Synthesis of [13C4]Baraclude (entecavir)

Article abstract of DOI:10.1002/jlcr.1664

Entecavir, labeled as 1H-[¹³C₄]purin-6(9H)-one, was prepared from commercially available [¹³C]guanidine HCl, 1 and diethyl [1,2,3-¹³C₃]malonate, 2. The reagents were condensed together to give 2-amino-4,6-dichloro[2,4,5,6-¹³C ₄]pyrimidine 3, which in turn was coupled to an optically active amino cyclopentanol derivative, 9. A further sequence of eight reaction steps completed the constructions of the purine ring system and the exocyclic olefin attachment on the cyclic pentyl portion, 18. The removal of the methoxide and benzyl protecting groups gave [¹³C₄]entecavir, 20 in an overall yield of 6.8%. The chemical purity of the title compound was determined by HPLC to be 99.23%. The percent isotopic [¹³C4] abundance was found by mass spectral analysis to be 96.7%. No detectable level of the unlabeled entecavir was found by LC-MS analysis. Copyright

Full text of DOI:10.1002/jlcr.1664

Research Article
Received 1 May 2009,
Revised 17 June 2009,
Accepted 22 June 2009
Published online 3 August 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1664
Synthesis of [13C4]Baracludes (entecavir)
Ã
Scott B. Tran, Ihoezo V. Ekhato, and J. Kent Rinehart
Entecavir, labeled as 1H-[13C4]purin-6(9H)-one, was prepared from commercially available [13C]guanidine HCl, 1 and diethyl
[1,2,3-13C3]malonate, 2. The reagents were condensed together to give 2-amino-4,6-dichloro[2,4,5,6-13C4]pyrimidine 3,
which in turn was coupled to an optically active amino cyclopentanol derivative, 9. A further sequence of eight reaction
steps completed the constructions of the purine ring system and the exocyclic olefin attachment on the cyclic pentyl
portion, 18. The removal of the methoxide and benzyl protecting groups gave [13C4]entecavir, 20 in an overall yield of 6.8%.
The chemical purity of the title compound was determined by HPLC to be 99.23%. The percent isotopic [13C4] abundance
was found by mass spectral analysis to be 96.7%. No detectable level of the unlabeled entecavir was found by LC-MS
analysis.
Keywords: carbon-13; entecavir; Baracludes; diethyl [1, 2, 3-13C3]malonate; antiviral; nucleotide
We report here the synthesis and characterization of [13C4]-
entecavir 20 (Figure 1).
Introduction
Hepatitis B virus (HBV) represents one of the most prevalent viral
diseases in the world and is a cause of serious liver disorders.
About 400 million people are thought to be infected with HBV,
Results and discussion
Among the many methods for making purine nucleosides, the
strategy that goes through a pyrimidine intermediate was
practical to prepare [13C4]entecavir, 20.5 In such a synthetic
sequence, 2-amino-4,6-dichloro[2,4,5,6-13C4]pyrimidine, 3 could
be coupled with (1S,2S,3S,5S)-5-amino-3-(benzyloxy)-2-(benzy-
loxymethyl)cyclopentanol, 9 to form the pyrimidinylamino
which left untreated can lead to cirrhosis and hepatocellular
carcinoma. HBV infection causes an alarming 0.5–1.2 million
deaths per year.1 Entecavir was approved under the trade name
Baracludes by the US Food and Drug Administration in March
2005 for the treatment of chronic HBV infection in adults.
Entecavir is an orally active, carbocyclic guanosine nucleoside
analog, with potent selectivity against HBV DNA polymerase.2
Prior to this preparation, our laboratories used the structural
guanosine analog Lobucavir, 21 as an internal reference
standard in LC-MS quantification analyses of drug material in
clinical samples.3 The failure rates in the bioanalytical assay gave
rise to concerns about the reliability of the internal standard.
