Synthesis of deuterium-labeled simvastatin

Article abstract of DOI:10.1002/jlcr.1906

This study describes the synthesis of deuterium-labeled simvastatin. The stable isotope-labeled compound was prepared starting from lovastatin in nine steps with 9% overall yield.

Full text of DOI:10.1002/jlcr.1906

Research Article
Received 11 January 2011,
Revised 26 April 2011,
Accepted 31 May 2011
Published online 19 July 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1906
Synthesis of deuterium‐labeled simvastatin
Lei Tian,a Jie Tao,a and Liqin Chenb
*
This study describes the synthesis of deuterium‐labeled simvastatin. The stable isotope‐labeled compound was prepared
starting from lovastatin in nine steps with 9% overall yield.
Keywords: deuterium‐labeled; lovastatin; simvastatin; hypercholesterolemia
ammonium salt derivative (5) by ammonia. Lactonization of
Introduction
ammonium salt derivative affords [2H3]simvastatin (6). However,
Coronary artery disease is the leading cause of death worldwide.
it is obvious that the [M + 3] internal standard is not sufficient to
meet typical thresholds for cross‐signal overlapping of a drug
with a molecular mass >400. Thus, the end‐user needed a target
product with more than three deuteriums as internal standard.
Therefore, we changed the synthesis strategy, and deuterium‐
labeled simvastatin could be obtained through esterification of
The primary risk factor for the disease is hypercholesterolemia.1,2
The level of the microsomal enzyme 3‐hydroxy‐3‐methylglutary‐
coenzyme A reductase (HMG‐CoA reductase) controls the rate‐
limiting step in the cholesterol biosynthetic pathway.3 This
enzyme is a prime target for pharmacological intervention. Sim-
vastatin, like all other statins, is an HMG‐CoA reductase inhibitor
deuterium‐labeled 2,2‐dimethylbutanoyl chloride (11) with mono-
silylated diol (13).16 Although 2,2‐dimethylbutanoyl chloride is a
used in the treatment of patients with hypercholesterolemia. It is
a synthetic derivate of a fermentation product of Aspergillus.4,5
Patients who receive treatment with simvastatin demonstrate
reductions in serums levels of total cholesterol and low‐density
lipoprotein cholesterol.6
commercial material, the synthesis of [2H6]2,2‐dimethylbutyryl
chloride has not been described. Scheme 2 presents the general
synthetic scheme for preparing compound (11). [2H6]Acetone (7)
was alkylated with ethyl magnesium bromide, and then
quenching with D2O gave [2H6]2‐methylbutane‐2‐ol (8). Solid
Na2CO3 was employed as stabilizer while the solvent was evapo-
rated and the product was distilled. Compound (8) was treated with
37% HCl solution to generate [2H6]2‐chloro‐2‐methylbutane (9).19
The reaction time and the quantity of concentrated HCl solution
are crucial to avoid the scrambling of deuterium in (9). Compound
(9) was treated with magnesium, and then gaseous CO2 passing
through a concentrated H2SO4 trap was bubbled to the reaction
solution to afford [2H6]2,2‐dimethylbutanoic acid (10).20,21 The
addition rate of (4) was very slow to avoid decomposition of tert‐
pentylmagnesium chloride. The flow rate of CO2 was regulated
so that the temperature of the solution did not rise above −5 °
C when rapid stirring was in progress. Compound (10) was
obtained in over 99% deuterium enrichment. Compound (10)
was treated with oxalyl chloride to form [2H6]2,2‐dimethylbutyryl
chloride (11).
Identification and quantification of drugs and metabolites by
liquid chromatography‐mass spectrometry (LC‐MS) relies largely
on stable isotope‐labeled analogs.7,8 A renewed interest has
been recently raised to develop a robust and validated LC‐MS
method to determine simvastatin in biological fluids. The prepara-
tion of the stable labeled version of the title compound with M + 6
was requested. Although 3H‐simvastatin and 14C‐simavastatin
have been prepared for pharmacological studies,9–11 the synth-
esis of its stable labeled internal standard has not been
described in detail.12 In this paper, the synthetic route to [2H6]
simvastatin is described in detail.
