Phosphoramidate protides of carbocyclic 2′,3′-dideoxy-2′, 3′-didehydro-7-deazaadenosine with potent activity against HIV and HBV

Article abstract of DOI:10.1081/NCN-200040678

Synthesis of phosphoramidate protides of carbocyclic D- and L-2′,3′-dideoxy-2′,3′-didehydro-7-deazaadenosine by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed s

Full text of DOI:10.1081/NCN-200040678

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Phosphoramidate Protides of Carbocyclic
2′,3′‐Dideoxy‐2′,3′‐Didehydro‐7‐Deazaadenosine with
Potent Activity Against HIV and HBV
Kristjan S. Gudmundsson a , Zhicheng Wang a , Susan M. Daluge a , Lance C. Johnson b ,
Richard Hazen b , Lynn D. Condreay b & Christopher McGuigan c
a Division of Chemistry , GlaxoSmithKline , 5 Moore Drive, Research Triangle Park, North
Carolina, 27709, USA
b Division of Virology , GlaxoSmithKline , 5 Moore Drive, Research Triangle Park, North
Carolina, 27709, USA
c Welsh School of Pharmacy , Cardiff University , Cardiff, UK
Published online: 17 Aug 2006.
To cite this article: Kristjan S. Gudmundsson , Zhicheng Wang , Susan M. Daluge , Lance C. Johnson ,
Richard Hazen , Lynn D. Condreay & Christopher McGuigan (2004) Phosphoramidate Protides of Carbocyclic
2′,3′‐Dideoxy‐2′,3′‐Didehydro‐7‐Deazaadenosine with Potent Activity Against HIV and HBV, Nucleosides, Nucleotides and
Nucleic Acids, 23:12, 1929-1937, DOI: 10.1081/NCN-200040678
To link to this article: http://dx.doi.org/10.1081/NCN-200040678
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 12, pp. 1929–1937, 2004
Phosphoramidate Protides of Carbocyclic
2’,3’-Dideoxy-2’,3’-Didehydro-7-Deazaadenosine
with Potent Activity Against HIV and HBV
1
1
Kristjan S. Gudmundsson,1, Zhicheng Wang, Susan M. Daluge,
Lance C. Johnson,2 Richard Hazen,2 Lynn D. Condreay,2
and Christopher McGuigan3
*
2
1Division of Chemistry and Division of Virology, GlaxoSmithKline,
Research Triangle Park, North Carolina, USA
3Welsh School of Pharmacy, Cardiff University, Cardiff, UK
ABSTRACT
Synthesis of phosphoramidate protides of carbocyclic D- and L-2’,3’-dideoxy-2’,3’-
didehydro-7-deazaadenosine by treatment of the nucleoside with phosphorochlo-
ridates in the presence of pyridine and t-BuMgCl is described. Several of these
protides showed significantly improved antiviral potency over the parent nucleosides
against both HIV and HBV.
Key Words: Carbocyclic 2’3’-dideoxy-2’3’-didehydro-7-deazaadenosine; Phosphor-
amidate protides; HIV; HBV.
*
Correspondence: Kristjan S. Gudmundsson, Division of Chemistry, GlaxoSmithKline, 5 Moore
Drive, Research Triangle Park, NC 27709, USA; Fax: (919) 483-6053; E-mail: Kristjan.
S.Gudmundsson@gsk.com.
1929
DOI: 10.1081/NCN-200040678
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