Isotope labeled versions of a drug are usually superior as
reference standards for LC-MS quantization and qualification
analyses. The confirmatory assurance that comes from the
co-elution of drug with its stable isotope labeled form is in part
accountable for the superior quality of the reference standard.4
Hence, we became interested in the synthesis of [13C4]entecavir.
derivative 10. If
a requisite 5-amino function could be
introduced by reduction of 5-[(p-chlorophenyl)azo derivative
11, a ring-closure reaction thereafter with triethyl orthoformate
would lead to the protected nucleoside 14. Although the
proposed sequence contains discouraging problematic trans-
formations, it has been extensively used in the literature to
access purine nucleosides and carbocyclic analogs.6 Fortunately,
[13C]guanidine HCl, 1 and diethyl [1,2,3-13C3]malonate, 2 are
useful labeled reagents and readily available. These reagents
provided 2-amino-4,6-dichloro[2,4,5,6-13C4]pyrimidine, 3 in a
two-step sequence.7 By this approach we condensed 1 and 2
in absolute ethanol containing sodium ethoxide to afford
2-amino[2,4,5,6-13C4]pyrimidine-4,6-diol. Subsequent chlorina-
tion using phosphorus oxychloride and N,N-dimethylaniline
gave 3 in 74% yield (Scheme 1).
O
O
The next phase of the synthesis required the preparation
of (1S,2S,3S,5S)-5-amino-3-(benzyloxy)-2-(benzyloxymethyl)cy-
clopentanol, 9.8,9 A sequence of steps in which Bom-Cl alkylation
N
N
N
HN
HN
*
*
*
N
H2N
N
H2N
N
*
OH
OH
Department of Chemical Synthesis, Radiochemistry Group, Bristol-Myers Squibb
Pharmaceutical Research and Development, P.O. Box 4000, Princeton, NJ 08543,
USA
HO
HO
* = 13
13C4]entecavir, 20
C
*Correspondence to: Scott B. Tran, Department of Chemical Synthesis, Radio-
chemistry Group, Bristol-Myers Squibb Pharmaceutical Research and Develop-
ment, P.O. Box 4000, Princeton, NJ 08543, USA.
Lobucavir, 21
[
Figure 1. [13C4]entecavir, 20 and Lobucavir, 21.
E-mail: scott.tran@bms.com
J. Label Compd. Radiopharm 2009, 52 485–489
Copyright r 2009 John Wiley & Sons, Ltd.

S. B. Tran et al.
Cl
construction of the target labeled guanosine nucleoside.
[13C4]Entecavir, 20 met our analytical specifications for use as
an internal reference standard.
.HCl
NH2
O
*
O
*
*
NH
*
N
*
*
a, b
EtO
OEt
H2N
+
*
H2N
N
3
* Cl
Experimental
2
1
Isotopic labeled diethyl [1,2,3-13C3]malonate with a 13C enrich-
ment of 99% was purchased from Cambridge Isotope Labora-
tories, Inc. and [13C]guanidine HCl with a 13C enrichment of
99.4% was purchased from Isotec.
Scheme 1. Synthesis of 2-amino-4,6-dichloro[2,4,5,6-13C4]pyrimidine, 3: (a) NaOEt,
EtOH, reflux, 1 h; (b) POCl3, N,N-dimethylaniline, 601C, 6 h, 74%.
High Performance Liquid Chromatography: HPLC methods
described below were used for in process and final product
analyses. Co-injections with authentic samples were used when
possible. All HPLC purities were measurements of UV purity and
performed on a Shimadzu SLC-10A system controller, dual
LC-10AT pumps, SPD-10A UV-VIS detector at 254 nm and
CLASS VP 5.0 integration software. Method: YMC-ODS-AQ C18,
3 mm, 4.6 Â 150 mm2, detected at 254 nm. Mobile phase A:
0.1% TFA in water, B: 0.1% TFA in acetonitrile. Gradient: 0 min
5% B, 7 min 50% B, 35 min 95% B, 40 min 95%B, 45 min 5% B,
flow rate = 1 mL/min. LCMS Method: Phenomenex C18, 5 mm,
4.6 Â 50 mm, detected at 220 nm. Mobile phase A: 0.1%
TFA in 10% MeOH, 90% water, B: 0.1% TFA in 90% MeOH,
10% water; oven temperature: 401C. Gradient: 0 min 0% B,
4 min 100% B, flow rate = 4 mL/min. NMR Spectra were recorded
on Bruker Avance NMR spectrometer. Only carbon-13 enriched
positions are reported for C-13 characterization purposes.
