Synthesis of [14C]-radiolabelled entecavir

Article abstract of DOI:10.1002/jlcr.955

Radiolabelled [^l4C]entecavir, (1), was prepared in 12 steps from (1S,2R,3S,5R)-3-(benzyloxy)-2-(benzyloxymethyl)-6-oxa-bicyclo[3.1.0]hexane 2. The chemical yield of [^l4C]entecavir was 14% from the epoxide 2. Introduction of [^14<

Full text of DOI:10.1002/jlcr.955

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 645–655.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.955
Research Article
Synthesis of [14C]-radiolabelled entecavir
Marc D. Ogan1,*, David J. Kucera2, Yadagiri R. Pendri2 and J. Kent
Rinehart1
1 Department of Chemical Synthesis, Radiochemistry Group, The Bristol-Myers Squibb
Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000, USA
2 Department of Process Research And Development, The Bristol-Myers Squibb
Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000, USA
Summary
Radiolabelled [14C]entecavir, (1), was prepared in 12 steps from (1S,2R,3S,5R)-3-
(benzyloxy)-2-(benzyloxymethyl)-6-oxa-bicyclo[3.1.0]hexane 2. The chemical yield of
[14C]entecavir was 14% from the epoxide 2. Introduction of [14C] radiolabel was
achieved by elaboration of 4,5-diaminopyrimidine 8 with triethyl[14C]orthoformate to
purine derivative 9. The radiochemical yield of [14C]entecavir from triethyl[14C]ortho-
formate was 11.3%. Radiochemical purity of [14C]entecavir determined by HPLC was
99.8%. The specific activity of [14C]entecavir was 108 mCi/mg (29.9 mCi/mmol).
Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: [14C]; triethyl[14C]orthoformate; entecavir; antiviral; nucleoside
Introduction
Available oral antiviral agents offer the potential for improved management of
chronic hepatitis B virus (HBV) infection which, left untreated, can lead to
cirrhosis and hepatocellular carcinoma.1,2 Entecavir is a carbocyclic guanosine
nucleoside analogue which is a potent selective inhibitor of hepatitis B virus.3–5
Preparation of [14C]entecavir (Figure 1) was required for clinical study of
absorption, distribution, metabolism and elimination (ADME).
The synthetic strategy for preparation of [14C]entecavir involved the
elaboration of the pyrimidine 8 to purine 9 by introducing the [14C] isotope
at the C-8 carbon. The radiolabelled precursor for the imidazole ring closure is
the readily available formate or orthoformate. This general strategy has been
achieved on the base as well as the nucleoside, and it is widely applicable in the
preparation of labelled nucleosides.6,7 This procedure has previously been used
*Correspondence to: Marc D. Ogan, Bristol-Myers Squibb, P.O. Box 4000, Princeton, NJ 08543-4000,
USA. E-mail: marc.ogan@bms.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 14 March 2005
Revised 23 March 2005
Accepted 23 March 2005

646
M. D. OGAN ET AL.
O
N
14
C
N
HO
NH
NH
N
HO
2
1, [14C] entecavir
Figure 1. 1, [14C]entecavir
within our laboratory to prepare [14C]lobucavir.8 This report details the
preparation and characterization of [14C]entecavir.
Results and discussion
The synthesis of [14C]entecavir was achieved starting with the chiral epoxide 29 as
shown in Scheme 1. The chiral epoxide was reacted with sodium azide in the
presence of NH4Cl to afford the trans-azidoalcohol 3 in quantitative yield. The
azide 3 was reduced using triphenylphosphine in wet THF to afford amine 4 as an
oil in 84% yield. The amine 4 readily reacted with 2-amino-4,6-dichloropyr-
imidine at 1158C to furnish 6-chloro diaminopyrimidine 5 in 82% yield.10,y
The pyrimidine 5 was reacted with p-chlorobenzenediazonium chloride,
generated in situ, to afford the bright yellow 5-diazopyrimidine 6 in 91% yield.
Reaction of 6 with aqueous potassium hydroxide in MeOH gave 4-methoxy-5-
(p-chlorophenylazo)pyrimidine 7 as an orange solid in 78% yield. Cleavage of
the diazo linkage of 7 with zinc in acetic acid gave the triaminopyrimidine 8 in
77% yield.y,z
Introduction of the [14C]radiolabel was achieved by elaboration of the
triaminopyrimidine 8 to purine 9. Reaction of a slight excess of 8 with high
specific activity triethyl[14C]orthoformate and catalytic toluenesulphonic acid
in dry acetonitrile afforded the purine 9. The volatile radioactive by-products
were isolated by distillation and 9 was then isolated in 86% yield after
chromatography. The specific activity of this isolated solid was 116 mCi/mg
(53 mCi/mmol).
