Synthesis of a 35S-labeled dinucleoside phosphorothioate prodrug, an orally bioavailable anti-HBV agent

Article abstract of DOI:10.1002/jlcr.2925

The ³⁵S-labeled, dinucleoside phosphorothioate 1, an orally available agent against hepatitis B virus, was prepared in eight steps with high specific activity and radiochemical purity. Radiolabeled 3H-benzodithiole-3- one-1,1-dioxide was synthe

Full text of DOI:10.1002/jlcr.2925

Research Article
Received 10 January 2012,
Revised 22 February 2012,
Accepted 2 April 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2925
35
Synthesis of a S-labeled dinucleoside
phosphorothioate prodrug, an orally
bioavailable anti-HBV agent
Sung-Whi Rhee,a Radhakrishnan P. Iyer,b John E. Coughlin,b
Seetharamaiyer Padmanabhan,b Jeremiah P. Malerich,a
a
*
and Mary J. Tanga
The 35S-labeled, dinucleoside phosphorothioate 1, an orally available agent against hepatitis B virus, was prepared in eight
steps with high specific activity and radiochemical purity. Radiolabeled 3H-benzodithiole-3-one-1,1-dioxide was synthesized
in four steps from 35S8 and was used as the sulfurizing reagent.
Keywords: dinucleoside; phosphorothioate; hepatitis B virus; solid-supported synthesis; antiviral
radiosynthesis (Scheme 1). Radiolabeled thiobenzoic acid was
Introduction
prepared by heating thiobenzoic acid (2) with 35S8. Treatment
Acute and chronic liver infections caused by hepatitis viruses
HBV and HCV constitute a major worldwide public health crisis
affecting nearly 550 million people worldwide. There are an esti-
mated 350 million chronic carriers of HBV worldwide of which
of labeled-2 with thiosalicylic acid and sulfuric acid gave the
cyclic disulfide 3. Trifluoroperoxyacetic acid, generated in situ
from trifluroacetic acid and hydrogen peroxide, was used to
oxidize 3, thereby providing access 35S-labeled 3H-BD (4).
about 1 million people die each year from chronic HBV.1 Liver
cancer patients as well as significant numbers of liver transplant
recipients have a continued need for effective anti-HBV therapy.
Although several anti-HBV drugs are in clinical use, significant
unmet medical need exists due to dose-limiting toxicities and
antiviral resistance. Safer and more effective anti-HBV drugs
with novel mechanisms of action that can be used alone and
in combination with other drugs are urgently needed.
The 35S-labeled dinucleoside phosphorothioate 11 was pre-
pared using solid-phase phosphoramidite chemistry (Scheme 2)9
via the intermediate solid support-bound dinucleoside phosphite
triester 9.10,11 Controlled-pore-glass (CPG) support was functiona-
lized with amino groups and succinoylated to give support-bound
carboxylic acid 5 to access 9. The NBz-protected deoxyadenosine
derivative 6 was loaded onto the solid support using carbodiimide
esterification conditions to yield 7. Removal of the 5’-dimethoxytrityl
group was accomplished using dichloroacetic acid (DCA) in
dichloromethane (DCM), and the resulting alcohol was coupled
to 2’-OMe-uridine phosphoramidite 8 in the presence of
5-(ethylthio)tetrazole to afford sulfurization precursor 9. In the
event, treatment of 9 with 35S-3H-BD (4) installed the radiolabel
on the thiophosphoryl triester [RP,SP]-10, which was isolated as a
mixture of diastereomers at phosphorus. This diastereomeric
mixture was carried to the target compound without separation.
The dimethoxytrityl group in [RP,SP]-10 was removed with DCA/
DCM. Subsequent treatment with 28% NH4OH resulted in the
cleavage of the dinucleotide from the solid support and simulta-
neous removal of the cyanoethyl and benzoyl protecting groups
Phosphorothioate dinucleotides and trinucleotides are a new
class of anti-HBV compounds with novel mechanism(s) of action
and potent antiviral activity in vitro and in vivo.2,3 We recently
reported that the alkyloxycarbonyl prodrug 1, a representative of
dinucleoside phosphorothioate, is an orally bioavailable anti-HBV
agent.4 In this paper, we report the synthesis of 35S-labeled 1 as
an [RP,SP]-diasteromeric mixture that was employed in absorption,
distribution, metabolism, and excretion (ADME) studies in rats.5
Results and discussion
A key step in the synthesis of the target compound is the oxidative
sulfurization of the support-bound dinucleoside phosphite triester
9. The use of elemental 35S8 in sulfurization reactions is cumber-
some and inefficient because it involves the use of toxic reagents
such as CS2, collidine, and lutidine as the reaction medium.6 Hence,
aSRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA
we considered the use of radiolabeled 3H-benzodithiole-3-one- bSpring Bank Pharmaceuticals, INC., S-7, 113 Cedar Street, Milford, MA 01757,
USA
1,1-dioxide (3H-BD), a commonly used sulfurizing reagent in
oligonucleotide synthesis.7
*Correspondence to: Mary J. Tanga, SRI International, 333 Ravenswood Avenue,
35S-Labeled 3H-BD (4) was prepared starting from thiobenzoic
Menlo Park, CA 94025, USA.