Optical rotation was measured with a polarimeter by use
of a 50 mm cell at 589 nm. 8.16 mg of the sample was
dissolved in 1.0 mL of MeOH. The reported specific rotation is
the average of ten individual measurements taken at ten-second
intervals.
of sodium cyclopentadiene 4, followed by (À)-IPC2BH hydro-
boration, alkaline peroxide oxidation, and stereospecific
epoxidation with tert-BuOOH/VO(acac)2 furnished (1S,2R,3S,5R)-
2-(benzyloxymethyl)-6-oxabicyclo[3.1.0]hexan-3-ol, 6. Benzylation
of 6 gave the protected alcohol 7 on which sequential azide
opening of the epoxide ring and reduction of the azide group to
the amino compound 9 were completed (Scheme 2). Coupling of
9 with 3 (Scheme 3) was conducted in hot butanol in the
presence of triethylamine to give 10 in 68%. In this synthesis,
we chose to introduce the required 5-amino group via
5-[(p-chlorophenyl)azo]-pyrimidine 11 prepared from 10 by its
reaction with p-chlorobenzenediazonium chloride. However, the
chloride substituent on the pyrimidine ring had to be replaced
with a methoxy group that would tolerate subsequent reaction
conditions. It was likely that dehalogenation would accompany
the Zn reduction of the azo functionality 11 to the amine and
potentially complicate the unmasking of the carbonyl of
guanosine down stream. Following the Zn reduction, compound
14 was made by a ring closure reaction by reacting tetra-
substituted pyrimidine 13 with triethyl orthoformate in the
presence of an acid catalyst. Labeled [13C4]intermediates 11, 12,
13, 15, 16, and 17 were prepared as described.8
To complete the synthesis of [13C4]entecavir, the exocyclic
olefin was installed. In preparation for this transformation, the
amino group of 14 was selectively protected by reaction with
4-monomethoxytrityl chloride (MMTrCl). The alcohol function-
ality in 15 was oxidized with Dess-martin periodinane to give the
ketone 16.9 Reaction of 16 with Nysted reagent installed the
requisite olefin group, and in the process afforded 17, fully
protected [13C4]entecavir. Removal of MMTr group was accom-
plished with 2 N HCl at room temperature to give 18 in 69%
yield. Demethylation was achieved with 3 N HCl at 601C cleanly
unmasking the carbonyl to afford the dibenzyl guanoside 19 in
75% yield. Debenzylation of 19 with excess BCl3 afforded 20
after crystallization from methanol. An analytical sample was
obtained by recrystallization from warm water in 89% recovery.
A total of 86 mg of [13C4]entecavir, 20 was synthesized in an
overall yield of 6.8% from 10. Mass spectrometric analysis of
[13C4]entecavir was performed using positive ion electrospray
on a Q-Tof Ultima mass spectrometer giving a molecular ion
[M1H]1 at 282.14 Da. This is consistent with the molecular
formula 13C4C8H15N5O3 for the free base. Isotopic purity was
2-Amino-4,6-dichloro[2,4,5,6-13C4]pyrimidine, 3
NaOEt (3.75 g, 55.2 mmol) was slowly added to a flask equipped
with a water cooled condenser containing a mixture of diethyl
[1,2,3-13C3]malonate (3 g, 18.4 mmol) and [13C]guanidine HCl
(1.77 g, 18.4 mmol) in EtOH (25 mL). The reaction was heated to
reflux for 1 h. The mixture was cooled to rt and filtered. A white
cake was obtained, and then dissolved in water (20 mL) and the
pH adjusted to 4 with 5% aqueous acetic acid. The resulting
solid was collected by vacuum filtration and washed with
additional water. The solid was dried under high vacuum for
16 h. The solid was placed in a dry flask, which was charged with
phosphorus oxychloride (11.3 g, 73.6 mmol). The mixture was
heated to 601C for 4 h. While maintaining this temperature,
N,N-dimethylaniline (4.46 g, 36.8 mmol) was added over 1 h. The
reaction mixture was stirred for an additional 1 h, then cooled to
01C and quenched with water (25 mL). The precipitated product
was collected by vacuum filtration. The filter cake was washed
with water and dried under vacuum to give a pale yellow solid
(2.29 g, 74%). This product was used in the next step without
further purification. MS [M1H]1 = 167.99, 37Cl35Cl [M1H]1
= 169.98, 37Cl2 [M1H]1 = 171.98. 1H NMR (400 MHz, CDCl3,
d):6.93, 6.47 (d, J13CH = 183 Hz), 5.35 (bs, 2H).
calculated to be 96.7%.