1930
Gudmundsson et al.
INTRODUCTION
2’,3’-Dideoxyribofuranonucleosides have received considerable attention as this
class of nucleosides includes several compounds with potent anti-HIV activity (e.g.
ddC, ddI and analogs).[1–3] Besides the synthesis of the natural purine and pyrimidine
analogs, several heterocycle-modified 2’,3’-dideoxyribofuranonucleosides, such as
pyrrolo[2,3-d]pyrimidines (7-deazaadenine) have been synthesized.[4–6] Several of
these have demonstrated antiviral activity. The preclinical research leading to the
selection of the carbocyclic nucleoside abacavir (Ziagen) involved the synthesis and
evaluation of several racemic carbocyclic analogs, including carbocyclic 2’,3’-dideoxy-
2’,3’-didehydro-7-deazaadenosine. As the racemic carbocyclic 2’,3’-dideoxy-2’,3’-
didehydro-7-deazaadenosine showed modest HBV activity, we became interested in
the synthesis of prodrug derivatives that could deliver its monophosphate intracellu-
larly. Several prodrug strategies have been described in the literature, these include
phosphoramidate derivatives,[7–14] cycloSal derivatives[15] and bis(SATE)phospho-
triester derivatives.[16,17] All of these have been shown to significantly improve the
potency in vitro of a variety of nucleosides against HIV and/or HBV.
Herein we describe the synthesis of several phosphoramidate prodrugs of
carbocyclic 2’,3’-dideoxy-2’,3’-didehydro-7-deazaadenosine along with their anti-HIV
and HBV activity.
RESULTS AND DISCUSSION
Several different prodrug strategies have been designed to deliver monophosphates
intracellularly. We decided to use the phosphoramidates as this approach is well
validated and we have demonstrated that it improves the HBV activity of 7-(2’,3’-
dideoxy-3’-fluoro-b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-4-amine.[18] An addition-
al advantage of the phosphoramidates is that the nucleoside can be converted to the
phosphoramidate prodrug in one chemical step. The D- and L-nucleosides (7) were
Scheme 1. Synthesis of carbocyclic L-2’,3’-dideoxy-2’,3’-didehydro-7-deazaadenosine (L-7,
D-analogs were synthesized in a similar fashion).

2’,3’-dideoxy-2’,3’-didehydro-7-deazaadenosine
1931
Scheme 2. Synthesis of phosphoramidates.
synthesized as outlined in Scheme 1 using a synthetic strategy similar to the one
previously described by Legraverend et al.[19,20] and Montgomery et al.[21]
We chose to synthesize four different phosphoramidate derivatives, the analogs of
which had previously been reported to give good improvement in activity against HIV
and HBV for other nucleosides.[7–14] Treatment of 2’,3’-dideoxy-2’,3’-didehydro-7-
deazaadenosine with the phosphorochloridate reagents[7–14] in pyridine in the presence
of t-BuMgCl (as outlined in Scheme 2) gave good yields of protides 8–11.
The activity of the protide derivatives 8–11 along with the activity of the parent
nucleosides (L-7 and D-7) against HIV and HBV is shown in Tables 1 and
2, respectively.
Phosphoramidate delivery improves the anti-HIV activity of the L-nucleoside
significantly, up to 3000 fold for the L-alanine methyl ester derivative L-8. Activity of
the D-nucleoside against HIV was not improved by protide delivery. Significant
Table 1. Anti-HIV-1 (3B) activity and cytotoxicity in MT-4 lymphocytes[22] of carbocyclic 2’,
3’-dideoxy-2’,3’-didehydro-7-deazaadenosine derivatives and their corresponding phosphoramidates.
Compound numbera
IC50 (mM)b
CC50 (mM)b
Selectivity indexb
L-7 (nucleoside)
L-8
280
>500
26
>2
0.091
0.175
0.3
>285
>57
>33
105
–
L-9
L-10
>10
>10
31.5
>200
>25
78
L-11
D-7 (nucleoside)
0.3
>200
>10
>78
>490
29
D-8
D-9
ꢀ1
ꢀ1
ꢀ1
ꢀ1
D-10
D-11
490
42
aAll protides were tested as 1:1 mixtures of diastereomers at phosphorus.
bIC50 is 50% inhibitory concentration, CC50 is 50% cytotoxic concentration, selectivity index is
CC50/IC50, results are averages of two or more experiments, each in duplicate.