The 2-amino group of the guanine 9 was protected in the quantitative yield
as the monomethoxytritylamine to afford 10 and then was oxidized with Dess–
Martin periodinate11,12 to give the labile ketone 11.} The crude ketone 11 was
converted to the exocyclic methylenic compound 12 by the reaction with excess
Nysted reagent in the presence of titanium tetrachloride in 75% yield. The
radiochemical purity of isolated 12 was 92%.
y MS and elemental analysis was consistent for the expected product.
z Manipulation of the diamine 8, including filtration and chromatography, were conducted under a blanket
of argon.
} Ketone 11 is a very labile compound prone to b-elimination of the 30-benzyloxy group to give the
corresponding cyclopentenone.
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF [14C]-RADIOLABELLED ENTECAVIR
647
Cl
OH
OH
H
N
O
X
BnO
BnO
BnO
BnO
a, b
c
N
N
BnO
3, X = N3
BnO
NH
2
2
5
4, X = NH2
Cl
N
N
X
H N
OMe
N
2
OH
OH
H
N
H
N
d, e
f
BnO
BnO
N
BnO
BnO
N
N
NH
NH
2
2
8
6, X = Cl
7, X = OMe
OMe
N
OMe
N
N
N
14
X
14
HO
C
N
C
N
N
g, h
i, j
BnO
BnO
BnO
BnO
N
NHMMT
NHR
11, X = O
12, X = CH2
9, R = H
10, R = MMT
O
N
14
C
N
RO
NH
NH
k, l
N
RO
2
13, R = Bn
1, R = H; [14C] entecavir
Reagents: a. NaN3, NH4Cl, EtOH, 99%; b. Ph3P, H2O, THF, 84%; c.
2-amino-4,6-dichloropyrimidine/Et3N/n-BuOH, 82%; d. NaNO2/H2O/p-chloroaniline, CH3CN, 91%; e. KOH,
MeOH, 78%; f. Zn, HOAc, H2O, MeOH, 77%; g. H[14C]C(OEt)3, TsOH, CH3CN, 86%; h. 4-methoxytrityl
chloride, Et3N, DMAP, CH2Cl, 104%; i. Dess-Martin periodinate, t-BuOH, CH2Cl2, 113%; j. Nysted reagent,
TiCl4, CH2Cl2, THF, 75%; k. 2N HCl, MeOH, THF, 88%; l. BCl3, CH2Cl2, 67%
Scheme 1. Synthesis of [14C]entecavir (1)
Conversion of 12 to [14C]entecavir was achieved by two consecutive
deprotection steps. The 2-monomethoxytritylamine and 6-methoxy protecting
groups were removed concurrently in good yield by reaction treatment with
2 N hydrochloric acid in methanol-tetrahydrofuran. The isolated penultimate
intermediate 13 had a radiochemical purity of 96%.}
} Structural identity of 13 was confirmed by high resolution mass spectrometry and by comparison to
authentic standard.
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M. D. OGAN ET AL.
Debenzylation of the penultimate 13 was achieved with boron trichloride in
dichloromethane. The crude product was isolated by extractive work up and
then further purified and desalted by chromatography on HP20 resin. The
[14C]entecavir obtained from the HP20 chromatography was crystallized from
water to afford crystalline [14C]entecavir. The specific activity of 1 was diluted
by addition of unlabelled entecavir followed by final crystallization from water
to afford [14C]entecavir in 67% yield from 13. The specific activity of 1 was
108 mCi/mg and the radiochemical purity was 99.8%.
Experimental
General: Radioactivity was determined with a Wallac Model 1409 liquid
scintillation counter (Wallac-LKB Instruments, Inc.). Counting efficiency was
determined by the channels ratio method. Analytical samples were weighed on
a Sartorius Model MC-5 microbalance. Mass spectra were obtained with a
Finnigan TSQ or a Finnigan LCQ mass spectrometer. Exact mass spectra were
obtained on a Micromass LCT TOF mass spectrometer. Proton NMR spectra
were recorded on a Varian Unity/Inova 500 MHz spectrometer. RadioTLC
was detected with a Bioscan System 200 Imager. TLC was performed on 60
F
254 silica gel plates (Merck). Flash chromatography was conducted on KP-Sil
silica gel (Biotage). HP-20 resin was obtained from Mitsubishi Chemical
Company.