A molecular ion of 281.13 Da,
corresponding to the empirical formula 13C3C9H15N5O3, was
3.3%. Unlabeled entecavir was below levels of detection.
In short, we have achieved the synthesis of entecavir bearing
13C4-labeled purine base from commonly available diethyl
[1,2,3-13C3]malonate and [13C]guanidine HCl. At the start,
these compounds were transformed into 2-amino-4,6-dichloro-
(1S,2S,3S,5S)-5-Amino-3-(benzyloxy)-2-(benzyloxymethyl)-
cyclopentanol, 9
[2,4,5,6-13C4]pyrimidine. Coupling with substituted amino Product 98 (clear oil, 3.10 g). TLC (10% MeOH/CH2Cl2, Product
cyclopentanol followed by additional steps to ultimately Rf = 0.05), HPLC (Product, Rt = 7.77 min), LCMS (LCMS Method,
install the exocyclic olefin on the carbocycle completed the Product Rt = 2.89 min). MS [M1H]1 = 328.3. 1H NMR (400 MHz,
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 485–489

S. B. Tran et al.
O
O
BnO
BnO
BnO
BnO
e
a, b, c
d
: Na
HO
HO
4
7
5
6
OH
OH
NH2
N3
BnO
g
BnO
f
BnO
BnO
9
8
Scheme 2. Synthesis of amino cyclopentanol intermediate, 9: (a) Bom-Cl, DMF, À401C, 15 min; (b) (À)-IPC2BH, THF, À78 to À101C, 48 h; (c) NaOH/H2O2, rt, 12 h; 75% over
three steps; (d) VO(acac)2, tert-BuOOH, DCM, rt, 2.5 h; (e) NaH, 60% dispersion, BnBr, TBAI, DMF, rt then À501C, 18 h; 83% over two steps; (f) NaN3, NH4Cl, EtOH, 851C, 22 h,
99%; (g) Ph3P, water, THF, rt, 5 h, 84%.
Cl
Cl
Cl
OMe
*
*
Cl
N
*
*
N
N
*
N
N
OH
N
*
N
*
*
*
H2N
N
NH
10
*
c
b
NH2
3, a
H2N
N
* NH
H2N
N
* NH
BnO
HO
HO
HO
BnO
BnO
OBn
9
BnO
OBn
BnO
OBn
11
12
OMe
OMe
*
OMe
N
N
N
N
*
*
NH2
*
N
*
*
g, h
*
N
f
*
e
d
N
MMTrHN
N
*
H2N
N
*
H2N * N * NH
HO
HO
HO
OBn
OBn
BnO
BnO
BnO
OBn
15
14
13
OMe
N
OMe
O
N
N
N
N
j, k
i
N
*
N
HN
*
*
*
*
*
*
N
MMTrHN
N
*
H2N
N
H2N
N
*
*
*
*
X
OBn
OBn
OR
BnO
BnO
RO
19, R = Bn
20, R = H, [13C4]entecavir
16, X = O
17, X = CH2
18
Scheme 3. Synthesis of [13C4]entecavir, 20: (a) Et3N, n-BuOH, 1151C, 18 h, 68%; (b) NaNO2, water, 4-chloroaniline, 3 N HCl, 01C, 30 min then 3, KOAc, acetic acid, CH3CN/
water (2:1), 24 h, 78%; (c) 5 N NaOH, MeOH, 801C, 2 h, 95%; (d) Zn, AcOH, water, MeOH, 801C, 2 h, 95%; (e) HC(OEt)3, TsOH, CH3CN, 70%; (f) MMTrCl, DMAP, Et3N, CH2Cl2,
88%; (g) Dess-martin periodinane, t-BuOH, CH2Cl2, 84%; (h) Nysted reagent, TiCl4, CH2Cl2, THF, 60%; (i) 2 N HCl, MeOH, THF, rt, 4 h, 69%; (j) 3 N HCl, MeOH, THF, 601C, 22 h,
75%; (k) 1M BCl3, CH2Cl2, À78 to À201C, 1.5 h, 89%.