1932
Gudmundsson et al.
Table 2. Anti-HBV activity and cytotoxicity in HepG2-2.2.15 cells[23] of carbocyclic 2’,
3’-dideoxy-2’,3’-didehydro-7-deazaadenosine derivatives and the corresponding phosphoramidates.
Compound numbera
IC50 (mM)b
CC50 (mM)b
Selectivity indexb
L-7 (nucleoside)
L-8
2
50
25
0.09
0.1
0.05
0.055
>200
54
230
>11
>2
>2500
>110
>40
>650
–
L-9
L-10
L-11
D-7 (nucleoside)
>36
>200
81
D-8
D-9
ꢀ1
11
73
18
64
ꢀ1
D-10
D-11
ꢀ1
20
51
ꢀ2
aAll protides were tested as 1:1 mixtures of diastereomers at phosphorus.
bIC50 is 50% inhibitory concentration, CC50 is 50% cytotoxic concentration, selectivity index is
CC50/IC50, results are averages of two or more experiments, each in duplicate.
increases in HIV potency (above 3 orders of magnitude) for the L-isomers suggest that
formation of the monophosphate is limiting for carbocyclic L-2’,3’-dideoxy-2’,3’-
didehydro-7-deazaadenosine.
Similarly, the activity of the L-enantiomer (L-7) against HBV was significantly
improved by protide delivery. Thus, all four protides improved activity against HBV
about 20-fold. Again the D-isomers proved much less active than the correspond-
ing L-isomers.
The improvements in potency are significantly greater for HIV than for HBV.
The SAR for the protide enhancement in activity with variation of the amino acid and
ester appear to differ between the HIV (L-alanine methyl ester phosphoramidate gave
the best potency) and HBV assay (dimethylglycine methyl ester phosphoramidate gave
the best potency), suggesting that different anabolic pathways predominate in liver
versus lymphocytes and that the optimal protide structure will vary with the target
cell type.
In conclusion, protides of L-2’,3’-dideoxy-2’,3’-7-deazaadenosine significantly
improved activity against HIV-1 (about 3 orders of magnitude) and against HBV
(about 1–2 orders of magnitude). This was not the case for the corresponding D-analogs.
We continue to investigate improvement in activity of nucleosides by protide delivery.
EXPERIMENTAL
Chemistry
Nuclear magnetic resonance (NMR) spectra were obtained at 300 MHz on Varian
Unity Plus NMR spectrophotometer. The chemical shifts are reported in parts per
million (ppm) relative to tetramethylsilane as an internal standard. Elemental analysis

