Synthesis of deuterium-labelled fosamprenavir calcium

Article abstract of DOI:10.1002/jlcr.1741

This study describes the synthesis of deuterium-labelled fosamprenavir calcium. The stable isotopic-labelled compound was prepared starting from L-phenylalanine in 18 steps with a 9% overall yield. Copyright

Full text of DOI:10.1002/jlcr.1741

Research Article
Received 29 October 2009,
Revised 8 December 2009,
Accepted 10 December 2009
Published online 25 January 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1741
Synthesis of deuterium-labelled
fosamprenavir calcium
Lei Shi,a Liping Chen,a Ryan Chen,b and Liqin Chenc
Ã
This study describes the synthesis of deuterium-labelled fosamprenavir calcium. The stable isotopic-labelled compound
was prepared starting from L-phenylalanine in 18 steps with a 9% overall yield.
Keywords: protease inhibitor; HIV; deuterium-labelled; fosamprenavir
and K2CO3 to yield N,N-dibenzyl-(S)-2-amino-3-phenyl-1-pro-
panol (10). The oxidation of compound (10) with pyridine-
Introduction
The human immunodeficiency virus (HIV) has been identified as
the causative agent of acquired immune deficiency syndrome
(AIDS). HIV protease, an essential enzyme for viral maturation,1–4
is now a well recognized target for therapeutic intervention
against the deadly disease. Fosamprenavir is an effective
therapeutic agent for the treatment of HIV infection. As the
phosphate ester pro-drug of the protease inhibitor amprenavir
(APV), fosamprenavir has improved solubility and bioavailability
compared with amprenavir.5 It was approved by the FDA and
came onto the market in October 2003. Patients who received
treatment with fosamprenavir demonstrated protease gene
mutations different from those commonly seen with other
protease inhibitors (except APV). Fosamprenavir Calcium
labelled with radioactive and stable isotopes of hydrogen was
required for drug metabolism (excretion, distribution, and
absorption) studies and to develop bioanalytical methods to
support clinical studies.
sulfurtrioxide complex produced N,N-dibenzylamino aldehyde
(11).12 The crude product (11) was utilized for the next step
without further purification. Treatment of (11) with lithium
and bromochloromethane, and then quenching with 6 M HCl
gave N,N-dibenzyl-(S)-3-amino-(S)-2-hydroxy-4-phenyl-1-chlor-
obutane hydrochloride. Recrystallization from MeOH gave
(2S,3S)-N,N-dibenzyl-3-amino
chlorohydrin
hydrochloride
(12).13 Yields for this reaction were found to be highly
dependent on careful temperature control (if the reaction
temperature was allowed to rise above À551C, yields
decreased significantly). Hydrogenolysis of (12) under stan-
dard conditions (1 atm H2, MeOH, 20% Pd(OH)2/C) gave
amino chlorohydrin hydrochloride (13) in excellent yield. The
use of the salt form is crucial to the success of the
deprotection. It accelerates hydrogenolysis of the benzylic
C–N bonds and prevents decomposition of (12) and (13).14
Protection of the amine of (13) with di-tert-butyl dicarbonate
and triethylamine in THF, followed by in situ epoxide
ring closure with methanolic potassium hydroxide, gave N-
Boc-aminoalkyl epoxides (14). HPLC analysis on a chiral
column (Chiralcel OD) indicated that the isomeric purity of
compound (14) was 499.5%.14 Treatment of (14) with
isobutyl amine gave, after epoxide ring opening, (2R,3S)-3-
tert-butoxycarbonylamino-1-isobutylamino-4-phenyl-2-butanol
Results and discussion
Although fosamprenavir calcium and p-nitrobenzenesulfonyl
chloride have been readily prepared via several synthetic
routes,6–8 the synthesis of [2H4]-fosamprenavir calcium and
p-[2H4]-nitrobenzenesulfonyl chloride have not been described
previously. Scheme 1 presents the general synthetic scheme for
preparing p-[2H4]-nitrobenzenesulfonyl chloride (7). Reduction
of compound (1) with Pd/C and ammonium formate produced
[2H5]-aniline (2). Treatment of (2) with acetic anhydride gave
[2H5]-acetanilide (3), which was then reacted with chlorosulfonic
acid (HSO3Cl) and gave [2H4]-N-acetylsulfanilyl chloride (4).9
Compound (4) was treated with 10% NaOH to generate p-[2H4]-
aminobenzene sulfonic acid (5). The oxidation of (5) with 30%
H2O2 in acetic acid yielded p-[2H4]-nitrobenzenesulfonic (6).10
This compound (6) was treated with PCl5 to form p-[2H4]-
nitrobenzenesulfonyl chloride (7).
