-1]. This paper describes the syntheses of formed or the yield was so minor it could not be isolated; there-
[D4]-4b-hydroxycholesterol [D4-1], [D7]-4b-hydroxycholesterol fore, other literature procedures that could provide both 4b- and
[D7-1], and unlabeled 4a-hydroxycholesterol 2.
4a-hydroxycholesterol isomers 1 and 2 were examined.
Marquet et al. detail a synthetic route that could provide both
4b- and 4a-hydroxycholesterol 1 and 2 isomers using similar
chemistry (Scheme 3).14 Cholesteryl benzoate 7 was oxidized
with selenium dioxide to provide the 4b-allylic alcohol 8 in low
to moderate yield. It is worth noting that none of the 4a-allylic
alcohol was observed. Alcohol 8 was deprotected to give 4b-
hydroxycholesterol 1 or oxidized, reduced, and deprotected to
provide 4a-hydroxycholesterol 2. This route could yield both
4b- and 4a-hydroxycholesterol isomers 1 and 2; however, in
our hands, the overall yields were relatively low and were not
suitable for an isotopically labeled synthesis. Even though the
yields were low, this was the only viable synthetic route that
Results and discussion
Standards of [D4]-4b-hydroxycholesterol [D4]-1, [D7]-4b-
hydroxycholesterol [D7]-1, and unlabeled 4a-hydroxycholes-
terol 2 needed to be synthesized in order to develop a liquid
chromatography–mass spectrometry assay to quantify 4b-
hydroxycholesterol in clinical samples. The SIL [D4]-4b-hydro-
xycholesterol [D4]-1 and [D7]-4b-hydroxycholesterol [D7]-1
analogs were required as either an internal standard or
calibrant in the assay, respectively. 4a-hydroxycholesterol 2 was
required during the development of the LC-ESI-MS/MS method
D
D
D3C
D3C
CD3
H
H
H
H
H
H
H
H
H
HO
HO
HO
OH
OH
OH
[D4]-4β-hydroxycholesterol
[D7]-4β-hydroxycholesterol
4α-hydroxycholesterol
2
[D4]-1
[D7]-1
Figure 1. Stable isotope labeled 4b-hydroxycholesterols [D4]-1 and [D7]-1 and unlabeled 4a-hydroxycholesterol 2.
www.jlcr.org
Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 61–65

W. A. Turley et al.
Selenium Dioxide,
Formic Acid
+
H
H
H
Dioxane
90oC
H
H
H
H
H
H
AcO
AcO
AcO
OH
OH
3
4
5
LAH
+
H
H
THF
0oC to RT
H
H
H
H
HO
HO
OH
OH
1
2
Scheme 1. Shibuya’s reported synthesis of 4b- and 4a-hydroxycholestrol 1 and 2.
Selenium Dioxide,
Formic Acid
+
H
H
H
Dioxane
90oC
H
H
H
H
H
H
3
3
4
4
HO
AcO
AcO
OAc
OH
3
4
6
Scheme 2. Observed products from oxidation reaction following Shibuya’s procedure.
Selenium Dioxide,
Formic Acid
5% KOH in MeOH
RT
H
H
H
Dioxane
90oC
O
O
H
H
H
8
H
H
H
HO
O
O
OH
OH
Ph3BiCO3, Na2CO3
7
1
wet THF
reflux
NaBH3CN, 10% HCl
Sodium carbonate
H
H
H
THF
RT
MeOH
RT
O
O
H
H
H
9
H
H
H
HO
O
O
OH
O
OH
10
2
Scheme 3. Synthesis of unlabeled 4b- and 4a-hydroxycholesterol 1 and 2.
was found to give 4a-hydroxycholesterol 2. We ultimately prepared the C4 position of cholesterol.15 This route was more attractive
65 mg of 4a-hydroxycholesterol 2 following Marquet’s procedure from a labeling standpoint because [D4]-4b-hydroxycholesterol
(Scheme 3) in an overall yield of 1% with a chemical purity of 97%. [D4]-1 or [D7]- 4b-hydroxycholesterol [D7]-1 could be prepared
An additional review of the literature revealed a more direct from labeled cholesterol in fewer steps and in higher yield. In
route to 4b-hydroxycholesterol. Poza et al. described using addition, no protection of the b-hydroxyl group at the C3
Petrows’ re-arrangement to incorporate a b-hydroxyl group at position of cholesterol would be needed. However, it should be
J. Label Compd. Radiopharm 2012, 55 61–65
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

W. A. Turley et al.
noted that this route leads only to 4b-hydroxycholesterol; none
of the 4a-hydroxycholesterol was observed. The synthesis of
deuterium labeled [D4]-4b-hydroxycholesterol [D4]-1 is illu-
strated in Scheme 4. Commercially available [D4]-cholesterol
[D4]-11 was reacted with bromine in chloroform followed by
treatment with silver acetate in pyridine to provide the acetate
protected intermediate [D4]-6. The intermediate was purified
by flash column chromatography and analyzed by 1H NMR.
NMR results on isolated acetate intermediate [D4]-6 compared
favorably with literature values for unlabeled compound.11
Acetate intermediate [D4]-6 was then hydrolyzed with
methanolic KOH to provide [D4]-4b-hydroxycholesterol
[D4]-1 after purification by flash column chromatography.
1H NMR results compared favorably with unlabeled com-
pound.15 Correlation spectroscopy (COSY) experiment was
conducted to unambigously assign protons C3 and C4 and
Experimental procedure
Reagents were obtained from Aldrich Chemical Co., EMD
Chemical Inc., and Mallinckrodt Inc. SIL [D7]-cholesterol [D7]-1
was obtained from Cambridge Isotope Laboratories, Inc. SIL
[D4]-cholesterol [D4]-1 was obtained from CDN Isotopes. All
reactions were magnetically stirred. Solvent removal under vacuum
was performed using a Buchi R-124 rotary evaporator. Column
chromatography was performed using a Biotage flash chromato-
graphy system. Proton NMR spectra were recorded on 400 MHz
Bruker DPX400A spectrometer (Buchi, Biotage, and Bruker). Proton
NMR COSY experiments were performed to determine the stereo-
chemistry at C3 and C4. Chemical purity was assessed by HPLC/UV/
Corona Charged Aerosol Detection using the following conditions:
instrument= Shimadzu HPLC, column = Unison C18 (3Â 150 mm),
temperature = 25 ꢀC, mobile phase = 90/10 MeCN/water (isocratic
for 25 min), and flow rate= 1 mL/min. Molecular weight was ver-
ified using GC/MS electron ionization (EI) using the following
conditions: instrument= Varian 3800, column= Agilent NP-5MS
(L = 30 m, ID = 250 mm, film= 0.25 mm), carrier gas = helium,
oven= 200 ꢀC for 1 min, 200–350 at 10 ꢀC/min, 350 ꢀC for 4 min,
detection = electron impact, and flow rate= 2 mL/min.
the 1H NMR H-H coupling constant of 3.27 Hz then confirmed
16
the cis orientation at C3 and C4.
We ultimately prepared
158 mg of [D4]-4b-hydroxycholesterol [D4]-1 in 15% overall yield
with a chemical purity of 94%.
Synthesis of [D7]-4b-hydroxycholesterol [D7]-1 was achieved
following the same two steps to provide [D4]-4b-hydroxycholesterol
[D4]-1 (Scheme 4). Commercially available [D7]-cholesterol [D7]-11
was converted to acetate intermediate [D7]-6, then the intermediate
was hydrolyzed to form the [D7]-4b-hydroxycholesterol [D7]-1.
Both acetate intermediate [D7]-6 and [D7]-4b-hydroxycholesterol
[D7]-1 were purified by flash column chromatography. Each
product was analyzed by 1H NMR and compared with literature
values for unlabeled compounds.15 COSY experiment on and
[D7]-4b-hydroxycholesterol [D7]-1 assigned protons C3 and
C4 and 1H NMR H-H coupling constant of 3.02 Hz confirmed
the cis orientation at C3 and C4.16 We ultimately prepared
345 mg of [D7]-4b-hydroxycholesterol [D7]-1 in 28% overall
yield with a chemical purity of 93%.
[D4]-4b-Acetoxycholest-5-en-3b-ol ([D4]-6)
An aluminum foil wrapped round-bottomed flask was charged
with [D4]-cholesterol [D4]-11 (954 mg, 2.44 mmol) and chloro-
form (13.3 mL). The reaction mixture was cooled to À10 ꢀC
and stirred under nitrogen atmosphere. Bromine (0.133 mL,
2.58 mmol) was added, and the mixture was allowed to stir at
À10 ꢀC for 30 min. A suspension of silver acetate (1.77 g,
10.6 mmol) in pyridine (3.98 mL, 49.2 mmol) was added, and
the reaction was warmed to room temperature. The reaction
mixture was stirred at room temperature for 17 h before being
filtered. The filtrate was diluted with 5% HCl (20 mL) and water
D
D
D3C
R
D3C
R
Br2,
silver acetate in pyridine
CHCl3
RT
H
H
H
H
H
H
HO
HO
OAc
[D4]-11: R = CH3
[D7]-11: R = CD3
[D4]-6: R = CH3
[D7]-6: R = CD3
D
D3C
R
5% KOH in MeOH
RT
H
H
H
3
4
HO
OH
[D4]-1: R = CH3
[D7]-1: R = CD3
Scheme 4. Synthesis of stable isotope labeled [D4]- and [D7]-4b-hydroxycholesterols [D4]-1 and [D7]-1.
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 61–65

W. A. Turley et al.
(40 mL). The biphasic mixture was partitioned, and the aqueous was performed on dipicolinic ester derivatives.6 Isotopic purity:
layer was extracted with CH2Cl2 (4 Â 30 mL). The combined [M + 7] 93.8%, [M + 6] 5.9%, [M + 5] <0.5%, all others below limit
organic extracts were washed with saturated NaHCO3 (100 mL) of detection.
and concentrated under reduced pressure. The resulting gray
solid was purified by flash column chromatography (75%
Conclusion
hexanes/25% EtOAc; TLC conditions: 75% hexanes/25%
We successfully synthesized two SIL 4b-hydroxycholesterol
EtOAc, stained with anisaldehyde, product Rf = 0.28) to provide
([D4]-1 and [D7]-1) analogs to assist in the development of a
[D4]-4b-acetoxycholest-5-en-3b-ol [D4]-6 as a white solid in 20%
yield (214 mg, 0.476 mmol). 1H NMR (400 MHz, CDCl3) d ppm:
new LC-ESI-MS/MS method to quantify 4b-hydroxycholesterol
levels in clinical plasma samples. Starting with the appropriate
5.83 (1H, dd, J = 5.0, 2.0 Hz), 5.38 (1H, d, J = 3.5 Hz), 3.53–3.76
commercial SIL cholesterols, [D4]- and [D7]-4b-hydroxycholesterols
(1H, m), 2.08 (3H, s), 1.11–2.05 (23H, m), 1.09 (3H, s), 0.91 (3H, d,
([D4]-1 and [D7]-1) were prepared following a two-step procedure
J = 6.5 Hz), 0.85 (6H, d, J = 1.5 Hz), 0.67 (3H, s).
in 15%–28% yield. Although the yields could likely be improved
through additional optimization, this work provided a quick and
relatively inexpensive route to [D4]- and [D7]-4b-hydroxycholesterols
[D4]-4b-Hydroxycholesterol ([D4]-1)
A round-bottomed flask was charged with [D4]-4b-acetoxychol-
est-5-en-3b-ol [D4]-6 (213 mg, 0.474 mmol) and a solution of
5% KOH in methanol (12 mL). The solution was stirred at room
temperature while being monitored by TLC (TLC conditions:
50% hexanes/50% EtOAc, stained with anisaldehyde, product
Rf = 0.60). After 2.5 h of stirring, the reaction mixture was concen-
trated under reduced pressure to half its original volume. The
remaining solution was poured into cold water (15 mL). The
aqueous solution was extracted with EtOAc (3 Â 15 mL). The com-
([D4]-1 and [D7]-1. This new MS assay has the potential to be used as
a general method to assess DDIs earlier in the drug development
process, potentially saving time and money compared with the
current techniques used to determine DDIs.
Acknowledgements
The authors would like to thank the BMS Pharmaceutical Candi-
date Optimization and Discovery Analytical Sciences Groups and
bined organic extracts were washed with water (2 Â 25 mL), satu- members of the BMS Radiochemical Synthesis Group. Special
thanks to David Wang-Iverson, Christopher Poronsky, Xiaohua
Huang, Yingru Zhang, Michael Witkus, and Jun Dai for all their
help.
rated brine (25 mL), and concentrated under reduced pressure.
The resulting light yellow solid was purified by flash column
chromatography (50% hexanes/50% EtOAc) to provide [D4]-4b-
hydroxycholesterol [D4]-1 as a white solid in 75% yield (158 mg,
0.354 mmol) with 94.4% chemical purity. A two step overall yield
of 15% starting form [D4]-cholesterol [D4]-11 was obtained. 1H
NMR (400 MHz, CDCl3) d ppm: 5.60–5.74 (1H, m), 4.14 (1H, d,
J= 3.3 Hz), 3.44–3.64 (1H, m), 1.18 (3H, s), 0.94–2.15 (24H, m), 0.91
(3H, d, J= 6.5 Hz), 0.85 (6H, d, J= 1.5 Hz), 0.68 (3H, s). GCMS (EI)
found m/z of 406.22. Isotopic distribution was performed on dipico-
linic ester derivatives.6 Isotopic purity: [M + 4] 97.5%, [M + 3] 2.4%,
all others below limit of detection.
Conflict of Interest
The authors did not report any conflict of interest.
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[D7]-4b-Hydroxycholesterol ([D7]-1)
[D7]-4b-hydroxycholesterol [D7]-1 was prepared following the
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Acetoxycholest-5-en-3b-ol [D7]-6 (588 mg, 1.30 mmol) gave by
flash column chromatography (50% hexanes/50% EtOAc) [D7]-
4b-hydroxycholesterol [D7]-1 as a white solid in 55% yield
(345 mg, 0.727 mmol) with 93.3% chemical purity. A two step
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1
obtained. H NMR (400 MHz, CDCl3) d ppm: 5.68 (1H, dd), 4.14
(1H, d, J = 3.0 Hz), 3.56 (1H, dt, J = 11.6, 4.2 Hz), 1.18 (3H, s),
0.95–2.14 (21H, m), 0.91 (3H, d, J = 6.5 Hz), 0.84–0.89 (6H, m),
0.68 (3H, s). GCMS (EI) found m/z of 409.13. Isotopic distribution
J. Label Compd. Radiopharm 2012, 55 61–65
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