2’,3’-dideoxy-2’,3’-didehydro-7-deazaadenosine
1933
were performed by Atlantic Microlab Inc. Flash column chromatography was
performed using Merck Silica gel 60 (230–400 mesh), and the stated solvent system
under pressure. Mass spectra were obtained on Micromass Platform mass spectrometers
from Micromass Ltd. Altrincham, UK, using Electrospray Ionization.
(+)-(1R,4S)-4-[4-Chloro-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-
methanol (L-6): (+)-(1S, 4R)-4-amino-2-cyclopentene-1-methanol hydrochloride (4,
3.96 mmol), triethylamine (1.20 g, 11.8 mmol), and 4,6-dichloro-5-(2,2-diethoxy-
ethyl)pyrimidine (3, 1.05 g, 3.96 mmol) were refluxed in absolute ethanol (5 mL) for
4 hours. The solution was cooled, 1 N NaOH (8 mL) added, and volatiles removed
under reduced pressure. The residue was chromatographed on silica gel with 10%
methanol-chloroform. Product-containing fractions (L-5) were combined and concen-
trated to an oil. This oil (L-5) was dissolved in dioxane (10 mL)—1 N hydrochloric
acid (3 mL) and the solution stirred at ambient temperature for 2 days, neutralized with
ammonium hydroxide, and volatiles evaporated under reduced pressure. The residue
was chromatographed on silica gel and title compound (L-6) eluted with 5–10%
1
methanol-chloroform as a light tan oil (0.60 g, 60%); H-NMR (DMSO-d6) d: 8.67 (s, 1
H), 7.70 and 6.69 ( two d, J = 3.65 Hz, 1 H each), 6.10 (m, 1 H), 6.0–5.8 (m, 2 H),
4.77 (m, 1 H), 3.5 (m, 2 H), 2.9 (m, 1 H), 2.7 (m, 1 H), 1.6 (m, 1 H); MS (API+):
252(35), 250(100), 156(24), 154(73). [a] 589+ 43.6° (c 0.0045, methanol).
(+)-(1R,4S)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-
methanol (L-7): (+)-4-[4-Chloro-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-
methanol (L-6, 600 mg, 2.36 mmol) was heated in liquid ammonia (50 mL) in a Parr
bomb at 65°C for 3 days. Volatiles were evaporated with 1 N sodium hydroxide (2.3
mL) and the residue chromatographed on silica gel. Title compound was eluted with
10% methanol-chloroform as a colorless oil. Evaporation of an acetonitrile solution
gave title compound (L-7) as white powder which was washed with hexanes (291 mg,
1
52%); m.p. 165–168°C; H-NMR (DMSO-d6) d: 8.07 (s, 1H), 7.06 and 6.56 ( two d,
J = 3.5 Hz, 1 H each), 7.0 (br s, 2 H), 6.10 (m, 1 H), 5.80 (m, 2 H), 4.75 (m, 1 H), 3.45
(m, 2 H), 2.85 (m, 1 H), 2.6 (m, 1 H), 1.45 (m, 1 H) ; MS (CI): M + 1, 231(100),
163(37), 135(45). Anal. Calcd. for C12H14N4O Â 1/2 H2O: C, 60.24; H, 6.32; N, 23.41.
Found: C, 60.54; H, 5.96; N, 23.73. [a]
+ 42.9° (c 0.0028, methanol).
589
(1R,4S)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (methoxy L-alaninyl)]phosphoramidate (L-8): To (+)-(1R, 4S)-4-[4-
amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (L-7, 0.19 g, 0.8
mmoles) was added anhydrous pyridine (4 mL) and anhydrous tetrahydrofuran (3 mL).
Subsequently, tert-butylmagnesiumchloride (0.9 mL, 1 M solution in tetrahydrofuran)
was added and the reaction stirred under nitrogen for 10 minutes at room temperature.
A solution of phenyl methoxy-L-alaninyl phosphochloridate (1.2 mmol in anhydrous
tetrahydrofuran) was added and the reaction stirred at room temperature for 12 hours.
The resulting mixture was concentrated to a syrup under reduced pressure. This syrup
was dissolved in dichloromethane (60 mL) and the organic phase washed with water
(2 Â 30 mL), dried (magnesium sulfate), filtered and concentrated to a foam. This
foam was purified by flash column chromatography on silica gel. The title compound
(L-8) was eluted with 5 % methanol in chloroform to give, after removal of volatiles,