Specific activity was determined gravimetrically by dissolution of two
weighed samples (ca 0.5 mg ea.) into DMF in 10- and 25-ml volumetric flasks.
Aliquots of 25 and 50 ml for each sample were diluted into Ecolite cocktail
(10 ml) and counted.
Materials: Triethyl [14C]orthoformate was purchased from Amersham
Pharmacia Biotech (radiochemical purity 98.4%, specific activity 58 mCi/
mmol). The chiral epoxide 2, penultimate standard 13 and unlabelled entecavir
were prepared by reported methods (9). MilliPore water was used for all
reactions except the final conversion and crystallization where B/Braun USP
water was employed.
High Performance Liquid Chromatography: HPLC was performed on a
YMC-ODS-A, 4.6 ꢀ 150 mm, S3 column using a Rainin Model SD-200
HPLC, a Rainin model UV-1 UV detector and a Beta-Ram radiometric
detector with a 0.5 ml flow cell (IN/US Systems Inc). Method A consisted of
isocratic elution using a mobile phase of CH3CN/H2O (60:40) and a flow rate
1.0 ml/min. UV detection was monitored at 254 nm. Method B consisted of
mobile phases A: H2O and B: CH3CN and a flow rate of 1.0 ml/min. A
gradient of 40% B from 0–10 min, 40–90% B from 10–20 min, and 90% B
from 20–30 min was used. UV detection was monitored at 254 nm. Method C
consisted of mobile phases A: 0.2% H3PO4/H2O and B: 0.2% H3PO4/CH3CN
and a flow rate of 1.0 ml/min. A gradient of 0–10% B from 0–15 min, 10% B
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF [14C]-RADIOLABELLED ENTECAVIR
649
from 15–20 min, and 10–90% B from 20–30 min was used. UV detection was
monitored at 254 nm.
(1S,2S,3S,5S)-5-azido-3-(benzyloxy)-2-(benzyloxymethyl)cyclopentanol ð3Þ
A solution of epoxide 2 (19.9 g, 64.1 mmol) in anhydrous EtOH (50 ml) was
added dropwise over 10 min to a suspension of NH4Cl (10.3 g, 0.19 mol) and
NaN3 (12.6 g, 0.19 mol) in anhydrous EtOH (100 ml). The reaction was carried
out at 858C under argon for 22 h when TLC (EtOAc/hexanes 1:1) showed that
the reaction was complete, and evaporated under vacuum. The residue was
dissolved in CH2Cl2 (75 ml) and extracted with H2O (3 ꢀ 50 ml). The combined
aqueous layer was back-extracted with CH2Cl2 (2 ꢀ 25 ml). The combined
CH2Cl2 layer was dried over MgSO4, filtered and evaporated under vacuum to
afford 22.4 g (99% yield) of compound 3 as a light brown oil. TLC (EtOAc/
hexanes 1:1) Rf 0.55. HPLC (Method A) RT 12.3 min, AP 95.6%. Mass spec:
1
352.3 (MꢁH)ꢁ. H and 13C NMR (CDCl3) were consistent with structure.
(1S,2S,3S,5S)-5-amino-3-(benzyloxy)-2-(benzyloxymethyl)cyclopentanol ð4Þ
Solid triphenylphosphine (20.0 g, 76.2 mmol) was added in small portions over
30 min to a solution of the azide 3 (22.4 g, 63.4 mmol) and H2O (1.7 ml)
dissolved in THF (100 ml). The resulting solution was stirred under argon at
room temperature for 5 h when TLC (EtOAc/hexanes 1:1) showed that the
reaction was complete. The solution was evaporated under vacuum and the
residue was chromatographed on silica gel (150 g) packed in CH2Cl2. The
column was eluted with CH2Cl2 (1900 ml) and then with a stepwise gradient of
CH2Cl2/MeOH (9:1) (600 ml), CH2Cl2/MeOH (7:3) (300 ml), and CH2Cl2/
MeOH (1:1) (300 ml) to afford 9.28 g of 4 as an oil. Impure fractions were
rechromatographed as described to afford an additional 8.16 g of product.