aCollege of Material Science and Technology, Nanjing University of Aeronautics
and Astronautics, 29 Yudao Street, Nanjing, Jiangsu 210016, Peoples’ Republic of
China
bDepartment of Biology, University of Western Ontario, London, Ont., Canada
N6A 5B7
cHi-Tech Research Institute and State Key Laboratory of Materials-Oriented
Chemical Engineering, Nanjing University of Technology, 5 Xinmofan Road,
Nanjing, Jiangsu 210009, Peoples’ Republic of China
Scheme 2 presents the preparation of [2H4]-fosamprenavir
calcium salt (20). L-phenylalaninol (9) was prepared by
reacting L-phenylalanine with sodium borohydride and
iodine.11 Compound (9) was treated with benzyl bromide
*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, Peoples’ Republic of
China.
E-mail: liqin_chen@hotmail.com
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L. Shi et al.
Scheme 1.
(15). Conversion of (15) into (16) using p-[2H4]-nitrobenzene- was heated carefully to reflux for 20 min. The catalyst was
sulfonyl chloride (7) and triethylamine in toluene and removed by filtration and washed with methanol. The filtrate
subsequent treatment with concentrated hydrochloric acid was distilled at atmospheric pressure through a short fractionat-
to remove the N-Boc group.6 The resulting amine (16) was ing column to afford (2) as a colorless liquid (8.8 g, 98.3%).
treated with (S)-3-hydroxytetrahydrofuran and bis(trichloro-
methyl)carbonate to produce (2S,3R)-N-(3-amino-2-hydroxy-4- Synthesis of [2H5]-acetanilide (3)
phenyl)-N-isobutyl-4-[2H4]-nitrobenzene sulphonamide hydro-
Compound (2) (8.8 g, 89.8 mmol) was cooled in ice-bath under
chloride (17) in a 82% yield. The reaction of compound (17)
nitrogen. Acetic anhydride (20.2 g, 197.8 mmol) was added
dropwise with stirring. After addition, the reaction mixture was
and POCl3, followed by the hydrolysis using 6 M HCl afforded
(3S)-tetrahydro-3-furanyl-(1S,2R)-3-[[(4-[2H4]-nitrophenyl)-sulfo-
warmed to room temperature and stirred for 2.5 h. It was added
nyl] (isobutyl)amino]-1-benzyl-2-(phosphonooxy)propylcarba-
to water (60 mL), neutralized with saturated NaHCO3 solution.
mate (18).15 Reduction of the nitro group using palladium
The aqueous phase was extracted with dichloromethane
on carbon gave the aryl amine (19) in 91.5% yield. This
(3 Â 80 mL). The combined organic phases were washed with
brine (150 mL), dried over Na2SO4, and concentrated under
hydrogenation step required very carefully controlled condi-
tions to avoid cyclization of the product. Compound (19) was
reduced pressure to afford (3) as a white solid (12.5 g, 99.2%).
treated with calcium acetate monohydrate to produce [2H4]-
fosamprenavir calcium (20) in a 61% yield.
Synthesis of [2H4]-N-acetylsulfanilyl chloride (4)
After purification by recrystallization, the desired product (20)
was obtained with 98.5% chemical purity. Mass spectrometry
analysis of compound (20) revealed that the compound has over
98% deuterium enrichment. The compound provided an
excellent internal standard in LC-MS-MS studies.
Compound (3) (6.0 g, 42.9 mmol) was cooled in an ice-salt bath
under nitrogen. Chlorosulfonic acid (53.6 g, 460 mmol) was
added dropwise with stirring. The reaction mixture was stirred at
room temperature for 3 h, then added carefully to the ice/water
mixture. The precipitate was filtered and washed with water
(50 mL) to afford (4) as a white solid (6.7 g, 65.7%). 1H NMR
(300 MHz, CD3OD):d 2.18 (s, 3 H).
Experimental
General
Synthesis of p-[2H4]-aminobenzene sulfonic acid (5)
All reagents were obtained from Sigma-Aldrich and CDN
Isotope. Mass spectra were recorded using a Quattro micro Compound (4) (13.0 g, 57 mmol) was mixed with 10% aqueous
API mass spectrometer. 1H NMR spectra were recorded on a sodium hydroxide (300 mL) and the mixture was heated at 951C
Bruker 300 MHz instrument. Chemical purities were determined for 3 h. The reaction mixture was then cooled in an ice bath and
by an Agilent 1200 HPLC with a XDB-C18 column, 5 mm, acidified with 10% HCl until pH 1–2. Some precipitates started to
4.6 Â 150 mm.
form. The precipitate was filtered, washed with cold water
(20 mL) and then dried in vacuo to afford (5) as a white solid
(8.3 g, 87%).