1934
Gudmundsson et al.
1
290 mg (77%) of a white foam: H-NMR (CDCl3) d: 8.23 (s, 1 H), 7.22 (m, 2 H), 7.10
(m, 3 H), 6.88 (2 Â d, 1 H), 6.27 (d, 1 H), 5.98 (m, 1 H), 5.87 (m, 1 H), 5.80 (m, 1 H),
5.35 (bs, 2 H), 3.85–4.10 (m, 4 H), 3.61 (s, 3 H), 3.06 (m, 1 H), 2.7 (m, 1 H), 1.50 (m,
1 H), 1.28 (m, 3 H); 31P-NMR (CDCl3): 2.99 and 2.80; MS (EI): M + 23 (Na), 494.
Anal. Calcd. for C22H26N5O4P Â 3/2 H2O: C, 54.77; H, 6.06; N, 14.52; Found: C,
54.32; H, 5.79; N, 14.22.
(1R,4S)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (benzyloxy L-alaninyl)]phosphoramidate (L-9). (+)-(1R, 4S)-4-[4-
Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (L-7, 0.20 g, 0.9
mmoles) was treated with phenyl benzyloxy-L-alaninyl phosphochloridate (2.5 mL of 1
M solution in tetrahydrofuran, 2.5 mmol) as described above to give after silica gel
chromatography with 5% methanol in chloroform 330 mg (69%) of the title compound
1
L-9 as a white foam: H-NMR (CDCl3) d:8.34 (s, 1 H), 6.9–7.4 (m, 10 H), 6.95(2 Â d,
1 H), 6.35 (m, 1 H), 5.8 –6.1 (3 Â m, 3 H), 5.45 (broad s, 2 H), 5.13 (m, 2 H), 4.0–4.2
(m, 4 H), 3.1 (m, 1 H), 2.75 (m, 1 H), 1.58 (m, 1 H), 1.4 (m, 3 H); 31P-NMR
(CDCl3):3.2 and 3.0; MS (EI): M + 1, 548; Anal. Calcd. for C28H30N5O5P Â 1/3 H2O:
C, 60.75; H, 5.58; N, 12.65; Found: C, 60.93; H, 5.65; N, 12.58.
(1R,4S)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (methoxy L-dimethylglycinyl)]phosphoramidate (L-10): (+)-(1R,
4S)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (L-7,
0.20 g, 0.9 mmoles) was treated with phenyl methoxy-L-dimethylglycinyl phospho-
chloridate (2.5 mL of 1 M solution in tetrahydrofuran, 2.5 mmoles) as described for
preparation of L-8 to give, after silica gel chromatography with 5% methanol in
chloroform, 310 mg (73%) of the title compound L-10 as a white foam: 1H-NMR
(CDCl3) d:8.35 (s, 1 H), 7.1–7.4 (m, 5 H), 6.97 (2 Â d, 1 H), 6.38 (m, 1 H), 6.08 (m,
1 H), 5.97 (m, 1 H), 5.90 (m, 1 H), 5.40 (broad s, 2 H), 4.00–4.20 (m, 3 H), 3.73 (s,
3 H), 3.15 (m, 1 H), 2.8 (m, 1 H), 2.25 (s, 1 H), 1.55 (m, 6 H); 31P-NMR (CDCl3):1.73;
MS (EI): M + 1, 486; Anal. Calcd. for C23H28N5O5P Â 1/2 H2O: C, 55.87; H, 5.91; N,
14.16; Found: C, 55.93; H, 5.91; N, 14.16.
(1R,4S)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (methoxy L-phenylalaninyl)]phosphoramidate (L-11): (+)-(1R, 4S)-
4-[4-amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (L-7, 0.20 g,
0.9 mmoles) was treated with phenyl methoxy-L-phenylalaninyl phosphochloridate (2.5
mL of 1 M solution in tetrahydrofuran, 2.5 mmoles) as described above to give after
silica gel chromatography with 5% methanol in chloroform 320 mg (67%) of the title
compound L-11 as a white foam: 1H-NMR (CDCl3) d: 8.22 (s, 1 H), 6.9–7.3 (m, 10 H),
6.85(m, 1 H), 6.25 (2 Â d, 1 H), 5.7–6.0 (m, 3 H), 5.35 (broad s, 2 H), 4.15 (m, 1 H),
3.55–4.0 (m, 2 H), 3.55 (2 Â s, 3 H), 2.6–2.9 (m, 5 H), 1.4 (m, 1 H); 31P-NMR
(CDCl3):3.0; MS (EI): M + 1, 548; Anal. Calcd. for C28H30N5O5P Â 0.7 MeOH: C,
60.48; H, 5.80; N, 12.29; Found: C, 60.49; H, 5.51; N, 11.98.
(À)-(1S,4R)-4-[4-Chloro-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-
methanol (D-6): Spectral data identical to L-6; [a]589À44° (c 0.05, methanol).