Total yield of the amine product 4 was 17.4 g (84%). This amine 4 solidified
upon storage at ꢁ208C. TLC (CH2Cl2/MeOH 9:1) Rf 0.2, on spot by UV.
1
Mass spec: 328.1 (M+H)+. H and 13C NMR (CDCl3) were consistent with
structure.
(1S,2S,3S,5S)-5-(2-amino-6-chloropyrimidin-4-yl)amino-3-(benzyloxy)-2-(benzy-
loxymethyl)cyclopentanol ð5Þ
A solution of the cyclopentylamine 4 (19.2 g, 58.6 mmol), 2-amino-4,6-
dichloropyrimidine (11.1 g, 67.7 mmol) and Et3N (40 ml, 0.29 mol) dissolved
in n-BuOH (100 ml) were heated at 1158C under argon overnight. The solvent
was evaporated under vacuum. The residue was dissolved in EtOAc, extracted
with H2O (2 ꢀ 50 ml), dried over MgSO4, filtered and evaporated under
vacuum to afford crude product. This crude material was chromatographed on
silica gel (170 g) eluting with EtOAc/hexanes (1:1) (750 ml) followed by EtOAc
Copyright # 2005 John Wiley & Sons, Ltd.
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650
M. D. OGAN ET AL.
(750 ml). Fractions containing the desired product were combined and
evaporated under vacuum to afford 22.0 g (82% yield) of compound 5. TLC
(EtOAc/hexanes 1:1) Rf 0.2, one spot by UV. Mass spec: 455.2 (M+H)+,
453.4 (MꢁH)ꢁ. Analytical Calculated for C24H27ClN4O3. 0.25EtOAc: C, 62.95;
H, 6.13; N, 11.75; Cl, 7.43. Found: C, 62.87; H, 5.96; N, 12.03; Cl, 7.68.
2-Amino-4-chloro-6-[(1a,2b,3a,4b)-2-hydroxy-4-(phenylmethoxy)-3-[(phenyl-
methoxy)methyl]cyclopentyl]amino-5-(4-chlorophenyl)diazopyrimidine ð6Þ
A solution of NaNO2 (3.44 g, 49.9 mmol) dissolved in H2O (20 ml) was added
dropwise over 10 min to an ice cold solution of 4-chloroaniline (6.08 g,
47.7 mmol) in 3 M HCl (80 ml). The resulting solution was stirred at 08C for
30 min. This solution of p-chlorobenzenediazonium chloride was added
dropwise, over 20 min, to a solution of pyrimidine 5 (19.6 g, 43.1 mmol),
KOAc (65.0 g, 0.66 mol) and HOAc (150 ml, 2.4 mol) dissolved in CH3CN/
H2O (2:1) (450 ml). The reaction was stirred at room temperature for 24 h. The
suspension was then diluted with H2O (300 ml) and stirred for 1 h in an ice
bath. The solid was isolated by filtration, washed 3 ꢀ 50 ml H2O, and dried
under vacuum to afford 23.3 g (91% yield) of 6 as a solid. TLC (CHCl3/
MeOH/HOAc 95:5:0.1) Rf 0.8. HPLC (Method B) RT 33.4 min. Mass spec:
593.1 (M+H)+, 591.3 (MꢁH)ꢁ.
2-Amino-4-methoxy-6-[(1a,2b,3a,4b)-2-hydroxy-4-(phenylmethoxy)-3-[(phe-
nylmethoxy)methyl]cyclopentyl]amino-5-(4-chlorophenyl)diazopyrimidine ð7Þ
A solution of 5 M KOH (3.4 ml, 17.0 mmol) was added to a suspension of 5-
diazopyrimidine 6 (5.00 g, 8.43 mmol) in MeOH (100 ml). The suspension was
stirred at 808C under a blanket of nitrogen. After 2 h, TLC (CH2Cl2/MeOH/
HOAc 95:5:0.1) showed the reaction was complete. The suspension was cooled
to room temperature and evaporated to dryness under vacuum. The solid was
dissolved in CH2Cl2 (100 ml) and extracted with half-saturated brine
(3 ꢀ 50 ml). The combined aqueous phase was back-extracted with CH2Cl2
(2 ꢀ 20 ml). The combined CH2Cl2 phase was dried over MgSO4, filtered and
evaporated under vacuum to afford an orange foam. The foam was dissolved
in CH3CN and evaporated under vacuum to a solid, redissolved in CH3CN
(70 ml) and allowed to stand at room temperature overnight. The crystalline
solid was collected by filtration, washed with CH3CN (5 ml), and air dried
under suction to afford 3.88 g (78% yield) of compound 7 as an orange solid.
TLC (CH2Cl2/MeOH 95:5) Rf 0.9, one spot by UV.
2-Amino-4-methoxy-6-[(1a,2b,3a,4b)-2-hydroxy-4-(phenylmethoxy)-3-[(phe-
nylmethoxy)methyl]cyclopentyl]amino-5-aminopyrimidine ð8Þ
The diazopyrimidine 7 (2.27 g, 3.86 mmol) was suspended in MeOH/H2O/
HOAc (40 ml:2.8 ml:1.65 ml) and heated to 808C under a blanket of argon.
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF [14C]-RADIOLABELLED ENTECAVIR
651
Solid Zn (2.52 g, 38.5 mmol) was added over 1 min via a solid addition funnel
maintained under argon. After 2 h, TLC (EtOAc/hexanes 4:1) showed the
reaction was complete. The reaction was cooled to room temperature and the
supernatant solution was transferred under argon via cannula to a dry
recovery flask. The solution was evaporated under vacuum to afford a yellow
residue. The residue was suspended in CHCl3 containing 0.1% HOAc (30 ml)
and the insoluble Zn salts were removed by filtration. The solid was washed
with 2 ꢀ 10 ml of CHCl3 containing 0.1% HOAc and the combined filtrate was
evaporated under vacuum to afford a residue. The residue was chromato-
graphed on a Biotage 40M column. The column was eluted with EtOAc/
hexanes (4:1) (500 ml) and then with EtOAc (900 ml). Fractions containing the
product were combined and evaporated under vacuum, redissolved in CH3CN
(20 ml) and evaporated under vacuum to afford 1.39 g (77% yield) of 8 as a
1
solid. TLC (EtOAc/hexanes 4:1) Rf 0.2, one spot by UV. H NMR was
consistent with the structure.
2-Amino-6-methoxy-1,9-dihydro-9-[(1a,2b,3a,4b)-2-hydroxy-4-(phenylmethoxy)
-3-[(phenylmethoxy)methyl]cyclopentyl]-6H-purine-8-[14C] ð9Þ
A solution of the triaminopyrimidine 8 (933.1 mg, 2.00 mmol) and TsOH
(30.4 mg, 0.16 mmol) dissolved in CH3CN (2 ml) was added to a solution of
triethyl [14C]orthoformate (100 mCi, 1.75 mmol) dissolved in CH3CN (5 ml).
The resulting solution was heated at 908C under a blanket of argon. HPLC
showed the reaction to be near completion after 2 h. The reaction was cooled
to room temperature and the solvent evaporated via a closed system vacuum
transfer to recover 6 mCi of volatile radioactivity. The solid residue was
chromatographed on a Biotage 40M column. The column was eluted with
EtOAc/hexanes (8:2) (1000 ml) followed by EtOAc:hexanes (9:1) (1000 ml).
Fractions containing the desired product were combined and evaporated
under a stream of nitrogen at 408C to afford 716.9 mg (86% yield) of 9 as a
light pink solid. HPLC (Method B) RT 16.1 min, Radiochemical purity
99.3%. TLC (EtOAc/hexanes 4:1) Rf 0.15, one spot by radiometric detection.
Specific activity was determined to be 116.3 mCi/mg (53.3 mCi/mmol).
[1S-(1a,2b,3a,4b)]-9-[2-hydroxy-4-(phenylmethoxy)-3-[(phenylmethoxy)methyl]-
cyclopentyl]-2-[[(4-methoxyphenyl)diphenylmethyl]amino]-6-(methoxy)-9H-pur-
ine-8-[14C] ð10Þ
DMAP (32.2 mg, 0.26 mmol), Et3N (312.3 mg, 3.09 mmol) and p-anisyl
chlorodiphenylmethane (611.6 mg, 1.98 mmol) were added to a solution of
purine 9 (716.9 mg, 1.51 mmol, 83.4 mCi) dissolved in CH2Cl2 (8 ml). The
resulting solution was protected from light and stirred under argon at room
temperature. After 3.5 h, HPLC (Method B) and TLC (CHCl3/MeOH/ 95:5)
showed the reaction was complete. The solution was evaporated under a
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M. D. OGAN ET AL.
stream of argon to afford a residue. The residue was chromatographed on a
Biotage 40L column eluting with CHCl3/MeOH (97:3). Fractions containing
the desired product were combined and evaporated under a stream of
nitrogen. The resulting solid was dissolved in CH3CN (30 ml), evaporated
under a stream of nitrogen, and dried under high vacuum to afford 1.17 g
(104% yield) of 10 as an orange foam.