(1S,2S,3S,5S)-5-(2-Amino-6-chloro[13C4]pyrimidin-4-ylamino)-3-
(benzyloxy)-2-(benzyloxymethyl)cyclopentanol, 10
DMSO-d6, d): 7.36–7.23 (m, 10H), 4.88 (bs, 1H), 4.47 (s, 2H), 4.41
(d, 2H, J = 4.0 Hz), 3.74–3.71 (m, 1H), 3.54 (dd, 1H, J = 4.0, 8.0 Hz),
3.43 (dd, 1H, J = 4.0, 8.0 Hz), 3.33 (bs, 2H), 3.18 (t, 1H, J = 8.0 Hz),
3.03–2.97 (m, 1H), 1.92–1.81 (m, 2H), 1.45–1.37 (m, 1H).
[a]2D0132.301.
Triethylamine (2.04 g, 20.21 mmol) was added to a solution
of 9 and 2-amino-4,6-dichloro[2,4,5,6-13C4]pyrimidine, 3 (0.70 g,
J. Label Compd. Radiopharm 2009, 52 485–489
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

S. B. Tran et al.
4.16 mmol) in n-butanol (15 mL). The solution was heated to dried over Na2SO4 and concentrated to dryness. The crude
1151C for 18 h. The reaction was monitored by TLC (35% EtOAc/ product was purified by silica gel flash chromatography eluting
Hex, Product Rf = 0.2). The solution was evaporated to dryness, with 25% EtOAc/Hexane to 100% EtOAc to 0.25% MeOH/EtOAc
and the residue dissolved in EtOAc (3 Â 10 mL) and washed with to afford a white foam 14 (0.66 g, 70%). LCMS (LCMS Method,
water (15 mL). The layers were partitioned and the organic layer Product Rt = 3.13 min). MS [M1H]1 = 480.2. 1H NMR (500 MHz,
was washed with brine, dried over Na2SO4, and concentrated to CDCl3, d 7.54–7.50 (dd, 1H, J = 4.9, 6.5 Hz), 7.31–7.24 (m, 10H),
dryness. The crude product was purified by silica gel flash 4.88 (bs, 2H), 4.64–4.55 (m, 1H), 4.52–4.49 (m, 5H), 4.23 (t, 1H,
chromatography eluting with 5À50% EtOAc/Hexane to afford a J = 8.3 Hz), 4.12–4.01 (m, 4H), 3.67–3.60 (m, 2H), 2.53 (m, 1H),
white solid 10 (1.31 g, 68%). LCMS (LCMS Method, Product 2.43 (m, 1H), 2.32–2.25 (m, 1H). 13C NMR (CDCl3, d 162.27 (dd,
Rt = 3.07 min). MS [M1H]1 = 459.2, 37Cl35Cl [M1H]1 = 461.2. 1H J = 7.6, 86.5 Hz), 158.28 (d, J = 7.6 Hz), 153.65 (d, J = 66.1 Hz),
NMR (400 MHz, CDCl3, d 7.39–7.28 (m, 10H), d, J13CH = 173 Hz), 115.98 (m).