2’,3’-dideoxy-2’,3’-didehydro-7-deazaadenosine
1935
(-)-(1S,4R)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-
methanol (D-7): (À)-4-[4-Chloro-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-
methanol (D-6, 4 g, 16 mmol) was treated as described above for L-7 to give 2.5 g,
68% of D-7 as a white solid: Spectral data identical to L-7. Anal. Calcd. for
C12H14N4O.: C, 62.59; H, 6.13; N, 24.33. Found: C, 62.70; H, 6.25; N, 24.52. [a]589
43.2° (c 0.004, methanol).
À
(1S,4R)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (methoxy L-alaninyl)]phosphoramidate (D-8): To (À)-(1R, 4S)-4-
[4-amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (D-7, 0.5 g,
2.2 mmoles) was added anhydrous pyridine (2 mL) and anhydrous tetrahydrofuran
(10 mL). Subsequently, tert-butylmagnesiumchloride (2.8 mL, 1 M solution in tetra-
hydrofuran) was added and the reaction stirred under nitrogen for 10 minutes at room
temperature. A solution of phenyl methoxy-L-alaninyl phosphochloridate (4.3 mmol in
anhydrous tetrahydrofuran) was added and the reaction stirred at room temperature for
12 hours. Workup as described above gave 450 mg (44%) of D-8 as a white foam:
Spetral data identical to L-8. Anal. Calcd. for C22H26N5O4P Â 3/2 H2O: C, 54.77; H,
6.06; N, 14.52; Found: C, 54.62; H, 5.99; N, 14.13.
(1S,4R)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (benzyloxy L-alaninyl)]phosphoramidate (D-9): (À)-(1R, 4S)-4-[4-
Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (D-7, 0.28 g, 1.2
mmoles) was treated with phenyl benzyloxy-L-alaninyl phosphochloridate (1 M
solution in tetrahydrofuran, 2.43 mmol) as described above to give after silica gel
chromatography with 5% methanol in chloroform 539 mg (81%) of D-9 as a white
foam: Spectral data identical to L-9. Anal. Calcd. for C28H30N5O5P Â H2O: C, 59.46;
H, 5.70; N, 12.38; Found: C, 59.18; H, 5.41; N, 12.37.
(1S,4R)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (methoxy L-dimethylglycinyl)]phosphoramidate (D-10): (À)-(1R,
4S)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (D-7,
0.28 g, 1.2 mmoles) was treated with phenyl methoxy-L-dimethylglycinyl phospho-
chloridate (1 M solution in tetrahydrofuran, 2.43 mmoles) as described above to give,
after silica gel chromatography with 5% methanol in chloroform, 400 mg of D-10 as a
white foam: Spectral data identical to L-10. Anal. Calcd. for C23H28N5O5P Â 1/2 H2O:
C, 55.87; H, 5.91; N, 14.16; Found: C, 55.45; H, 5.75; N, 14.39.
(1S,4R)-4-[4-Amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-metha-
nol-O-[phenyl (methoxy L-phenylalaninyl)]phosphoramidate (D-11): (À)-(1R, 4S)-
4-[4-amino-7H-pyrrolo(2,3-d)pyrimidin-7-yl]-2-cyclopentene-1-methanol (D-7, 0.41 g,
1.8 mmoles) was treated with phenyl methoxy-L-phenylalaninyl phosphochloridate (1 M
solution in tetrahydrofuran, 3.58 mmoles) as described above to give after silica gel
chromatography with 5% methanol in chloroform 350 mg (36%) of D-11 as a white
foam: Spectral data identical to L-11. Anal. Calcd. for C28H30N5O5P Â H2O C, 59.46;
H, 5.70; N, 12.38; Found: C, 59.42; H, 5.36; N, 12.09.

1936
Gudmundsson et al.
ANTIVIRAL ASSAYS
HBV
Antiviral potency and growth inhibition potential of compounds was determined
using the assay developed by Jansen et al.a Briefly, HepG2-2.2.15 cells constitutively
producing HBV[24] were seeded into 96 well microtiter plates at a density of 5 Â 103
per well and growth medium containing drug was replaced every other day for 9 days.
Supernatants were then collected and analyzed for HBV content. Samples were tested
in conjunction with both positive (0.448 fg/ul plasmid DNA) and negative (RPMI
medium supplemented with 2mM L-glutamine and 10% fetal calf serum) controls.
Data was normalized to non-drug treated cells, and expressed as a percent of control
for analysis.
Evaluation of toxicity (i.e. growth inhibition) was made by fixing monolayers with
70% ethanol, and staining with bisbenzimide H33342 for 1 hour at 37°C. Fluorescence
values of drug treated cells were compared to non-drug treated cells and expressed as a
percentage of control. HBV detection (and hence efficacy determination) was
performed by ