Synthesis of stable isotope labeled anacetrapib, its major metabolites and [14C]anacetrapib

Article abstract of DOI:10.1002/jlcr.3073

Cholesteryl ester transfer protein inhibitors are an important class of compounds designed to treat hypocholesterolemia and prevent cardiovascular disease. Anacetrapib (MK-0859) is currently in phase III trials for the treatment of elevated cholesterol levels and prevention of cardiovascular disease. In order to further support the development of anacetrapib, we prepared [M + 6]MK-0859, which was required in support of an absolute bioavailability study of the active pharmaceutical ingredient (API). Additional support included the synthesis of an internal standard [M + 13] and three stable isotope labeled metabolites, which were used to analyze clinical samples, and [ ¹⁴C]MK-0859 to support drug metabolism studies. A labeling strategy for the preparation of [M + 6], [M + 13], and [¹⁴C]anacetrapib is described. The preparation of stable isotope labelled metabolites of anacetrapib is also described.

Full text of DOI:10.1002/jlcr.3073

Research Article
Received 22 March 2013,
Revised 15 May 2013,
Accepted 16 May 2013
Published online 9 July 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3073
Synthesis of stable isotope labeled
anacetrapib, its major metabolites
14
and [ C]anacetrapib
*
*
Jeffrey T. Kuethe, Eric D. Soli, Pernilla Royster, and Catherine A. Quinn
Cholesteryl ester transfer protein inhibitors are an important class of compounds designed to treat hypocholesterolemia and
prevent cardiovascular disease. Anacetrapib (MK-0859) is currently in phase III trials for the treatment of elevated
cholesterol levels and prevention of cardiovascular disease. In order to further support the development of anacetrapib,
we prepared [M + 6]MK-0859, which was required in support of an absolute bioavailability study of the active pharmaceutical
ingredient (API). Additional support included the synthesis of an internal standard [M + 13] and three stable isotope labeled
metabolites, which were used to analyze clinical samples, and [14C]MK-0859 to support drug metabolism studies.
Keywords: anacetrapib; MK-0859; internal standard; CETP
we document the synthesis of stable isotope labeled
anacetrapib [M + 6] and [M + 13], the synthesis of the three
Introduction
Anacetrapib (MK-0859, 1) is a potent and selective cholesteryl
ester transfer protein (CETP) inhibitor that is under inves-
stable labeled major oxidative metabolites of anacetrapib and
the preparation of [14C]anacetrapib.
tigation for the prevention and treatment of hyper-
cholesterolemia and mixed hyperlipidemia.1 CETP works by
exchanging cholesteryl ester and triglycerides between various
lipoprotein particles and its mechanism of action has
been characterized.2 It has been demonstrated that inhibition
of CETP dramatically increases high-density lipoprotein-
cholesterol in both animal models and the clinic.3,4 Although
the development of Pfizer’s torcetrapib was halted in 2006
due to elevated blood pressure, anacetrapib has shown no
treatment-related effect on blood pressure.5 In phases I and II
clinical trials, dramatic effects on plasma lipid profiling
including increases in high-density lipoprotein-cholesterol
levels of ~130% with reduction of low-density lipoprotein-
cholesterol by levels of ~40% have been reported for
anacetrapib.5,6 The tolerability, safety, and favorable
pharmacokinetic and pharmacodynamic properties of
anacetrapib have been assessed in clinical studies.7 The
metabolism and excretion of anacetrapib in preclinical species8
and humans9 have also recently been described. In order to
further support the development of anacetrapib, stable isotope
labeled anacetrapib [M + 6] was required for an absolute
bioavailability study, as well as [M + 13]anacetrapib, which
was to be utilized as an internal standard. In addition, the
synthesis of stable labeled anacetrapib metabolites was
required for evaluation of certain clinical samples. The original
synthesis of anacetrapib2,10 and improved processes for its
Results and discussion
[13C3,15N](4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-4-methyl-1,3-
oxazolidin-2-one (9)
The synthesis of the labeled oxazolidinone core 9 of anacetrapib
began with [13C3,15N] L-alanine 5 and closely followed the estab-
lished procedures with minor modifications (Scheme 1).11,12
For example, reaction of 5 with benzyl chloroformate in the
Department of Process Chemistry, Labeled Compound Synthesis Group, Merck &
preparation have been reported.11 Retrosynthetic analysis of Co., Inc, Rahway, New Jersey 07065, USA
each of these routes leads to three possible precursors:
oxazolidinone 2, biaryl 3 bearing an appropriately substituted
leaving group, or functionalized aryl halide 4, also having a
*Correspondence to: Jeffrey T. Kuethe and Eric D. Soli, Department of Process
Chemistry, Labeled Compound Synthesis Group, Merck & Co., Inc., Rahway,
New Jersey 07065, USA.
suitable leaving group in the benzylic position. In this paper, E-mail: Jeffrey_Kuethe@merck.com; Eric_Soli@merck.com
J. Label Compd. Radiopharm 2013, 56 600–608
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J. T. Kuethe et al.
Scheme 1. Preparation of Labeled Oxazolidinone Core 9
presence of 2M NaOH furnished the N-protected amino acid, [13C5,15N] Anacetrapib (19)
which was converted to Weinreb amide 6 via standard
amide coupling conditions in 90% overall yield. Treatment of 6
with the Grignard reagent of 7 (generated by reaction of 7 with
isopropylmagnesium chloride) afforded ketone 8 in 93% yield.
Diastereoselective reduction of ketone 8 was accomplished
with aluminum isopropoxide, Al(Oi-Pr)3, at 50 ꢀC in a mixture of
toluene/isopropyl alcohol. Direct addition of potassium
hydroxide resulted in cyclization and formation of [13C3,15N]
oxazolidinone 9 in enantiomerically pure form and in 88% yield
for the one-pot procedure.