[1S-(1a,2b,3a,4b)]-9-[2-oxo-4-(phenylmethoxy)-3-[(phenylmethoxy)methyl]cy-
clopentyl]-2-[[(4-methoxyphenyl)diphenylmethyl]amino]-6-(methoxy)-9H-pur-
ine-8-[14C] ð11Þ
The alcohol 10 (1.17 g, 1.57 mmol) was dissolved in anhydrous CH2Cl2 (10 ml)
and placed under argon. To this solution was added tert-BuOH (220.1 mg,
2.98 mmol) followed by solid Dess–Martin periodinate (1.34 g, 3.16 mmol).
The resultant light green solution was stirred at room temperature under
argon. After 1 h, HPLC (Method B) showed the reaction was complete. A
solution of 10% Na2SO3/10% NaHCO3/brine (5 ml:10 ml:5 ml) was added and
the reaction was stirred under argon for 15 min. The aqueous layer was
removed and the CH2Cl2 layer was further extracted with H2O (15 ml) and
brine (15 ml). The combined aqueous layer was back-extracted with CH2Cl2
(5 ml). The combined CH2Cl2 layer was dried over Na2SO4, filtered, and
evaporated under a stream of nitrogen. The residue was dried under high
vacuum to afford 1.32 g (113% yield) of crude 11 as a brown foam having a
radiochemical purity of 84%. This crude product was used without further
purification.
[1S-(1a,3a,4b)]-9-[2-methylene-4-(phenylmethoxy)-3-[(phenylmethoxy)methyl]-
2-[[(4-methoxyphenyl)(diphenyl)methyl]amino]-6-(methoxy)-9H-purine-8-[14C]
ð12Þ
The ketone 11 (1.32 g, 1.57 mmol) was dissolved in CH2Cl2 (18 ml), placed
under a blanket of argon and cooled to ꢁ788C. Nysted reagent (6.0 ml, 1.42 g,
3.12 mmol) was added to the reaction dropwise over 5 min. To the resultant
suspension was added a solution of 1 M TiCl4 in CH2Cl2 (3 ml, 3 mmol)
dropwise over 5 min. The reaction was allowed to warm to room temperature
and stirred under argon. After 1 h, HPLC (Method B) showed the reaction was
complete. The reaction was cooled in an ice bath and saturated NaHCO3
(2.5 ml) was added dropwise over 15 min (Caution-gas evolution). The
suspension was filtered and the zinc colloid was washed with CH2Cl2
(3 ꢀ 10 ml). The combined filtrate was extracted with H2O (2 ꢀ 25 ml). The
combined aqueous layer was back-extracted with CH2Cl2 (10 ml). The
combined CH2Cl2 layer was dried over Na2SO4, filtered and the Na2SO4
was washed with CH2Cl2 (2 ꢀ 5 ml). The combined filtrate and washings were
evaporated under a stream of nitrogen and dried under high vacuum to afford
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SYNTHESIS OF [14C]-RADIOLABELLED ENTECAVIR
653
1.15 g of crude 12 as an orange foam. The crude product was chromato-
graphed on a Biotage 40M column eluting with EtOAc/hexanes (3:7) (1000 ml)
and then with EtOAc/hexanes (1:1) (1000 ml) to afford 670.6 mg (67%) of 12
with a radiochemical purity of 91%. Mixed chromatography fractions were
combined and rechromatographed as described to afford an additional
75.5 mg (7.6%) of compound 12 with a radiochemical purity of 98.5%.