Results and discussion
Although several methods are available for conversion of lovas-
tatin to simvastatin,13–18 the synthesis of deuterium‐labeled sim-
vastatin has not been described. Many approaches can be
followed to prepare stable labeled versions of simvastatin. Initi-
ally, we chose the synthesis of [2H3]simvastatin (6). Based on pro-
tocols from the literature,13–15 [2H3]simvastatin is synthesized in
nine steps starting from lovastatin (1) (Scheme 1). Treatment of
lovastatin (1) with butylamine under reflux followed by removal
of the excess butylamine at reduced pressure and silylation of
the resulting diol affords the protected amide (2) in one spot.
Enolization of amide (2) with 2.3 equivalents of lithium pyrroli-
dide is followed by quenching with [2H3]methyl iodide to afford
the [2H3]methylated derivative (3). Acid‐catalyzed silyl transfer
of [2H3]methylated derivative with methane sulfonic acid in
aqueous methanol affords the dihydroxy amide (4). Hydrolysis
of the amide (4) gives hydroxyl acid, which is converted to
Scheme 3 illustrates the synthesis of [2H6]simvastatin (15).
Treatment of lovastatin (1) with LiOH under reflux in water
resulted in rapid opening of the lactone ring followed by slow
aCollege of Material Science and Technology, Nanjing University of Aeronautics
and Astronautics, 29 Yudao Street, Nanjing, Jiangsu 210016, China
bHi‐Tech Research Institute and State Key Laboratory of Materials‐ Oriented
Chemical Engineering, Nanjing University of Technology, 5 Xinmofan Road,
Nanjing, Jiangsu 210009, China
*Correspondence to: Liqin Chen, Hi ‐Tech Research Institute and State Key
Laboratory of Materials‐Oriented Chemical Engineering, Nanjing University of
Technology, 5 Xinmofan Road, Nanjing, Jiangsu 210009, China.
E‐mail: liqin_chen@hotmail.com
J. Label Compd. Radiopharm 2011, 54 625–628
Copyright © 2011 John Wiley & Sons, Ltd.

L. Tian et al.
TBSO
TBSO
CONH(CH2)3CH3
OTBS
CONH(CH2)3CH3
OTBS
O
O
N
1) butylamine
; CD3I
Li
O
H
O
H
1
CD3
2) TBSCl;
imdazole
2
3
HO
HO
CONH(CH2)3CH3
OH
COONH4
OH
O
O
CH3SO3H
100°C
(-NH3)
1) 2N NaOH
O
O
H
4
H
5
2) HCl
3) NH4OH
CD3
CD3
HO
O
O
O
O
H
6
CD3
Scheme 1. Synthesis of [2H3]simvastatin.
Scheme 2. Synthesis of [2H6]2,2‐dimethylbutyryl chloride.
1
saponification of the 2‐methylbutyric ester over 68 h. Neutraliza- mass spectrometer. H NMR spectra were recorded on a Bruker
tion provided a dihydroxy acid, which when heated at reflux in 300‐MHz instrument. Chemical purities were determined with an
toluene for 3 h, relactonized to afford lactone (12). Selective Agilent 1200 HPLC with a XDB‐C18 column (5 µm, 4.6 × 150 mm).
protection of the diol lactone (12) hydroxyl was accomplished
Synthesis of [2H6]2‐methylbutan‐2‐ol (8)
by treatment with t‐butyldimethylchlorosilane and imidazole in
To a stirred solution of [2H6]acetone (7) (25 g, 0.369 mol) in
dry DMF to produce silyl ether derivative (13). Acylation of alco-
hol (13) was carried out with [2H6]2,2‐dimethylbutyryl chloride
freshly distilled diethyl ether (125 mL) was added ethyl magne-
sium bromide in diethyl ether (3 N, 136.5 mL) over 2.5 h while
the temperature was maintained at 8–12 °C by cooling with an
ice–water bath as necessary. White solid was formed upon the
(11) in anhydrous pyridine in the presence of 4‐dimethylamino‐
pyridine (DMAP) as catalyst to give [2H6]simvastatin derivative
(14). Efforts were made to improve the yield for this step by
optimizing reaction temperature and time. The best yield of
addition of ethyl magnesium bromide. After addition, the mix-
68% was obtained by heating the reaction at 90 °C for 8 h. Depro-
ture was stirred at 10 °C for 1 h. The reaction was quenched with
tection of (14) with tetrabutylammonium fluoride and acetic acid
D2O (10.5 mL), and the resulting solution was stirred at 10 °C for
1 h. Then the solution was allowed to stand for 30 min, and the
slightly milky diethyl ether solution on top was decanted. The
resulting solid was stirred with diethyl ether (120 mL) for
caused cleavage of the silyl ether protecting group to give [2H6]sim-
vastatin (15) in 70.5% yield.