In order to support an absolute bioavailability study, a labeling
strategy was devised with only 13C or 15N to achieve the required
[M + 6] of anacetrapib. The synthesis began with bromoanisole
10 (Scheme 2). Friedel–Crafts acylation of 10 with [13C2]acetyl
chloride 11 was conducted in the presence of aluminum
chloride in dichloroethane and gave 12 in 90% yield. Addition
of methyl magnesium chloride to a solution of 12 in THF yielded
tertiary alcohol 13. Reductive deoxygenation of the tertiary
alcohol of 13 with trifluoroactic acid/(HSiMe2)O2 in CH2Cl2
successfully installed the isopropyl moiety, giving 14 in 90%
Scheme 2. Preparation of [M + 6]Anacetrapib
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J. T. Kuethe et al.
13C5,D7,15N]anacetrapib 25 was
yield for two steps. Treatment of 14 with i-PrMgCl ꢁ LiCl in
the presence of tetramethylethylenediamine (TMEDA) and
subsequent quenching of the intermediate Grignard reagent with
triisopropyl borate yielded boronic acid 15 in 78%. Suzuki–Miyaura
cross-coupling of 15 with commercially available benzylalcohol
16 was performed in the presence of dichloro[1,10-bis(di-tert-
butylphosphino)ferrocene]palladium (II) (PdCl2DTBPF) and K2CO3
in DMF/water to furnish the coupled product 17 in 50% yield.
Conversion of 17 to benzyl chloride 18 by reaction with thionyl
chloride in DMF proceeded in excellent yield giving 18 in 97%
isolated yield. Finally, nucleophilic displacement of benzyl chloride
18 with oxazolidinone 9, using NaHMDS as the base, gave
[13C5,15N]anacetrapib 19 in 50% after crystallization from heptane.
Conversion of 23 to
accomplished in similar fashion as outlined in Scheme 2 and
gave 25 in 71% yield over the last two steps.
[
Stable isotope labeled metabolites of anacetrapib
The main pathway of elimination in humans has been
demonstrated to be unabsorbed parent.9 The majority of
absorbed anacetrapib involves oxidative metabolism from
systemic circulation. The mechanism of clearance is relatively
simple and only three oxidative metabolites have been observed
(Scheme 4).9 The metabolic pathway includes a CYP3A4-
catalyzed O-demethylation to give 26, which likely undergoes
further oxidation at the biphenyl and isopropyl moieties leading
to 27 and 28. These metabolites are >14-fold less potent in vitro
pharmacological activity than anacetrapib and likely do not
contribute to the pharmacological effects to any significant
extent. However, evaluation of clinical samples required the
stable isotope labeled synthesis of each of these metabolites to
be used as internal standards, which are outlined in the
succeeding text.
[13C5,D7,15N]Anacetrapib 25
In order to evaluate clinical samples of stable isotope
labeled anacetrapib [M + 6] by liquid chromatography–mass
spectrometry, an internal standard was required with
a
necessary mass increase of [M + 13]. The judicious choice of
suitably labeled starting materials for the synthesis of [M + 13]
anacetrapib is outlined in Scheme 3. Addition of 13CD3MgI,
generated in situ from magnesium turnings and 13CD3I, to readily
available ester 20, followed by hydrogenation of the
intermediate tertiary alcohol under a balloon pressure of
deuterium gas and palladium on carbon with added TFA, and
subsequent bromination with N-bromosuccinimide (NBS) gave
bromide 21 in 70% overall yield for three steps. Treatment of
21 with i-PrMgCl ꢁ LiCl in the presence of TMEDA and
subsequent quenching of the intermediate Grignard reagent
with triisopropyl borate yielded boronic acid 22 in 83%.
[13C5,15N]Metabolite 26a
The simplest metabolite, from a synthetic perspective, was the
preparation of [13C5,15N]metabolite 26; which was envisioned
arising via O-demethylation of 19 (Scheme 5). Treatment of 19
with a slight excess of BBr3 in CH2Cl2 followed by purification
by silica gel chromatography gave [13C5,15N]metabolite 26a in
70% yield.
[13C3,15N]Metabolite 27a
Suzuki–Miyaura cross-coupling of 22 with benzyl alcohol 16 The preparation of metabolite 27a began with the preparation
was performed with (PdCl2DTBPF) and K2CO3 in IPA/isoproypl of intermediates 30 and 34 (Scheme 6). Intermediate 30 was
acetate/water and afforded coupled product 23 in 78% yield. synthesized by starting with benzyl alcohol 16. Conversion of
Scheme 3. Preparation of [M + 13]Anacetrapib
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J. T. Kuethe et al.
Scheme 4. Major Metabolies of Anacetrapib
[14C]Anacetrapib 44
The preparation of [14C]-anacetrapib began with 41 (Scheme 8),
the unlabeled version of compound 39, which was prepared in
analogous fashion as shown in Scheme 7. Addition of 14CH3MgI,
prepared in situ from 14CH3I and magnesium turnings, gave the
intermediate tertiary alcohol 42, which was not isolated.
Reaction of 42 with MsCl in the presence of Hünigs base leads
to activation and subsequent elimination to give styrene 43 in
good overall yield. Catalytic hydrogenation of 43 over palladium
on carbon gave [14C]-anacetrapib (44).
In conclusion, the stable isotope labeled synthesis of both
[M + 6] and [M + 13] anacetrapib (MK-0859), the three oxidative
metabolites, and [14C]MK-0859 has been achieved. Effective
labeling strategies were employed that minimized the number
of synthetic steps and allowed for the rapid preparation of each
of the target compounds.
Scheme 5. Preparation of Metabolite 26a
16 to benzyl chloride 29 proceeded in near quantitative yield by
reaction of 16 with thionyl chloride in DMF. Alkylation of 9 with
29 was performed in DMF utilizing LiHMDS to give 30 in 95%
yield. Preparation of 34 started with bisphenol 31. Alkylation of
31 with excess MeI in the presence of K2CO3 was followed by
Friedel–Crafts acylation giving 32 in 79% over the two steps.