2-Amino-1,9-dihydro-9-[(1 S,3R,4 S)-2-methylene-4-(phenylmethoxy)-3-[(phenyl-
methoxy)methyl]cyclopentyl]-6H-purin-6-one-8-[14C] ð13Þ
A solution of the compound 12 (670.6 mg, 0.90 mmol) in MeOH/THF (1:1)
(6 ml) was heated under a blanket of argon at 608C and then a solution of 2 M
HCl (2 ml, 4 mmol) was added. The reaction was stirred at 608C overnight at
which time HPLC (Method C) showed 10% of the starting material 12
remained. The organic solvent was evaporated under a stream of nitrogen and
the resultant aqueous suspension was neutralized to pH 8 by addition of 5 M
KOH (0.75 ml). The aqueous phase was extracted with EtOAc (3 ꢀ 10 ml). The
combined EtOAc phase was evaporated under a stream of nitrogen and the
residue dried under high vacuum to afford 688.8 mg of a foam. This crude
material was chromatographed on Biotage 40M eluting with CHCl3 (300 ml)
followed by CHCl3/MeOH (93:7). Fractions containing the purified product
were combined and evaporated under a stream of nitrogen to afford 361.3 mg
(88% yield) of 13 as an orange solid. HPLC (Method B) RT 15.2 min,
radiochemical purity 95.8%. Mass spec (exact mass) 460.2199 (M+H)+
consistent for 14C112C25H27N5O3.
[14C]Entecavir ð1Þ
A solution of 13 (57.9 mg, 0.13 mmol) dissolved in CH2Cl2 (1.5 ml) was placed
under nitrogen and cooled to ꢁ658C. A 1 M solution of BCl3 (2.0 ml,
2.0 mmol) dissolved in CH2Cl2 was added dropwise over 2 min and the
solution was stirred at ꢁ658C for 5 min and then at ꢁ208C for 1 h. HPLC and
TLC showed the reaction was complete. The solution was cooled to ꢁ788C
and quenched by dropwise addition of MeOH (20 ml) over 2 min. The clear
solution was stirred at 08C for 45 min and then the solvent was evaporated
under a stream of nitrogen. The residue was dissolved in H2O (10 ml) and
extracted with CHCl3 (2 ꢀ 5 ml). The combined CHCl3 layer was back-
extracted with H2O (5 ml). The aqueous layer was neutralized by the addition
of 1 M NaOH and reduced in volume under a stream of nitrogen. This
aqueous solution was chromatographed in two batches on a column
(1.5 ꢀ 3 cm) of HP-20 resin eluting with H2O (10 ml), 2.5% CH3CN/H2O
(10 ml), 5% CH3CN/H2O (10 ml) and 10% CH3CN/H2O (55 ml). The product
which eluted in 10% CH3CN/H2O was evaporated under a stream of nitrogen
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 645–655

654
M. D. OGAN ET AL.
to afford 33.9 mg (97%) of [14C]entecavir as a white solid. This was combined
with an additional 45.5 mg of [14C]entecavir prepared similar to that described
above. HPLC radiochemical purity (Method C) of the combined product was
98.4%.
HP20 purified [14C]entecavir (79.4 mg) was dissolved in hot H2O (15 ml).
The solution was filtered hot (Whatman Autovial, 0.45 mm) and the filter
washed with hot H2O (3 ꢀ 2 ml) and CH3CN (5 ml). The combined filtrate and
washings were evaporated under a stream of nitrogen and the solid was
crystallized from hot H2O (2.5 ml). Following crystallization, the solvent was
reduced in volume to 0.5 ml under a stream of nitrogen. The crystals were
isolated, washed with CH3CN (0.5 ml), and dried under vacuum to afford
63.2 mg of [14C]entecavir as a white solid (77% yield from 13). This
crystalline[14C]entecavir (63.2 mg) and unlabelled entecavir (61.7 mg) were
dissolved in hot H2O (5 ml) and then evaporated to a solid under a stream of
nitrogen. A portion of this solid (107.5 mg) was dissolved in hot MeOH/H2O
(6:4) (5 ml). The hot solution was decolorized with charcoal (4.4 mg).
The charcoal was removed by hot filtration (Whatman Autovial, 0.45 mm)
and washed with hot MeOH/H2O (6:4) (3 ꢀ 1.5 ml). The combined filtrate and
washings were reduced in volume to 2 ml under a stream of nitrogen, heated to
afford a solution and allowed to crystallize at room temperature and then at
08C. The crystals were isolated, washed with CH3CN (0.5 ml), and dried under
vacuum to afford 96.8 mg of [14C]entecavir as a white solid (90% yield). HPLC
(Method C) RT 10.3 min, radiochemical purity 99.8%. Specific activity was
determined to be 108 mCi/mg.
Acknowledgements
The authors thank S.T. Chao of Discovery Chemistry for helpful discussions,
R. Gedamke, R. Shukla, K. Himes and C. Gerek of Analytial Research and
Development for their support with spectroscopic and analytical methods.
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