After purification by chromatography and recrystallization, the
desired product (15) was obtained with 98% chemical purity. MS
30 min, and the suspension was allowed to stand for 30 min;
analysis of compound (15) revealed that the compound has over
the slightly milky diethyl ether solution on top was decanted.
98% deuterium enrichment. Compound (15) provided an excellent
This process was repeated twice. The combined slightly milky
diethyl ether solutions were washed with saturated Na2CO3 solu-
internal standard for LC‐MS/MS studies.
tion. The organic layer was separated, dried over Na2SO4, and
filtered by gravity. The filtrate was concentrated to about 80 mL
Experimental
under reduced pressure after solid Na2CO3 (0.15 g) was added
General
as stabilizer. Then the residue was transferred to a 100‐mL
All reagents were obtained from Sigma‐Aldrich and CDN Iso- distilling flask, into which fresh solid Na2CO3 (0.15 g) was added.
topes. Mass spectra were recorded using a Quattro micro API The solution was distilled using a long water condenser. The
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J. Label Compd. Radiopharm 2011, 54 625–628

L. Tian et al.
Scheme 3. Synthesis of [2H6]simvastatin.
fraction boiling at 100–102 °C was collected to give (8) as a Synthesis of [2H6]2,2‐dimethylbutyryl chloride (11)
colorless liquid (27 g, 78.6%).
To a solution of (10) (2.00 g, 17.21 mmol) in CH2Cl2 (20 mL) was
1H NMR (CDCl3, 300 MHz): δ 1.47–1.53 (q, 2H), 1.23 (s, 1H),
0.90–0.94 (t, 3H).
added oxalyl chloride (6.55 g, 52 mmol). Two drops of DMF was
added, and carbon dioxide started to evolve immediately. The
reaction solution was stirred at room temperature for 1 h. The
reaction solution was evaporated to dryness under nitrogen to
give crude product (11) as a colorless oil (2.1 g, 95%).
Synthesis of [2H6]2‐chloro‐2‐methylbutane (9)
A mixture of compound (8) (27 g, 0.286 mol) in concentrated HCl
solution (12.2 M, 105.7 mL) was stirred at room temperature for
0.5 h. The mixture was cooled to 10 °C and diluted with H2O
(50 mL) and Et2O (60 mL). The mixture was stirred for 5 min and
then allowed to stand for 5 min. The organic layer was separated,
and the aqueous layer was extracted with diethyl ether (30 mL).
The combined organic layers were washed with 10% NaHCO3
solution (35 mL), water (30 mL), and brine (20 mL). The organic
layers were stirred with Na2SO4 (10 g) for 30 min and filtered by
gravity. The resulting solution was distilled using a long water
condenser. The fraction boiling at 85–86 °C was collected to give
(9) as a colorless liquid (25 g, 77.4%).
Synthesis of (4S,6R)‐4‐hydroxy‐6‐(2‐((1S,2S,6R,8S,8aR)‐8‐hydroxy‐2,6‐
dimethyl‐1,2,6,7,8,8a‐hexahydronaphthalen‐1‐yl)ethyl)tetrahydro‐2H‐
pyran‐2‐one (12)
To a suspension of LiOH (8.88 g, 0.371 mol) in H2O (900 mL) was
added lovastatin (1) (15 g, 0.037 mol). The reaction mixture was
stirred under nitrogen at reflux for 68 h. The reaction mixture
was cooled to 0 °C and acidified with conc. HCl to pH 2 and then
extracted with ether (3 × 300 mL). The combined organic layers were
washed with H2O (3 × 300 mL) and then with brine (300 mL),
dried over Na2SO4, and concentrated under reduced pressure
to give a yellow solid. The residue was recrystallized from ether
to give an off‐white solid (9.2 g, 73.7%). This solid was suspended
in dry toluene (165.6 mL) and heated at reflux for 3 h in a Dean‐
Stark apparatus for azeotropic removal of water. The suspension
became a clear off‐white solution. After evaporation of the
toluene, the residual oily solid was stirred at reflux in hexane
(92 mL) for 0.5 h. After being cooled to 0 °C, the hexane solution
was filtered and the desire product (12) was obtained as a beige
solid (7.2 g, 82.6%).
1H NMR (CDCl3, 300 MHz): δ 1.58–1.72 (q, 2H), 0.95–0.99 (t, 3H).