Addition of methyl magnesium chloride to 32 and subsequent
hydrogenation of the intermediate tertiary alcohol with Pd/C,
in the presence of TFA, followed by regioselective bromination
with NBS gave 33 in 75% overall yield. Treatment of 33 with n-
butyllithium under Barbier reaction conditions in the presence
of triisopropylborate furnished boronic acid 34 in a modest
50% yield. The mass balance of the reaction was proto-
dehalogenation of 33. Suzuki–Miyaura cross-coupling of 30
and 34 was achieved by reaction with PdCl2DTBPF and K2CO3
in THF/water. The isolated yield of 35 was 63% where the mass
balance was a result of proto-deborylation of 34. Reaction of
Experimental
General
All reactions were carried out under an atmosphere of nitrogen.
Purifications were carried out by flash column chromatography on
either a Teledyne Isco CombiFlash Rf or Biotage using a gradient elution
of 0–100% EtOAc/hexanes unless otherwise stated. All commercially
available reagents and solvents were used as received without
purification. Samples were analyzed on an Agilent 1100 CDMSD (Palo
Alto, Ca, USA) instrument in API-ES positive ionization mode. The stable
labeled raw materials, [13C3,15N]L-alanine (5), [13C2]acetyl chloride (11)
and 13CD3I, were purchased from Cambridge Isotope Laboratories, Inc.
35 with three equivalents of BBr3 in CH2Cl2 gave 27a in 69% (Andover, MA, USA) The deuterium gas was purchased from ISOTEC
Stable Isotopes (Sigma-Aldrich, St. Louis, MO, USA). The [14C]methyl
iodide was purchased from ViTrax Radiochemicals (Placentia, CA, USA).
isolated yield.
[13C3,D3]Metabolite 28a
All unlabeled products have previously been fully characterized else-
where, where noted in the succeeding paragraphs.
The preparation of 28a started with the synthesis of unlabeled
37 and labeled boronate ester 38 (Scheme 7). The preparation
Preparation of [13C N] (4S,5R)-5-(3,5-bis(trifluoromethyl)phenyl)-4-
15
3,
of 37 was similar to that outlined in Scheme 6 and employed
methyloxazolidin-2-one (9). A solution of 2.69 mL (5.38 mmol) of 2 M
unlabeled version of the same starting materials. Palladium- NaOH and 1.008 g (5.18 mmol) of benzyl chloroformate were
added dropwise simultaneously from two separate syringes to a solution
of 0.5 g (5.37 mmol) of [13C3,15N] alanine 5 in 2.59 mL (5.18 mmol) of 2 M
NaOH at 0 ꢀC. The mixture was stirred for 1 h at 0 ꢀC then warmed
to room temperature and stirred for 1 h. The solution was diluted
with water (5 mL), washed with Et2O (5 mL), acidified with 2 M HCl to
pH 2–3, and extracted with Et2O (3ꢂ 5 mL). The combined organic extracts
were dried over Na2SO4, filtered, and concentrated under reduced pressure
to give 1.3g of
acid as a white solid that was used as is in the next step.
To a solution of 1.2 g (5.28 mmol) of the aforementioned product
in 12 mL of THF was added 0.890 g (5.81 mmol) of HOBT and
mediated borylation of 12 with Pd(OAc)2/X-phos in the presence
of KOAc and bis(pinacolato)diboron gave 38 in 54% yield. The
mass balance was proto-deborylation of 38 under the reaction
conditions. Suzuki–Miyaura cross-coupling of 38 and 37 was
achieved by treatment with (PdCl2DTBPF) and K2CO3 in THF/
water and afforded 39 in 68% isolated yield. Addition of CD3MgI
to 39 gave 40 (99%), which was subjected to BBr3 in CH2Cl2 to
provide 28a in 36% overall yield form 39. The low yield for the
last step was attributed to elimination of the tertiary alcohol
under the acidic reaction conditions and during workup.
[
13C3,15N]-(S)-2-(((benzyloxy)carbonyl)amino)propanoic
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J. T. Kuethe et al.
Scheme 6. Preparation of Metabolite 27a
0.567 g (5.81 mmol) of N,O-dimethylhydroxylamine hydrochloride. To
stirred to room temperature for 1 h. The mixture was diluted with water
the resulting slurry was added 2.21 mL (12.68 mmol) of N,N- (10 mL) and 6 N HCl (0.7 mL) and concentrated under reduced pressure.
diisopropylethylamine followed by 1.215 g (6.34 mmol) of EDC, and the
mixture was stirred at room temperature overnight (15 h). The reaction
mixture was concentrated onto Celite and purified by silica gel
chromatography (hexanes/EtOAc) to afford 1.26 g (90% from 5) of [13C3, 15N]
(S)-benzyl (1-methoxy(methyl)amino-1-oxopropan-2-yl)carbamate 6 as
a white solid.
To a solution of 0.85 g (3.15 mmol) of Weinreb amide 6 and 0.807 ml
(3.93 mmol) of aryl bromide 7 in THF (10 mL) pre-cooled to 0 ꢀC was
added dropwise 3.93 mL (7.86 mmol) of a 2 M solution of isopropyl
magnesium chloride. The reaction mixture was allowed to warm to
room temperature and stirred for 20 h. The solution was diluted with
toluene (15 mL), cooled to 0 ꢀC, and quenched with 6 N HCl (1.5 mL).
The organic layer was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to give 1.24 g (93%) of [13C3,
15N]-(S)-benzyl (1-(3,5-bis(trifluoromethyl)phenyl)-1-oxopropan-2-yl)carbamate
8 as a pale yellow solid that was used as is in the next step without further
purification.
The residue was purified by silica gel chromatography to afford 790 mg
(88%) of 9 as a white solid.