5.00 (s, 2H), 4.55 (m, 3H), 4.25 (s, 1H), 3.95(s, 1H), 3.84 (t, 1H,
J = 7.2 Hz), 3.70–3.60 (m, 2H), 2.35–2.31 (m, 2H), 1.81–1.74 (1S,2S,3S,5S)-3-(Benzyloxy)-2-(benzyloxymethyl)-5-(6-meth-
(m, 1H). 13C NMR (CDCl3, d 164.31 (d, J = 60.37 Hz), 161.83 (m),
160.31 (d, J = 72.94 Hz), 94.81 (m).
oxy-2-((4-methoxyphenyl)diphenylmethylamino)-9H-
[
13C4]purin-9-yl)cyclopentanol, 15
Product 158 (light-yellow solid, 0.9 g, 88%), LCMS (LCMS
(1S,2S,3S,5S)-5-(2-Amino-6-chloro-5-((E)-(4-chlorophenyl)-
diazenyl)[13C4]pyrimidin-4-ylamino)-3-(benzyloxy)-2-(benzy-
loxymethyl)cyclopentanol, 11
1
Method), MS [M1H]1 = undetectable. H NMR (500 MHz, CDCl3,
d 7.55–6.70 (m, 25H), 6.25 (s, 1H), 4.53 (m, 5H), 4.11 (bs, 1H), 3.77
(m, 5H), 3.67–3.60 (m, 3H), 2.34 (bs, 2H), 2.11 (bs, 1H), 1.64 (bs,
Product 118 (yellow solid, 1.32 g, 78%), LCMS (LCMS Method, 2H). 13C NMR (CDCl3, d 161.27 (dd, J = 86.0 Hz), 157.00 (m),
Product Rt = 4.40 min). MS [M1H]1 = 597.20. H NMR (400 MHz, 153.65 (m), 115.00 (m).
1
CDCl3, d 10.72 (s, 1H), 7.69–7.66 (dd, 2H, 4.49, 9.56 Hz), 7.47–7.41
(dd, 2H, 4.49, 9.56 Hz), 7.38–7.27 (m, 9H), 5.59 (bs, 2H), 4.58–4.50 (2R,3S,5S)-3-(Benzyloxy)-2-(benzyloxymethyl)-5-(6-meth-
oxy-2-((4-methoxyphenyl)diphenylmethylamino)-9H-
[
(m, 5H), 4.0 (m, 1H), 3.99 (t, 1H, J = 4.2 Hz), 3.94–3.90 (t, 1 H,
J = 7.6 Hz), 3.69–3.62 (m, 2H), 2.44–2.34 (m, 2H), 1.94–1.86 (m,
1H), 1.60 (bs, 2H). 13C NMR (CDCl3, d 165.86 (m), 159.39 (m),
156.50 (m), 119.47 (m).
13C4]purin-9-yl)cyclopentanone, 16
Product 168 (light-yellow foam, 0.90 g, 84%), LCMS (LCMS
Method), MS (observed mol. ion of the unprotected,
Rt = 3.36 min) [M1H]1 = 478.20. 1H NMR (400 MHz, CDCl3,
ddd, 1H, J = 1.26, 8.31 Hz), 7.39–7.17 (m, 24H), 6.77–6.75
(d, 2H, J = 8.81 Hz), 6.21 (bs, 1H), 4.53 (bs, 2H), 4.41 (m, 2H),
4.15–4.25 (bs, 1H), 3.76 (m, 4H), 3.58 (bs, 2H), 2.71 (s, 1H),
(1S,2S,3S,5S)-5-(2-Amino-5-((E)-(4-chlorophenyl)diazenyl)-6-
methoxy[13C4]pyrimidin-4-ylamino)-3-(benzyloxy)-2-(benzy-
loxymethyl)cyclopentanol, 12
Product 128 (orange solid, 1.25 g, 95%), LCMS (LCMS Method, 1.59 (s, 3H).
Product Rt = 4.10 min). MS [M1H]1 = 593.30, 37Cl35Cl [M1H]1
595.3. 1H NMR (400 MHz, CDCl3, d 10.95 (s, 1H), 7.62–7.58 9-((1S,3R,4S)-4-(Benzyloxy)-3-(benzyloxymethyl)-2-methyle-
=
necyclopentyl)-6-methoxy-N-((4-methoxyphenyl)diphenyl-
(dd, 2H, J = 4.7, 9.5 Hz), 7.42–7.26 (m, 12H), 6.20 (bs, 1H), 5.16
(s, 2H), 4.55 (s, 3H), 4.45 (s, 1H), 4.06 (d, 2H, J = 3.8 Hz), 4.02
(m, 1H), 3.90 (t, 1H, J = 7.8 Hz), 3.70–3.59 (m, 2H), 2.41–2.33
(m, 2H), 1.96–1.88 (m, 1H), 1.57 (s, 2H). 13C NMR (CDCl3, d
169.58–168.66 (dd, J = 8.25 Hz, 83.81 Hz), 161.32 (m), 156.50 (m),
113.0 (m).
methyl)-9H-[13C4]purin-2-amine, 17
Product 178 (off-white solid, 0.54 g, 60%). MS [M1H]1 = 748.36.