acid (2) by adapting the method previously described8 for E-mail: mary.tanga@sri.com
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

S.-W. Rhee et al.
Scheme 1. Synthesis of 35S-3H-benzodithiole-3-one-1,1-dioxide (4).
Scheme 2. Synthesis of 35S-1 (DMT = dimethoxytrityl).
to afford the phosphorothioate [RP,SP]-11. The solution-phase concentrations of iodide 12. In our optimal procedure, upon
alkylation of 7 with iodomethyl isopropyl carbonate (12) gave consumption of 11, as determined by analytical radio-HPLC,
the target [RP,SP]-35S-labeled 1.
the isopropanol was removed in vacuo. Extraction with hexanes
Careful work-up of the reaction mixture from alkylation removed most of the excess iodide 12, leaving an aqueous
proved critical to obtaining pure 35S-1. After several attempts solution of 35S-1 and inorganic iodide salts. Application of this
of the final step gave poor purity of the target compound; it dilute solution directly to a preparative reversed-phase column
was determined that 35S-labeled 1 is very sensitive to high allowed us to obtain pure 35S-1.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012

S.-W. Rhee et al.
Microwave-assisted amination of CPG 500
Experimental
In a pressure reactor with a Teflon plug having a chemically resistant
O-ring (Chemraz), CPG 500 (135 g) and 3-aminopropyltriethoxysilane
(400 mL, ~3.5 mL/g) was placed. The reaction slurry was mixed well
and heated in a microwave oven (800 W) for 8 minutes in 1-minute
cycle with intermittent cooling. After each heating cycle, the contents
of the reaction slurry was mixed well and cooled. At the end of the
final heating cycle, the reaction slurry was cooled to RT and filtered.
The CPG was washed with toluene (2 ꢀ 125 mL), MeOH (2 ꢀ 250 mL),
CHCl3 (1 ꢀ 250 mL), DCM (2 ꢀ 250 mL), and hexanes (2 ꢀ 250 mL). The
aminated-CPG was air-dried in a glass tray and the amino loading was
determined by dimethoxytrityl analysis. Typical amino loadings were
found to be 90–113 mmol/g.
General methods
All reactions were carried out using commercial grade reagents and sol-
vents under argon atmosphere. NMR Spectra were recorded on a Varian
Mercury VMX 300-MHz spectrophotometer (Varian Medical Systems, CA,
USA) using tetramethylsilane as the internal standard. NMR multiplicities
are reported using the following abbreviations: s, singlet; d, doublet; m,
multiplet. Low-resolution mass spectra were obtained on a Finnigan
LCQDuo LC MS/MS instrument (Thermo Fisher Scientific, MA, USA) by
electrospray ionization (ESI). Reversed-phase HPLC data were obtained
using a Waters 2690 Separations Module (Waters Corporation, MA, USA)
with photodiode array detector and a Perkin Elmer radioactivity monitor
(PerkinElmer, Inc., MA, USA). Thin layer chromatography analyses were car-
ried out on a commercial pre-coated silica gel 60F254 plates (E. Merck;
5 ꢀ 10 and 5 ꢀ 20cm). The plates were scanned using a Bioscan System
200 Imaging Scanner (Bioscan, Inc., WA, USA). Specific activities were deter-
mined by the weighing and counting method utilizing an external standard
with a liquid scintillation counter.
Preparation of succinoylated CPG (5)
In a 500-mL pressure reactor with a Teflon screw cap stopper (with a
Chemraz O-ring), aminopropyl CPG (150 g) was placed followed by a
solution of succinic anhydride (60 g) and 4-dimethylaminopyridine
(DMAP; 7.2 g) in N,N-dimethylformamide (300 mL). Additional DMF
(150 mL) was added to facilitate the mixing of the slurry, and the con-
tents were heated in a microwave oven for eight cycles each of 30-
second duration. Caution! The reaction is exothermic and adequate
safety precautions should be taken in performing this step. The
resulting dark-colored reaction mixture was mixed well by shaking and
cooling between the heating cycles. The completion of succinoylation
was ascertained by taking a small aliquot of CPG and heating with a solu-
tion of ninhydrin in EtOH. Absence of purple color signified the comple-
tion of reaction, following which the colored slurry was filtered, and the
solid was washed with DCM, MeOH, and hexanes (2 ꢀ 200 mL each)
and dried to obtain succinoylated CPG (5).
35S-Labeled thiobenzoic acid (35S-2)
Unlabeled thiobenzoic acid (2; 2.0 mL, 1.0 mmol, 90%), 35S-elemental sul-
fur (0.95 mL, 95 mCi, 1 Ci/mg, Perkin Elmer Lot #04069), and toluene
(7 mL) were placed in a 10-mL flask. The mixture was heated for 22 hours
at 97ꢁC. The resulting yellow mixture was cooled at room temperature
(RT) and concentrated to dryness under a stream of argon. The residue
was used in the next step without further purification.
35S-Labeled 3H-1,2-benzodithiol-3-one (3)
Crude 35S-2 was cooled in an ice bath following in which thiosalicylic acid
(75.1 mg, 490 mmol) and concentrated sulfuric acid (1 mL) were added.
The reaction mixture was heated for 20 hours at 50ꢁC. The resulting dark
brown mixture was cooled in a dry ice/acetone bath, and ice water
(20 mL) and DCM (20 mL) were added. Additional water (20 mL) was
added and the resulting mixture was extracted with DCM (3 ꢀ 20 mL).
The organic extracts were combined and washed with 5% aqueous
Na2CO3 (3 ꢀ 25 mL). The organic phase (~75 mL) was passed over a
column anhydrous MgSO4, and concentrated to give of a dark yellow
solid (97 mg). The residue was redissolved in warm hexane, and the
hexane solution was passed again through anhydrous MgSO4, filtered,
and the filtrate was concentrated to give 35S-3H-1,2-benzodithiol-3-one
(3) as a pale-yellow solid (86 mg, 50 % chemical yield based on 1, total
activity 66.63 mCi, specific activity 130 mCi/mmole).
Loading of nucleoside on succinoylated CPG using LOTUS reactorW:
Loading of DMT-NBz-dA
For nucleoside loading of the support,10,11 succinoylated CPG (5; 100 g,
103 mmol/g amino loading), DMT-NBz-dA (6; 19.7 g, 3 equiv., 30 mmol),
and DMAP (3.66 g, 3 equiv., 30 mmol) were added to the LOTUS
reactorW. Anhydrous DMF (400 mL, freshly distilled from CaH2) was intro-
duced and contents were mixed using an orbital shaker. Then, anhydrous
Et3N (4.2 mL, 30 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodimide
hydrochloride (EDC; 5.76 g, 3 equiv., 30 mmol) were added sequentially,
and the reaction mixture was mixed under orbital shaking and recycling
for 8 hours. Orbital shaking was continued for 8 hours. Periodically, aliquots
of recycling liquid were withdrawn and trityl analysis was carried out to
determine the amount of 6 consumed. If needed, additional amount of
EDC (2.95 g), DMAP (3.7 g), and Et3N (5 mL) were added, and shaking was
continued. The contents of the reactor were filtered under vacuum, and
the filtrate was collected to recover the excess unreacted nucleoside. The
loaded support was washed twice with MeOH, DCM, and hexanes
(2 ꢀ 400 mL each), and the support was dried thoroughly under vacuum in
the reactor. A sample of solid support isolated showed a typical nucleoside
loading of 75–80 mmol/g by trityl analysis.
35S-Labeled 3H-1,2-benzodithiol-3-one-1,1-dioxide (35S-3H-BD, 4)
35S-3H-1,2-benzodithiol-3-one (3; 86 mg, 0.5 mmol, 66 mCi). The reaction
flask was immersed in an ice water bath, and trifluoroacetic acid
(0.6 mL) and hydrogen peroxide (0.3 mL, 30%) were added. The reaction
mixture was slowly warmed to 40ꢁC and maintained at this temperature
for 4 hours. The reaction was monitored by thin layer chromatography
(SiO2, chloroform, I2 visualization, 3: Rf = 0.9, mono-oxide intermediate:
Rf = 0.6, sulfone 4: Rf = 0.8). The mixture was cooled in an ice bath, and
the reaction was quenched by the addition of ice water (5 mL), producing
a white precipitate. The mixture was filtered, and the precipitate was
washed with cold water (3 ꢀ 6 mL). The solid was dried under vacuum
overnight at RT to give 35S-3H-BD (4; 20 mg, radiochemical yield 20.1%,
total activity 13.3 mCi, specific activity 132 mCi/mmol). The compound
was stored at ꢂ20ꢁC until used.
The nucleoside-loaded CPG was capped with CAP A and CAP B
mixtures (325 mL each) for 3 hours under orbital shaking, and the
capped support was filtered, washed with methanol, DCM, and finally
with hexanes (2 ꢀ 300 mL each). The loading of dried support was
typically around 70–80 mmol/g. The nucleoside-loaded support was
stored at 4ꢁC.
Preparation of CPG-bound dinucleoside phosphite triester (9)
The reaction was carried out in LOTUS reactorW.11 Nucleoside-loaded
support (7; 112 g, 89 mmol) was placed in the reactor and detritylated
using DCA in DCM (2.5%, DCA, 3 ꢀ 400 mL) with DCM washes
Solid-phase synthesis
The requisite CPG-bound dinucleoside phosphite triester 6 was synthesized
on a multimillimol scale using NBz-dA-loaded CPG support in conjunction with (3 ꢀ 400 mL) between each DCA/DCM treatment. The support was subse-
solid-phase phosphoramidite chemistry.9 For the synthesis, we employed a
quently washed thoroughly with DCM (5 ꢀ 400 mL) and then acetonitrile
specially fabricated LOTUS reactorW as previously described.11 The NBz-dA- (low water, <30 ppm). Detritylated nucleoside was coupled with
loaded CPG support was prepared through microwave-assisted amina- 2’-O-methyluridine phosphoramidite (8; 5 equiv.) in the presence of
tion and succinoylation of native CPG (500 Å) as summarized below.
5-ethylthiotetrazole (0.4 M, 10 equiv.) in anhydrous acetonitrile. After
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd. www.jlcr.org

S.-W. Rhee et al.
the removal of excess phosphoramidite by acetonitrile washings, mixture was maintained at RT in darkness. Radio-HPLC analysis after
the support was dried in the reactor at RT under a stream of argon.
20 hours showed unreacted 11. Additional quantities of 12 (80 mg,
Support-bound 9 was removed from the reactor and stored in an 0.326 mmol) were added. After an additional 40 hours at RT, 11 was not
argon-filled bag and stored at ꢂ20ꢁC until ready for use in the sulfurization observed by analytical radio-HPLC. Isopropanol was removed in vacuo,
step during radiolabeled synthesis.
and the remaining aqueous solution (10 mL) was extracted with hexane
(3 ꢀ 10 mL) to remove excess 12. Without further concentration, the
aqueous layer was passed through a reversed-phase column (Sep-Pak
Vac 35cc, C18 cartridge, eluent: water ! acetonitrile gradient). Fractions
containing [RP,SP]-35S-1 (monitored by radio-HPLC) were lyophilized to
give the target compound as an amorphous solid (16.5 mg, total activity
2.27 mCi, specific activity 96.4 mCi/mmol).
A second run was carried out concurrently on the same scale (from
95 mCi 35S8) to afford additional [RP,SP]-35S-1 (17.7 mg, total activity
2.14 mCi, specific activity 84.9 mCi/mmol). The [RP,SP]-diastereomeric ratio
was typically ~60:40 (ascertained by 31P-NMR) when identical conditions
were followed for non-radioactive synthesis of 1 from 9.4
CPG-bound 35S-labeled dinucleoside O-(2-cyanoethyl)
phosphorothioate (10)
To a silanized7 50-mL flask, 35S-3H-BD (4; 20 mg, 0.1 mmol, 13.3 mCi) in
acetonitrile (10 mL) and CPG-bound dinucleoside phosphite triester 9
(904 mg, 0.9 mmol) were added. The reaction mixture was maintained
at RT for 18 hours. The slightly reddish mixture was filtered through a
sintered glass funnel (medium, 15 mL). The solid was washed with
acetonitrile (3 ꢀ 10 mL) and dried in vacuo (~10 torr).
35S-labeled dinucleoside phosphorothioate (11)
The support-bound dinucleotide 10 was treated with a solution of DCA in
DCM (15 mL, 2.5% v/v). The resulting bright orange mixture was stirred
for 5 minutes and then centrifuged for 5 minutes. The orange superna-
tant was removed carefully by pipette, and the solid was washed with
DCM (10 mL). This process (DCA treatment, centrifugation, separation,
and washing) was repeated three times until no orange mixture
appeared. The solid was then dried in vacuo to afford the support-bound
dimethoxytrityl deprotected compound (679 mg). It was subsequently
treated with NH4OH (3 mL, 28–30%) and maintained at RT for 22 hours.
The reaction mixture was cooled on an ice bath and neutralized with
careful addition of glacial AcOH (0.5 mL) to pH 6.5–7.0. The mixture was
filtered through a glass sintered funnel (medium, 15 mL), and the solid
was washed with water (3 ꢀ 10 mL). The filtrate and washings (~30 mL)
were combined, and neutral impurities were removed by extraction with
ethyl acetate (3 ꢀ 15 mL). The aqueous layer was lyophilized to give a vis-
cous residue (428mg), which was desalted by reversed-phase chromatogra-
phy (Sep-Pak Vac 35 cc/10 g C18 cartridge, eluent: water! 50% acetonitrile/
water gradient). Fractions containing compound 11 (monitored by
radio-HPLC) were lyophilized to afford 11 as a transparent residue (32mg,
total activity 2.4 mCi).
Acknowledgements
Financial support from the DMID (Contract N01-AI-60011, J.
Mirsalis, H. J. Parish, PIs) and the National Institutes of Health
under a Research Project Cooperative Agreement Grant Award
(UO1 AI058270, RPI, PI) is gratefully acknowledged.
Supporting Information
Radio-HPLC traces of 35S-1 are provided.
Conflict of Interest
The authors did not report any conflict of interest.
References
[1] Hepatitis B. Fact sheet WHO/204. Geneva: World Health Organization;
Oct, 2000. http://www.who.int/inf-fs/en/fact204.htm
[2] R. P. Iyer, Y. Jin, A. Roland, J. D. Morrey, S. Mounir, B. Korba, Antimicrob.
Agents Chemother. 2004, 48, 2199.
[3] R. P. Iyer, A. Roland, Y. Jin, S. Mounir, B. Korba, J. G. Julander,
J. D. Morrey, Antimicrob. Agents Chemother. 2004, 48, 2318.
[4] J. E. Coughlin, S. Padmanabhan, G. Zhang, C. J. Kirk, C. P. Govardhan,
B. E. Korba, K. O’Loughlin, C. E. Green, J. Mirsalis, J. D. Morrey,
R. P. Iyer, Bioorg. Med. Chem. Lett. 2010, 20, 1783.
Iodomethyl isopropyl carbonate (12)
To a solution of NaI (25 g, 166 mmol) in acetonitrile (40 mL) at RT, a solu-
tion of chloromethyl isopropyl carbonate (12.8 g, 84 mmol) in acetonitrile
(15 mL) was added drop wise. The reaction vessel was covered with
aluminum foil, and the mixture was maintained at RT. After 4 days, the
solid was removed using filtration and washed with DCM (2 ꢀ 50 mL).
The combined filtrates were concentrated. The residue was taken up in
water (50 mL) and extracted with DCM (3 ꢀ 30 mL). The combined DCM
extracts were washed with 5% sodium bisulfite (100 mL) and brine
(100 mL), successively. The organic phase was dried over Na2SO4, filtered,
[5] R. P. Iyer, et al. Manuscript in preparation.
[6] W. J. Stec, G. Zon, W. Egan, B. Stec, J. Am. Chem. Soc. 1984, 106, 6077.
[7] R. P. Iyer, L. R. Phillips, W. Egan, J. B. Regan, S. L. Beaucage, J. Org.
Chem. 1990, 55, 4693.
[8] R. P. Iyer, W. Tan, D. Yu, S. Agrawal, Tetrahedron Lett. 1994, 35, 9521.
[9] S. L. Beaucage, R. P. Iyer, Tetrahedron 1992, 48, 2223.
[10] S. Padmanabhan, J. E. Coughlin, R. P. Iyer, Tetrahedron Lett.
2005, 46, 343.
1
and concentrated to give 12 as a pale-yellow oil (14.63 g, 71%): H NMR
(300 MHz, CDCl3) d 1.2 (6H, d), 4.8 (1H, m), 5.8 (2H, s); 13C NMR (75 MHz,
CDCl3) d 22, 36, 76, 154; MS (ESI+) m/z 244.
35S-1. In a 50-mL flask covered with aluminum foil, 11 (32 mg,
0.0525 mmol, 2.4 mCi) in 4:1 water/isopropanol (5 mL) and iodomethyl
isopropyl carbonate (12; 40 mg, 0.163 mmol) was placed. The reaction
[11] R. P. Iyer, J. E. Coughlin, S. Padmanabhan, Org. Prep. Proced. Int.
2005, 37, 205.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012