Synthesis of [2H6]2,2‐dimethylbutanoic acid (10)
To a suspension of Mg (2.16 g) turnings in freshly distilled diethyl
ether was added (9) (0.5 g) and a crystal of iodine. The reaction
mixture was stirred at 26 °C for 10 min. The solution became
slightly milky. A solution of (9) (10 g, 89 mmol) in freshly distilled
ether (20 mL) was added to the reaction solution over a period of
3 h. After addition, the reaction mixture was stirred at 26 °C for
1 h. The reaction was cooled by an ice–salt bath to −5 °C. The
reaction mixture was stirred, and gaseous CO2 was bubbled into
the reaction after passing through two concentrated H2SO4
traps. This process was continued for 20 min. The flow rate of
CO2 was regulated so that the temperature of the mixture did
not rise above −5 °C. The reaction was cooled with ice–water
Synthesis of (4S,6R)‐4‐(tert‐butyldimethylsilyloxy)‐6‐(2‐((1S,2S,6R,
8S,8aR)‐8‐hydroxy‐2,6‐dimethyl‐1,2,6,7,8,8a‐hexahydronaphthalen‐
1‐yl)ethyl)tetrahydro‐2H‐pyran‐2‐one (13)
bath and acidified with 25% sulfuric acid (50 mL). The diethyl To a solution of (12) (9.2g, 28.71 mmol) in dry DMF (100 mL) was
ether layer was separated and the aqueous layer was extracted added imidazole (9.77 g, 0.144 mol) and TBDMSCl (10.82 g,
with Et2O (4 × 40 mL). The combined diethyl ether extracts were 71.8mmol). The reaction mixture was stirred at room temperature
washed with 25% NaOH solution (4 × 20 mL). The combined alka- for 20 h. The reaction mixture was diluted with ether (700 mL) and
line aqueous layers were cooled with an ice–water bath and washed successively with H2O (350 mL), 2% HCl solution (350 mL),
acidified with 25% sulfuric acid to pH 2. The aqueous layer was H2O (350 mL), and saturated NaHCO3 solution (350 mL). The
extracted with Et2O (4 × 50 mL). The combined organic layers organic layer was separated and concentrated under reduced
were dried over Na2SO4 and concentrated under reduced pressure to give an off‐white solid, which was recrystallized from
pressure to give (10) as a colorless oil (3.5 g, 33.9%).
hexane to afford (13) as a white solid (6.2 g, 49.7%).
1H NMR (DMSO‐d6, 300 MHz): δ 1.56–1.62 (q, 2H), 0.87–0.91 (t, 3H).
MS‐EI (m/z): 417 (42), 457 (MNa+, 100), 458 (38), 508 (30).
J. Label Compd. Radiopharm 2011, 54 625–628
Copyright © 2011 John Wiley & Sons, Ltd.
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L. Tian et al.
Synthesis of [2H6](1S,3R,7S,8S,8aR)‐8‐(2‐((2R,4S)‐4‐(tert‐butyldimethyl MS‐EI, (m/z): 447 (MNa+, 38), 871.5 (100), 872.5 (55). HPLC (XDB‐
silyloxy)‐6‐oxotetrahydro‐2H‐pyran‐2‐yl)ethyl)‐3,7‐dimethyl‐1,2,3,7,8, C18, ACN/H2O = 76/24, 1.0 mL/min): tR 5.35 min (98.6%). Isotopic
8a‐ hexahydronaphthalen‐1‐yl 2,2‐dimethylbutanoate (14)
enrichment determined by MS was over 98%.
A solution of (13) (1 g, 2.3 mmol) and DMAP (0.03 g, 1 mmol) in
dry pyridine (10 mL) was stirred at 0 °C. To this stirred solution
was added (11) (1.29 g, 9.2 mmol) in CH2Cl2 (10 mL) over 5 min.
The solution was stirred at 0 °C for 10 min and then heated at
90 °C for 8 h. The reaction mixture was diluted with Et2O
(150 mL) and washed successively with 2% HCl solution
(3 × 75 mL), saturated NaHCO3 solution (75 mL), and brine
(2 × 75 mL). The organic layer was separated and concentrated
under reduced pressure to give a yellow oil. The crude product
was purified by chromatography on a silica gel column and then
eluted with ether/hexane (2:8) to afford (14) as a light yellow
solid (0.83 g, 68.1%).
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J. Label Compd. Radiopharm 2011, 54 625–628