Preparation of [13C5,15N](4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-3-
{[40-fluoro-20-methoxy-50-isopropyl-4-(trifluoromethyl)[1,10-biphenyl]-
2-yl]-4-methyl}-1,3-oxazolidin-2-one (19). To a solution of 13.31 mL
(103.0mmol) of 2-bromo-5-fluoroanisole 10 in 85mL of CH2Cl2 cooled to
0 ꢀC was added 17.92g (134.0mmol) of AlCl3 followed by 8.84 mL
(124.0mmol) of [13C2]acetyl chloride 11. The mixture was stirred for
30 min and diluted with water and EtOAc. The layers were separated,
and the organic layer was washed with 1N HCl, sat. NaHCO3, brine, and
dried over MgSO4. The solution was filtered and concentrated under
reduced pressure. The residue was slurried in hexane and filtered to give
22.21 g
[ a
13C2]1-(5-bromo-2-fluoro-4-methoxyphenyl)ethanone 12 as
colorless solid. A second crop 1.01g was obtained to give a total of
23.22 g (90%) of 12 as a colorless solid.
To a solution of 26.8 mL (80 mmol) of a 3 M solution of MeMgCl at
ꢃ10 ꢀC was added dropwise 10.0 g (40.2 mmol) of 12 in 56 mL of THF.
The reaction mixture was stirred for 1 h and quenched by the addition
of 57.2 mL (57.2 mmol) of 1 N HCl. The mixture was extracted with EtOAc,
and the organic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was slurried in
To a solution of 1.5 g (2.83 mmol) of ketone 8 (from a separate
synthesis) in 25 mL of IPA was added 0.193 g (0.945 mmol) of aluminum
isopropoxide. The resulting solution was heated at 50 ꢀC for 20 h. The
reaction mixture was cooled at room temperature, and 0.476 mL
(5.66 mmol) of 11.9 M solution of potassium hydroxide was added and
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J. T. Kuethe et al.
Scheme 7. Preparation of Metabolite 28a
Scheme 8. Preparation [14C]Anacetrapib
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J. T. Kuethe et al.
hexane, filtered, and dried to provide 9.70 g (91%) of [13C2]2-(5-bromo-2-
fluoro-4-methoxyphenyl)propan-2-ol 13 as a colorless solid.
Preparation of [13C5, D7,15N](4S,5R)-5-(3,5-bis(trifluoromethyl)phenyl)-3-
((40-fluoro-50-isopropyl-20-methoxy-4-(trifluoromethyl)-[1,10-biphenyl]-2-
yl)methyl)-4-methyloxazolidin-2-one (25). To a suspension of 0.831g
(34.2mmol) of magnesium turnings in 10 mL of ether was added dropwise
2.134ml (34.2mmol) of iodomethane-[13C,D3] in 5mL of ether and the
mixture was refluxed for 1 h then cooled to room temperature. In a separate
flask was added 1.26 g (6.84 mmol) of ester 20 in 10 mL of ether, and the
solution was cooled to 0 ꢀC. The aforementioned Grignard was added
dropwise to the ester solution while maintaining the internal temperature
below 5 ꢀC. The reaction mixture was warmed to room temperature and
stirred for 15 h. The solution was cooled to 0 ꢀC and quenched with saturated
ammonium chloride, extracted with EtOAc (3ꢂ 15 mL). The combined
organic extracts were washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure to afford 785 mg of-[13C2, D7]2-(2-
fluoro-4-methoxyphenyl)propan-2-ol that was used as is in the next step.
To a solution of 785 mg (4.08 mmol) of the aforementioned alcohol in
3 mL of THF was added 157 mL (2.042 mmol) of TFA and 87 mg (0.041 mmol)
of 10% Pd/C. The reaction mixture was deuterated at 60 ꢀC under an
atmosphere of deuterium (50 psi) for 15 h then cooled to room temperature.
The catalyst was filtered, and the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel chromatography to afford
630 mg of [13C2,D7]-4-fluoro-5-isopropyl-2-methoxybenzene as an oil.
While shielding from light, 400mg (2.257mmol) the aforementioned
isopropylarene intermediate in 2 mL of THF was cooled to 0 ꢀC. To the
solution was added 442 mg (2.483mmol) of NBS, and the mixture was
stirred at 0 ꢀC for 4h then warmed to room temperature and stirred for
18 h. The crude reaction mixture was concentrated onto Celite and purified
via silica gel chromatography to afford 1.23 g (70% from 20) of [13C2, D7]
bromo-4-fluoro-5-isopropyl-2-methoxybenzene 21 as a white solid.
A solution of 456 mg (1.780 mmol) of bromoanisole 21 and 363 mL
(2.403 mmol) of TMEDA in 1mL of THF was sparged with nitrogen for
15 min. To the solution was added dropwise 1.849 mL (2.403mmol) of a
1.3M solution of i-PrMgCl-LiCl while maintaining an internal temperature
below 30 ꢀC. The resulting solution was stirred for 3h at 40 ꢀC. In a separate
flask was dissolved 653mL (2.85 mmol) of (i-PrO)3B in 1.5 mL of dry hexanes,
and this solution was cooled to ꢃ20 ꢀC. The Grignard solution was cooled to
room temperature and was added, via syringe, to the borate solution. After
addition, the mixture was stirred for an additional 30 min at ꢃ20 ꢀC. The
reaction mixture was quenched with 1.3mL of 3M H2SO4, warmed to room
temperature, and the aqueous layer was removed. The organic layer was
extracted with 2M KOH (2ꢂ 5mL). The combined aqueous extracts were
cooled to 0 ꢀC and acidified with 3M H2SO4. The resulting solid was filtered,
and the wet cake was washed with water (2ꢂ 5 mL) to give after drying
228mg (83%) of [13C2, D7] (4-fluoro-5-isopropyl-2-methoxyphenyl)boronic
acid 22 as a white solid.
To a solution of 6.47 mL (36.6 mmol) of 1,1,3,3-tetramethyldisiloxane
in 25 mL of dichloromethane cooled to ꢃ20 ꢀC was added 3.66 mL
(47.6 mmol) of TFA. To the mixture was added 9.70 g (36.6 mmol) of
13 at such a rate to keep the internal temperature less than ꢃ5 ꢀC.
After 15 min, the reaction was quenched with sat NaHCO3 and the
layers separated. The organic layer was washed with saturated
NaHCO3, brine, and dried over Na2SO4. The mixture was filtered and
concentrated under reduced pressure to give 9.13 g (99%) [13C2]1-
bromo-4-fluoro-5-isopropyl-2-methoxybenzene 14, which was used
without further purification.
To a mixture of 9.13 g (36.6 mmol) of 14 and 7.46 mL (49.5 mmol)
of TMEDA in THF was degassed by vacuum/nitrogen fills three times
and placed under a nitrogen atmosphere. To the mixture was added
dropwise 38.0 mL (49.5 mmol) of a 1.3 M solution of i-PrMgCl ꢁ LiCl. The
resulting solution was warmed to 40 ꢀC and stirred for 3 h and cooled
to room temperature. In
a separate flask was placed 13.53 mL
(58.6 mmol) of triisopropyl borate and 23 mL of heptane, and the solution
was cooled to ꢃ20 to ꢃ30 ꢀC. The aforementioned Grignard solution was
then added via cannula over 30 min while maintaining the internal
temperature less than ꢃ20 ꢀC. After the addition was complete, the
reaction mixture was stirred for 30 min and quenched with 62.9 mL
(189 mmol) of a 3 M solution of H2SO4 while allowing the reaction
mixture to warm at room temperature. The biphasic mixture was
separated, and the organic layer was extracted with 30 mL and then
15 mL of 2M KOH. The combined aqueous extracts were cooled to
10 ꢀC and acidified (pH <2) with 3M H2SO4. The resulting slurry was
cooled and filtered. The wet cake was washed with 45 mL of water,
45 mL of 5% NaHCO3, 90 mL of water, and dried under vacuum/nitrogen
sweep to give 6.11 g (78%) [13C2](4-fluoro-5-isopropyl-2-methoxyphenyl)
boronic acid 15 as a colorless solid.
To a mixture of 5.88 g (27.9 mmol) of 16 and 6.11 g (28.5 mmol) of 15
in 18 mL of 2-propanol and 18 mL of isopropylacetate was added 23.3 mL
(69.9 mmol) of a 3M solution of K2CO3. The mixture was sparged with
nitrogen for 30 min, and PdCl2DTBPF (1.82 g, 2.8 mmol, 10 mol%) was
added, and sparging was continued for 15 min. The reaction mixture
was heated to 75 ꢀC for 18 h and diluted with isopropylacetate. The layers
were separated, and the organic layer was dried over Na2SO4, filtered
through a pad of Celite, and concentrated under reduced pressure.
The residue was purified by silica gel chromatography to give 4.8 g
(50%)
[
13C2](40-fluoro-50-isopropyl-20-methoxy-4-(trifluoromethyl)-[1,10-
biphenyl]-2-yl)methanol 17 as a tan solid.
To a solution of 4.78 g (13.87mmol) of 17 in 24 mL of DMF cooled to 0 ꢀC
was added dropwise 1.31mL (18.03mmol) of thionyl chloride. The reaction
mixture was stirred for 5 h and quenched with 25 mL of water at 0ꢀC and
stirred for 1 h. An additional 25ml of water was added, and mixture was
A solution of 228 mg (1.032 mmol) of boronic acid 22 and 213 mg
(1.011 mmol) of aryl chloride 16 in a solution of 910 mL (2.73 mmol) of a 3 M
allowed to warm to room temperature. The biphasic solution was extracted potassium carbonate, 700 mL of IPA, and 700 mL of isopropylacetate was
sparged for 15 min with nitrogen. To the solution was added 2.99 mg
(5.06 mmol) of PdCl2DTBPF, and sparging was continued for 10 min. The
reaction mixture was heated at 75 ꢀC for 2 h and cooled to room temperature.
The mixture was diluted with isopropylacetate/water (10 mL, 1:1), the layers
separated. The organic layer was washed with water (5 mL), brine (5 mL),
dried over Na2SO4, filtered, and concentrated under reduced pressure to
afford 275 mg (78%) of [13C2, D7] (40-fluoro-50-isopropyl-20-methoxy-4-
(trifluoromethyl)-[1,10-biphenyl]-2-yl)methanol 23 as a pale yellow solid.
To a solution of 275 mg (0.78 mmol) of benzyl alcohol 23 in 1.4 mL of
DMF at 0 ꢀC was added dropwise 74.3 mL (1.017 mmol) of SOCl2, and the
mixture was stirred at 0 ꢀC for 6 h. The reaction mixture was quenched
with water (300 mL) and stirred at 0 ꢀC for 1 h at which point an additional
300 mL of water was added and stirred at 0 ꢀC for an additional hour and
warmed to room temperature. The biphasic mixture was extracted with
EtOAc (2ꢂ 10 mL), concentrated under reduced pressure, and purified
with isopropylacetate, dried over Na2SO4, filtered, and concentrated under
reduced pressure to give 4.90g (97%) [13C2] 20-(chloromethyl)-4-fluoro-5-
isopropyl-2-methoxy-40-(trifluoromethyl)-1,10-biphenyl 18 as an oil that
was sufficiently pure for use without further purification.
To a mixture of 3.57 g (9.93 mmol) of 18, 3.15 g (9.93 mmol) of 9 (from
a
separate synthesis), in 3.60 mL of DMF was added 19.67 mL
(19.67 mmol) of a 1 M solution of NaHMDS. The mixture was stirred for
18 h. The reaction mixture was cooled to room temperature and diluted
with 7 mL of heptane and 6.75 mL of 3 N HCl and warmed to 60 ꢀC and
the layers were separated. The organic layer was sequentially washed
with 10 mL of 3:2 DMF/water (2ꢂ), water (2ꢂ 10 mL), and concentrated
under reduced pressure. The crude residue was purified by silica gel
chromatography. The pure fractions were combined and concentrated
under reduced pressure to give 5.17 g (82%) 19 as a viscous oil. A portion
of this material (3.167 g) was crystallized by dissolving in heptane and
cooling to 0 ꢀC. The crystals were filtered and washed with cold heptane by silica gel chromatography to afford 265 mg (92%) of [13C2, D7] (2’-
to afford 2.65 g of analytically pure 19 as a colorless solid. Anacetrapib 1: (chloromethyl)-4-fluoro-5-isopropyl-2-methoxy-4’-(trifluoromethyl)-1,1’-
m/z 638.1 (M + H)+; [M + 6] 19 m/z 644.1 (M + H)+. The spectroscopic data biphenyl 24 as a pale yellow solid.
for 19 and all other intermediates was in complete agreement with that
A solution of 242 mg (0.763 mmol) of oxazolidinone 9 in 3.30 mL of
reported in the literature.11c
DMF was cooled to ꢃ15 ꢀC, and 667 mL (0.667 mmol) of 1 M NaHMDS
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Copyright © 2013 John Wiley & Sons, Ltd.
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J. T. Kuethe et al.
was added dropwise followed by 235 mg (0.635 mmol) of biaryl chloride the solution was added dropwise 3.60 g (18.16mmol) of 32 in 20 mL of
24 in DMF (600 mL). The reaction mixture was warmed to 10 ꢀC and THF at such a rate that the internal temperature was maintained below
stirred for 20 h. The reaction was quenched with 5 N HCl (250 mL) and 0 ꢀC. After 30min, the reaction mixture was inversely quenched into a
diluted with water/EtOAc. The layers were separated and the aqueous mixture of sat. NH4Cl and MTBE. The layers were separated, and the
layer back extracted with EtOAc (2ꢂ 15 mL). The combined organic aqueous layer was back extracted with 25mL of MTBE. The combined
extracts were evaporated to give yellow oil, which was purified by silica extracts were dried over MgSO4, filtered, and concentrated under reduced
gel chromatography to afford 210 mg (71% from 23) of 25 as a white pressure. The crude product was dissolved in 30mL of isopropyl acetate,
solid. Anacetrapib 1: m/z 638.1 (M + H)+; [M + 13] 25 m/z 651.1 (M + H)+. and 387mg of 10% Pd/C was carefully added followed by 0.70mL
The spectroscopic data for 25 and all other intermediates was in
(9.08 mmol) of TFA. The mixture was hydrogenated at 50ꢀC under an
atmosphere of 60 psi hydrogen for 12h. The reaction mixture was cooled
to room temperature, filtered through a pad of Celite, and concentrated
under reduced pressure. The crude product was dissolved in 20mL of
MeCN, and 3.56g (19.98 mmol) of NBS was added as a solid. The mixture
was stirred at room temperature for 5 h, concentrated under reduced
pressure, and purified by silica gel chromatography to give 3.77 g (75%)
of 1-bromo-4-fluoro-5-isopropyl-2,3-dimethoxybenzene 33 as an oil.
To a mixture of 3.77 g (13.6mmol) of 33 and 3.45g (18.37mmol) of
triisopropyl borate in 30 mL of THF cooled to ꢃ75 ꢀC was added dropwise
6.80 mL (17.00 mmol) of a 2.5M solution of n-BuLi. The reaction was stirred
for 30 min and was quenched with 25 mL of 1M aqueous H3PO4 and
allowed to warm at room temperature. The mixture was extracted with
50 mL of MTBE. The MTBE layer was then washed with 1 N NaOH
(2ꢂ 25mL). The aqueous washes were acidified (pH = 1) with 1M aqueous
H3PO4 and extracted with MTBE (2ꢂ 25mL). The combined extracts were
dried over MgSO4 and concentrated under reduced pressure. The residue
was purified by silica gel chromatography employing MTBE/hexane as the
eluent to give 1.65g (50%) of (4-fluoro-5-isopropyl-2,3-dimethoxyphenyl)
boronic acid 34 as a colorless solid.
complete agreement with that reported in the literature.11c
Preparation of [13C5,15N](4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-
3-{[40-fluoro-20-hydroxy-50-isopropyl-4-(trifluoromethyl)[1,10-biphenyl]-
2-yl]}-4-methyl-1,3-oxazolidin-2-one (26a). To a stirred solution of
240mg (0.373 mmol) of 19 in 4 mL of CH2Cl2 at 0 ꢀC was added dropwise
53 mL (0.560 mmol) of BBr3. After 1 h, the reaction mixture was diluted with
15mL of EtOAc and 10 mL of sat. NH4Cl. The layers were separated, and the
organic layer was washed with brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by silica
gel chromatography to afford 164mg (70%) of 26a as oil. LC-MS (EI+)
Anacetrapib metabolite 26: m/z 624.2 (M + H)+; [M+ 6] 26a m/z 630.2
(M+ H)+. The spectroscopic data for 26a and all other intermediates was
in complete agreement with that reported in the literature.11c
Preparation of [13C3,15N](4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-
3-{[40-fluoro-20,30-dihydroxy-50-isopropyl-4-(trifluoromethyl)[1,10-
biphenyl]-2-yl]}-4-methyl-1,3-oxazolidin-2-one (27a). To a solution of
500mg (2.37 mmol) of 16 in 8mL of DMF pre-cooled to 0ꢀC was added
dropwise 0.225 (3.09mmol) of thionyl chloride. The reaction mixture was
stirred for 1 h and was diluted with 20mL of methyl tert-butyl ether (MTBE)
and 20mL of water. The layers were separated and the aqueous layer
back extracted with 10 mL of MTBE. The combined extracts were washed
with brine, dried over MgSO4, filtered, and concentrated under reduced
pressure to give 543mg (99%) of 1-chloro-2-(chloromethyl)-4-
(trifluoromethyl)benzene 29, which was used in the next step without
further purification.
In a 40 mL vial was added 180mg (0.353mmol) of 30, 85 mg
(0.353 mmol) of 34, and 127 mg (0.918mmol) of K2CO3. To the mixture
was added 3 mL of THF and 0.31mL of water, and the resulting slurry was
degassed by bubbling nitrogen through the solution for 25 min. To the
mixture was then added 12 mg (0.018 mmol) of PdCl2DTBPF and degassing
was continued for an additional 15 min. The reaction mixture was warmed
to 40ꢀC and stirred for 22 h. The mixture was cooled to room temperature
and diluted with 15 mL of water and extracted with 25mL of MTBE. The
organic layer was dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by silica gel chromatography to afford
To a solution of 586mg (1.85mmol) of 9 in 8 mL of DMF at to 0ꢀC was
added dropwise 1.85mL (1.85 mmol) of a 1 M solution of LiHMDS. The
resulting mixture was stirred for 10 min, and 423 mg (1.85mmol) of 29 in
5mL of DMF was added. The reaction mixture was allowed to warm to room
temperature and stirred for 3.5h and quenched with 5mL of 6 N HCl. The
mixture was extracted with 10% isopropylacetate in heptane. The layers
were separated, and the organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. The residue
was purified by silica gel chromatography to give 895mg (95%) of
150 mg (63%)
[
13C3,15N](4S,5R)-5-(3,5-bis(trifluoromethyl)phenyl)-3-((40-
fluoro-50-isopropyl-20,30-dimethoxy-4-(trifluoromethyl)-[1,10-biphenyl]-2-yl)
methyl)-4-methyloxazolidin-2-one 35 as a colorless oil.
To a stirred solution of 150 mg (0.223 mmol) of 35 in 3 mL of CH2Cl2 at
0 ꢀC was added dropwise 63 mL (0.67 mmol) of BBr3. After 1 h, the
reaction mixture was diluted with 15 mL of EtOAc and 10 mL of saturated
NH4Cl. The layers were separated, and the organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel chromatography to
give 100 mg (69%) of 27a as oil. LC-MS (EI+) Anacetrapib metabolite 27:
m/z 638.1 (M + H)+; [M + 4] 27a m/z 642.0 (M + H)+. The spectroscopic
data for 27a and all other intermediates was in complete agreement with
that reported in the literature.11c
[
13C3,15N](4S,5R)-5-(3,5-bis(trifluoromethyl)phenyl-3-(2-chloro-5-
(trifluoromethyl)benzyl-4-methyloxazolidin-2-one 30 as a colorless foam.
To a slurry of 3.75g (29.3mmol) of 31 and 16.18 g (117.0 mmol) of
powdered K2CO3 in 60 mL of acetone was added dropwise 12.47 g
(88.0mmol) of MeI. The resulting mixture was warmed to 40 ꢀC and stirred
for 20 h. The reaction mixture was cooled to room temperature, diluted with
100mL of MTBE, and filtered. The filtrate was concentrated under reduced
pressure and re-dissolved in 50mL of MTBE, washed with 15 mL of 1 N
H3PO4, 15 mL of brine, and dried over MgSO4. The solution was filtered then
concentrated under reduced pressure. The residue was purified by silica gel
chromatography to give 4.00 g (88%) of 1-fluoro-2,3-dimethoxybenzene.
To a mixture of 4.44 g (33.3 mmol) of AlCl3 in 30 mL of CH2Cl2 cooled
to ꢃ20 ꢀC was added dropwise 3.02 g (38.4 mmol) of acetyl chloride
followed by 4.00 g (25.6 mmol) of the aforementioned dimethylated
product. The reaction mixture was warmed to room temperature and
stirred for 5 h. The mixture was poured onto ice, and the layers were
Preparation of [13C2,2H3]4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-
3-{[40-fluoro-20-hydroxy-50-(2-hydroxypropan-2-yl)-4-(trifluoromethyl)
[1,10-biphenyl]-2-yl]}-4-methyl-1,3-oxazolidin-2-one (28a). To
a
solution of 1.64 g (5.23 mmol) of 36 in 8 mL of DMF at 0 ꢀC was added
dropwise 5.00 mL (5.00 mmol) of a 1M solution of LiHMDS. The resulting
mixture was stirred for 10min and 1.09 g (4.75mmol) of 29 (from a separate
synthesis) in 5 mL of DMF was added. The reaction mixture was allowed
to warm to room temperature and stirred for 3.5h and quenched with
15 mL of 6 N HCl. The mixture was extracted with 10% isopropylacetate
separated. The aqueous layer was back extracted with 30 mL of CH2Cl2, in heptane. The layers were separated, and the organic layer was washed
and the combined extracts were washed with brine, dried over MgSO4, with brine, dried over MgSO4, filtered, and concentrated under reduced
filtered, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography to give 3.60 g (90%) of 1-(2-
fluoro-3,4-dimethoxyphenyl)ethanone 32 as a slight yellow solid.
To a 100-mL round bottom flask was added 13.3 mL (40.0 mmol) of
MeMgCl and 20 mL of THF, and the mixture was cooled to ꢃ10 ꢀC. To
pressure. The residue was purified by silica gel chromatography to
give 2.26g (94%) (4S,5R)-5-(3,5-bis(trifluoromethyl)phenyl-3-(2-chloro-5-
(trifluoromethyl)benzyl-4-methyloxazolidin-2-one 37 as a colorless oil.