Synthesis of [2H5]-aniline (2)
To a solution of [2H5]-nitrobenzene (1) (12.0 g, 102 mmol) in
MeOH (120 mL) was added ammonium formate (30.7 g,
Synthesis of p-[2H4]-nitrobenzenesulfonic acid (6)
487 mmol). After stirring for 10 min under nitrogen, 10% To a suspension of (5) (0.7 g, 4 mmol) in glacial acetic acid
palladium on charcoal (1.2 g) was added. The reaction mixture (5.6 mL), 30% H2O2 (5.6 mL) was added dropwise under nitrogen.
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J. Label Compd. Radiopharm 2010, 53 147–151

L. Shi et al.
Scheme 2.
J. Label Compd. Radiopharm 2010, 53 147–151
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www.jlcr.org

L. Shi et al.
The reaction mixture was refluxed at 761C for 4.5 h and cooled
to room temperature. The reaction mixture was concentrated to 3.74 (d, 4H), 3.45 (t, 1H), 3.00 (m, 3H).
1H NMR (300 MHz, DMSO-d6): d9.70 (s, 1H), 7.38-7.14 (m, 15H),
afford a yellow solid that was dried under vacuum overnight.
Crude (6) (0.76 g, 92.7%) was used without further purification. Synthesis of N,N-dibenzyl-(S)-3-amino-(S)-2-hydroxy-4-
MS-EI (m/z): 205.0 (8), 206.0 (MÀ, 100), 207.0 (8), 208.0 (5).
phenyl-1-chlorobutane hydrochloride (12)
A solution of (11) (1.2 g, 3.6 mmol) in dry THF (20 mL) was cooled
to À751C under an argon atmosphere. Four alternating portions
Synthesis of p-[2H4]-nitrobenzenesulfonyl chloride (7)
of lithium shot (4 Â 0.1 g, 54.6 mmol) and bromochloromethane
(4 Â 0.13 g, 4.0 mmol) were added while maintaining an internal
temperature below À651C at all times. The suspension
To a solution of crude (6) (0.5 g, 2.4 mmol) in 1,2-dichloroethane
(7 mL) was added PCl5 (1.0 g, 4.8 mmol). The reaction mixture
was stirred at 651C for 2.5 h and cooled to room temperature.
was mechanically stirred vigorously for 3 h. TLC analysis
The reaction mixture was concentrated to afford (7) as a yellow
indicated disappearance of starting material. The solution was
solid that was dried under vacuum overnight. Crude (7) (0.51 g,
separated from floating lithium residues (the excess metal was
94.3%) was used without further purification.
washed with THF, dried under vacuum, and recycled) and
transferred into 6 M HCl (4 mL). The mixture was concentrated in
Synthesis of L-phenylalaninol (9)
vacuo until a clear aqueous layer separated from a gummy,
brown residue. The aqueous layer was removed and the residue
crystallized from hot methanol (2.5 mL). The crude product was
collected, washed with 10% methanol in ether (1.5 mL) and
recrystallized from methanol (3 mL) to afford (12) as a white solid
(0.48 g, 32%).
To a suspension of L-phenylalanine (8) (60 g, 364 mmol) and
sodium borohydride (34.53 g, 913 mmol) in dry THF (400mL)
cooled in an ice bath was added dropwise a solution of iodine
(92.36 g, 364 mmol) in dry THF (200 mL). After addition gas
evolution had ceased. The mixture was heated to reflux for 18 h.
The reaction mixture was cooled in ice-bath, and methanol was
added cautiously until the mixture became clear. After stirring for
30 min, the solvent was removed to give a white paste which was
dissolved by addition of 20% aqueous KOH (600mL). The solution
was stirred for 30 min and extracted with dichloromethane
(3 Â 200 mL). The organic layer was washed with brine (200mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure to afford a white semisolid which was recrystallized from
toluene to afford (9) as a white solid (41.8 g, 76.0%).
Synthesis of (S)-3-Amino-(S)-2-hydroxy-4-phenyl-1-
chlorobutane hydrochloride (13)
To a mixture of (12) (1.0 g, 2.4 mmol) in MeOH (10 mL) was
added 20% palladium hydroxide on charcoal (0.1 g). The slurry
was stirred under 1 atm of hydrogen at room temperature for
4 h. The catalyst was filtered and washed with methanol. After
concentration the residue was triturated with ether, filtered, and
dried under vacuum to give (13) as a white solid (0.54 g, 94.7%).