1H NMR (500 MHz, CDCl3, d 7.60 (bs, 1H), 7.38–7.12 (m, 22H), 6.73
(d, 2H, J = 8.8 Hz), 6.25 (s, 1H), 5.15 (s, 2H), 4.72 (s, 1H), 4.45 (m,
4H), 3.98 (s, 1H), 3.72 (s, 3H), 3.65–3.50 (m, 3H), 2.90 (s, 1H),
1.65–1.45 (m, 2H). 13C NMR (CDCl3, d 162.27 (m), 158.28 (m),
153.50 (m), 114.0 (s).
(1S,2S,3S,5S)-3-(Benzyloxy)-2-(benzyloxymethyl)-5-(2,5-dia-
mino-6-methoxy[13C4]pyrimidin-4-ylamino)cyclopentanol, 13
Product 138 (off-white foam, 0.94 g, 95%), LCMS (LCMS Method,
9-((1S,3R,4S)-4-(Benzyloxy)-3-(benzyloxymethyl)-2-methyle-
1
Product Rt = 2.79 min). MS [M1H]1 = 470.20. H NMR (500 MHz,
necyclopentyl)-6-methoxy-9H-[13C4]purin-2-amine, 18
CDCl3, d 7.28–7.37 (10H, m), 5.26 (1H, br. s.), 4.38–4.61 (4H, m),
4.18 (1H, m), 3.95 (2H, m), 3.81–3.89 (3H, m), 3.76 (2H, t,
J = 7.97 Hz), 3.63–3.71 (2H, m), 3.58 (2H, m), 2.20–2.40 (3H, m),
1.83 (1H, ddd, J = 13.33, 10.58, 6.87 Hz).
Compound 17 (505 mg, 0.67 mmol) was dissolved in THF/MeOH
(1:1) (6 mL). To this solution, 2 N HCl (1.7 mL, 3.38 mmol) was
slowly added. The reaction was stirred at rt for 9 h. The reaction
progress was monitored by TLC (70% EtOAc/Hexane, Rf SM = 0.8,
Rf Product = 0.35). The solution was concentrated to dryness.
The pH was adjusted to 8 with 2.5 N KOH and extracted
with EtOAc (3 Â 15 mL). After the layers were separated, the
organic layer was washed with brine, dried over Na2SO4, and
concentrated to dryness. The crude product was purified by
silica gel flash chromatography eluting with 15–75% EtOAc/Hex
to afford a colorless oil 18 (221 mg, 69%). MS [M1H]1 = 476.18.
1H NMR (500 MHz, CDCl3, d 7.65 (m, 1H), 7.35–7.28 (m, 10H),
5.54 (bm, 1H), 5.18 (s, 1H), 4.80 (m, 3H), 4.57–4.51 (m, 4H), 4.17
(m, 1H), 4.08 (m, 3H), 3.67–3.65 (m, 2H), 3.03 (s, 1H), 2.41–2.34
(m, 2H).
(1S,2S,3S,5S)-5-(2-Amino-6-methoxy-9H-[13C4]purin-9-yl)-3-
(benzyloxy)-2-(benzyloxymethyl)cyclopentanol, 14
p-Toluenesulfonic acid monohydrate (20 mg, 0.1 mmol) was
added to a solution of 13 (0.93 g, 1.98 mmol) and anhydrous
triethyl orthoformate (0.44 g, 2.98 mmol) in acetonitrile (20 mL)
under nitrogen. The solution was heated to 901C for 1 h. The
reaction was monitored by TLC (75% EtOAc/Hex, Product
Rf = 0.2). The solution was evaporated to dryness, dissolved in
EtOAc (3 Â 10 mL), and washed with water (15 mL). After the
layers were separated, the organic layer was washed with brine,
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J. Label Compd. Radiopharm 2009, 52 485–489

S. B. Tran et al.
13C NMR (DMSO-d6, d 156.82 (dd, J = 7.7, 86.5 Hz), 153.18 (s),
150.91 (dd, J = 7.7, 61.0 Hz), 115.81 (m). Elemental analysis for
13C4C8H15N5O3.1.5 equiv. water, calculated (%) C = 48.05;
H = 4.86; N = 22.71, found (%) C = 47.56; H = 5.17; N = 22.96.