A
mixture of 88 mg (0.394 mmol) of Pd(OAc)2 and 375 mg
(0.787 mmol) of X-phos were dissolved in 2 mL of dioxane and sparged
J. Label Compd. Radiopharm 2013, 56 600–608
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J. T. Kuethe et al.
with nitrogen for 15 min. In a separate flask was added 1.96 g (7.87 mmol)
A solution of 20.2mCi (241mg, 0.37 mmol) of alcohol 42 in 3mL of
of 12, 2.20 g (8.66 mmol) of bis(pinacolato)diboron, 2.32 g (23.61 mmol) methylene chloride was cooled to 0ꢀC followed by the addition of
of KOAc, and 24 mL of dioxane. The mixture was sparged with nitrogen 0.289 mL (1.65 mmol) of N,N-diisopropylethylamine and 0.26 mL
for 15 min and the aforementioned catalyst/ligand mixture was added. (3.31 mmol) of methanesulfonyl chloride. The reaction was stirred at room
The reaction mixture was heated to 100 ꢀC for 2 h and cooled to temperature for 18 h and quenched with brine/EtOAc (1:1, 20 mL). The
room temperature. The reaction mixture was diluted with MTBE and layers were separated, and the organic layer was dried over Na2SO4, filtered,
filtered through a pad of Celite. The filtrate was concentrated under and concentrated under reduced pressure. The residue was purified via
reduced pressure and the residue purified by silica gel chromatography. silica gel chromatography to give 17.2mCi of 43 (199 mg, 85%, 98.6%
The first product to elute from the column was identified as the proto- RCP) as a colorless oil.
deborylated product and was not quantified. The second product to elute
In a hydrogenation vessel was suspended 133mg (0.062 mmol) of 5%
from the column, [13C2]1-(2-fluoro-4-methoxy-5-(4,4,5,5-tetramethyl-1,3,2- Pd/C, 17.2mCi (199mg, 0.31 mmol) of 43 in 4 mL of THF. The suspension
dioxaborolan-2-yl)phenyl)ethanone 38 (1.26 g, 54%) was isolated as a wax. was stirred for 15h at 60ꢀC under an atmosphere of hydrogen (60 psi)
In a 40 mL vial was added 1.26 g (2.49 mmol) of 37, 738 mg (2.49 mmol) and cooled to room temperature. The catalyst was filtered, and the filtrate
of 38, and 895 mg (6.48 mmol) of K2CO3. To the mixture was added 20 mL concentrated under reduced pressure. The residue was purified via silica
of THF and 2.20 mL of water, and the resulting slurry was degassed by gel chromatography to give 11.18 mCi (55.3mCi/mmol, as determined by
bubbling nitrogen through the solution for 25 min. To the mixture was liquid chromatography–mass spectrometry of 44 (130 mg, 65%, 98.8%
then added 81 mg (0.125 mmol) of PdCl2DTBPF, and degassing was RCP) as a colorless solid. Anacetrapib 1: m/z 638.1 (M+ H)+; [14C]-44m/z
continued for an additional 15 min. The reaction mixture was warmed 640.1 (M + H)+. The spectroscopic data for 44 and all other intermediates
to 40 ꢀC and stirred for 22 h. The mixture was cooled to room temperature was in complete agreement with that reported in the literature.11c
and diluted with 15 mL of water and extracted with 25 mL of MTBE. The
organic layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel chromatography
to afford 1.08 mg (68%) of
Conflict of Interest
[
13C2](4S,5R)-3-((50-acetyl-40-fluoro-20-
methoxy-4-(trifluoromethyl)-[1,10-biphenyl]-2-yl)methyl)-5-(3,5-bis
(trifluoromethyl)phenyl)-4-methyloxazolidin-2-one 39 as a colorless oil.
A solution of 680 mg (1.06mmol) of 39 in 7 mL of THF was added
dropwise to a pre-cooled (ꢃ20 ꢀC) solution of CD3MgI. The resulting mixture
was stirred for 30min and quenched with 10 mL of 10% aqueous citric acid.
The mixture was extracted with EtOAc, washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. The residue
was purified by silica gel chromatography (10–60% MTBE/hexane) to give
700 mg (99%) of [13C2, D3](4S,5R)-5-(3,5-bis(trifluoromethyl)phenyl)-3-((40-
fluoro-50-(1-hydroxyethyl)-20-methoxy-4-(trifluoromethyl)-[1,10-biphenyl]-2-
yl)methyl)-4-methyloxazolidin-2-one 40 as a colorless oil.
To a stirred solution of 700 mg (1.06 mmol) of 40 in 8 mL of CH2Cl2 at
0 ꢀC was added dropwise 151 mL (1.60 mmol) of BBr3. After 1 h, the
reaction mixture was diluted with 25 mL of EtOAc and 18 mL of sat.
NH4Cl. The layers were separated, and the organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel chromatography to give
250 mg (36%) of 28a as clear oil. LC-MS (EI+) Anacetrapib metabolite
28: m/z 622.2 (M + H)+; [M + 5] 28a m/z 627.2 (M + H)+. The spectroscopic
data for 28a and all other intermediates was in complete agreement with
that reported in the literature.11c
The authors did not report any conflict of interest.
References
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isopropyl-20-methoxy-4-(trifluoromethyl)-[1,10-biphenyl]-2-yl)methyl)-
4-methyloxazolidin-2-one (44). A suspension of 12.5 mg (0.50 mmol) of
magnesium turnings in 3 mL of ether was frozen in a liquid nitrogen bath.
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noted by the manufacturer) of iodomethane-[14C] was transferred, and
the resulting mixture was heated at reflux for 1 h and cooled to room
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dried over Na2SO4, filtered, and concentrated under reduced pressure to
afford 20.2mCi (241 mg, 97.4 radiochemical purity (RCP)) of alcohol 42,
which was use as is in the next step.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 600–608