1H NMR (300 MHz, DMSO-d6): d 8.09 (br, s, 3H), 7.38–7.30 (m,
5H), 6.12 (d, 1H), 3.96 (m, 1H), 3.66 (m, 1H), 3.51 (m, 2H), 2.99 (dd,
1H), 2.82 (dd, 1H).
Synthesis of N,N-dibenzyl-(S)-2-amino-3-phenyl-1-propanol
(10)
To a suspension of potassium carbonate (20.1 g, 146 mmol) in
methanol (150 mL) and water (30 mL) was added (9) (11.0 g,
72.7 mmol). The reaction mixture was refluxed for 10 min. Benzyl
bromide (31.1 g, 181.9 mmol.) was added. After stirring for 1 h,
the reaction mixture was cooled to room temperature and water
(100 mL) was added. The mixture was extracted with dichlor-
omethane (3 Â 150 mL). The combined organic layers were
washed with brine (150 mL), dried over Na2SO4, and concen-
trated under reduced pressure to afford a white solid which was
then washed with diethylether (2 Â 50 mL) to afford (10) as a
white solid (22.4 g, 93%).
Synthesis of (2S,3S)-N-Boc-3-amino-1,2-epoxy-4-phenylbu-
tane (14)
Di-tert-butyl dicarbonate (1.8 g, 8 mmol) and triethylamine (1.6 g,
1.6 mmol) were dissolved in THF (20 mL), and the solution was
cooled in an ice bath. Solid amino chlorohydrin hydrochloride
(13) (1.8 g, 7.6 mmol) was added in portions. The cooling bath
was removed and the reaction mixture was stirred for 3.5 h at
room temperature. TLC analysis showed that the reaction was
complete. The white slurry was cooled again in an ice bath, and
a solution of KOH (2.6 g, 46 mmol) in methanol (20 mL) was
added. The ice bath was removed and stirring was continued for
an additional 1 h. TLC analysis indicated that the reaction was
complete. The reaction mixture was poured into 50 mL of water
and the white precipitates were collected by suction filtration.
The crude product was purified by chromatography on silica gel
(80 g) column, eluted with EtOAc/Hexanes (5:95), to afford (14)
as white solid (1.75 g, 87.5%).
1H NMR (300 MHz, DMSO-d6): d7.20 (s, 13H), 7.09 (d, 2H), 4.43
(t, 1H), 3.71-3.62 (m, 5H), 3.43 (m, 1H), 2.83 (m, 3H).
Synthesis of N,N-dibenzyl-(S)-2-amino-3-phenylpropanal (11)
To a solution of (10) (2.8 g, 8.4 mmol) in dry DMSO (20 mL) was
added dropwise triethylamine (2.56 g, 25.3 mmol). The solution
was immersed in an ice bath. Pyridine-sulfurtrioxide complex
(4.03 g, 25.3 mmol) in DMSO (14 mL) was added in small portions
over 5 min (the complex can also be added as the solid). After
stirring for one hour at 10–151C, the reaction was quenched
Synthesis of (2R,3S)-3-tert-butoxycarbonylamino-1-
isobutylamino-4-[2H4]-phenyl-2- butanol (15)
with ice-water (100 mL) and extracted with dichloromethane To a solution of (14) (1.2 g, 5 mmol) in anhydrous ethanol
(3 Â 30 mL). The combined organic layers were washed with (15 mL) was added isobutyl amine (6.6 mL, 7.5 mmol). The
brine (30 mL), dried over anhydrous Na2SO4, filtered and reaction mixture was refluxed at 781C under nitrogen for 1 h.
concentrated under reduced pressure. The orange oil was dried TLC analysis showed that the reaction was complete. The
further under high vacuum for 12 h. The crude aldehyde (2.6 g, reaction mixture was concentrated to afford (15) as a white solid
94.2%) was used without further purification.
(1.53 g, 99.8%).
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J. Label Compd. Radiopharm 2010, 53 147–151

L. Shi et al.
1H NMR (300 MHz, DMSO-d6): d 7.17 (m, 5H), 6.74 (d, 1H), 3.50 (2 Â 30 mL). The combined methyl isobutyl ketone extracts
(m, 1H), 3.36 (m, 1H), 2.98 (d, 1H), 2.60–2.40 (m, 2H), 2.30 (d, 2H), were washed with brine, dried over Na2SO4, and concentrated
1.64 (m, 1H), 1.26–1.10 (d, 9H), 0.87 (d, 6H).
under reduced pressure to afford (18) as a yellow foam (1.0 g,
84.7%).
Synthesis of (2S,3R)-N-(3-amino-2-hydroxy-4-phenyl)-N-
isobutyl-4-[2H4]-nitrobenzene sulphonamide hydrochloride
(16)
Synthesis of (3S)-tetrahydro-3-furanyl-(1S,2R)-3-[[(4-[2H4]-
aminophenyl)-sulfonyl](isobutyl)amino]-1-benzyl-2-
(phosphonooxy)propylcarbamate (19)
To a solution of (15) (1.27 g, 3.8 mmol) in toluene (9 mL) heated
to 801C was added triethylamine (0.42 g, 4.2 mmol). The mixture To a solution of (18) (0.3 g, 0.5 mmol) in MeOH (7.5 mL) was
was heated to 901C and a solution of (7) (1.0 g, 4.5 mmol) in added 10% palladium on charcoal (0.15 g). The slurry was stirred
toluene (4 mL) was added and stirred for 2 h. The reaction under hydrogen at room temperature for 5 h. The catalyst was
mixture was then cooled to 801C and then concentrated removed by filtration and washed with methanol. After
hydrochloric acid (0.4 mL, 4.8 mmol) was added slowly. The concentration, it gave (19) as a yellow foam (0.27 g, 91.5%).
mixture was heated to reflux (approx 881C) for 1 h. Another
portion of concentrated hydrochloric acid (0.3 mL, 3.6 mmol) Synthesis of [2H4]-fosamprenavir calcium (20)
was added. Solvent was removed from the reaction mixture by
To a suspension of (19) (0.5 g, 0.9 mmol) in MeOH (10 mL) was
using the rotary evaporator. EtOAc (6 mL) was added. The
added a solution of calcium acetate monohydrate (0.27 g,
1.7 mmol) in water (2 mL). The reaction mixture was stirred at
bath. The product was isolated by filtration, washed with cold
mixture was stirred at 601C for 30 min, and then cooled in an ice
room temperature for 15 min and then treated slowly with 5%
EtOAc (3 mL), and dried under vacuum to afford (16) as a white
NaOH until pH 8–9. Product started to precipitate. It was filtered,
solid (1.4 g, 80.5%).
washed with EtOH/H2O (6 mL, 1:1), and then dried in vacuo at
35–401C to afford desired product (20) as a white solid (0.34 g,
64.2%).
1H NMR (300 MHz, Solvent 0.1N DCl in D2O): d 7.36–7.28 (m,
5H), 4.96–4.79 (m, 1H masked by HOD signal), 4.49 (m, 1H),
Synthesis of (2S,3R)-N-(3-amino-2-hydroxy-4-phenyl)-N-
isobutyl-4-[2H4]-nitrobenzene sulphonamide hydrochloride
(17)
To a solution of bis(trichloromethyl)carbonate (0.56 g, 1.9 mmol)
in dichloromethane (15 mL) cooled in an ice-salt bath was added
dropwise (S)-3-hydroxytetrahydrofuran (0.5 g, 5.7 mmol) and
triethylamine (0.57 g, 5.7 mmol). The reaction mixture was stirred
at room temperature for 3 h. TLC analysis showed that the
reaction was complete. The reaction mixture was evaporated to
dryness under nitrogen. The white residue was dissolved in
dichloromethane (50 mL). Amine hydrochloride (2.3 g, 5 mmol)
was added dropwise. The suspension was cooled in ice-water.
Triethylamine (1.4 g, 14 mmol) was added dropwise. The
reaction mixture was stirred at room temperature for 1 h. TLC
analysis showed that the reaction was complete. The reaction
mixture was evaporated to dryness. Water (30 mL) was added.
The reaction mixture was extracted with dichloromethane
(3 Â 25 mL). The combined dichloromethane extracts were
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure to afford a white solid which was recrystallized
from EtOAc/EtOH (16 mL, 4:1), to afford (17) as a white solid
(2.2 g, 82.1%).
4.29–4.16 (m, 1H), 3.89–3.55 (m, 4H), 3.42–3.33 (m, 2H), 3.16–2.90
(m, 3H), 2.75–2.60 (m, 1H), 2.11–1.85 (m, 2.5H), 1.30–1.15 (m,
0.5H), 0.85–0.79 (m, 6H). MS-EI (m/z): 587.1 (7), 588.1 (MÀ, 100),
589.1 (30), 590.1 (10). HPLC (XDB-C18, CH3OH/10 mmol/L
NaH2PO410.1% H3PO4 = 50/50, 1.0 mL/min): tR 5.8 min (498.5%).
MS-EI analysis of compound (20) revealed that the compound has
98.2% deuterium enrichment, which is about the same as
compound (6).
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Copyright r 2010 John Wiley & Sons, Ltd.
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