2-Amino-9-((1S,3R,4S)-4-(benzyloxy)-3-(benzyloxymethyl)-2-
methylenecyclopentyl)-1H-[13C4]purin-6(9H)-one, 19
Of total, 0.5 mL of 3 N HCl was added to a solution of 18
(73.5 mg, 0.154 mmol) in THF/MeOH (1:1) (3 mL) under N2. The
reaction was heated to 601C for 18 h. The reaction was
monitored by TLC (70% EtOAc/Hexane, SM Rf = 0.8 and 10%
MeOH/CH2Cl2, Product Rf = 0.2) and HPLC. The solution was
evaporated to dryness and the residue was dissolved in EtOAc
(15 mL) and sat NaHCO3 (5 mL). The pH was adjusted with 5 N
KOH to 7.5–8. After the layers were separated, the organic layer
was washed with brine, dried over Na2SO4, and concentrated to
dryness. The crude product was purified by silica gel flash
chromatography eluting with 0–5% MeOH/CHCl3 to afford a
pure white solid 19 (53.5 mg, 75%). LCMS (LCMS Method,
Product Rt = 3.16 min). MS [M1H]1 = 462.1. 1H NMR (400 MHz,
CDCl3, d 12.11 (s, 1H), 7.80 (s, 1H), 7.55–7.20 (m, 10H), 6.02
(s, 2H), 5.52 (m, 1H), 5.23 (s, 1H), 4.87 (s, 1H), 4.63–5.52 (m, 6H),
4.20 (s, 1 H), 3.69 (m, 2H), 3.06 (s, 1H), 2.41 (m, 2H).
Acknowledgement
We gratefully acknowledge the Bristol-Myers Squibb scientists
Samuel Bonacorsi, Jr. of Radiochemistry, Edward Ruediger of
Discovery Chemistry, Siva Prasad of Process R and D, and Bang-
Chi Chen of Chemical Synthesis, for discussions detailing the
synthesis of [13C4]entecavir. Also thanks are due to Brad Maxwell
of Radiochemistry for his review of the manuscript and Richard
Gedamke of Analytical R and D, for providing the analytical mass
spectrometry results.
References
[
13C4]Entecavir, 20
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Compound 19 (158 mg, 0.34 mmol) was placed in a 25 mL
reaction flask and dissolved in dichloromethane (3 mL). The flask
was cooled to À781C, and BCl3 (1 M in CH2Cl2, 2.05 mL,
2.05 mmol) was added slowly. The solution was stirred at
À781C for 0.5 h, then at À201C for 1 h with monitoring by HPLC
(HPLC Method, Product Rt = 23.33 min). The reaction flask was
cooled back to À781C and MeOH (6 mL) was slowly added. The
resulting clear solution was warmed to rt, then evaporated to
dryness, and the crude product was crystallized from MeOH
(1 mL). The white solid was collected by vacuum filtration and
washed with CH2Cl2 (1 mL) to give 74 mg of product. The
mother liquor was dissolved in 50:50 saturated NaHCO3/water
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low solubility in CH2Cl2). After the two layers were separated,
the aqueous layer was concentrated to dryness under high
vacuum to give a crude product (HPLC purity = 93.8%, 28 mg).
This crude product was crystallized from warm water (4 mL) to
give an additional 12 mg of product (total amount synthesized,
20: 86 mg, yield: 89%; overall yield: 6.8% from 10). HPLC
chemical purity = 99.23%. HPLC coinjected with the authentic
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J. Label Compd. Radiopharm 2009, 52 485–489
Copyright r 2009 John Wiley & Sons, Ltd.
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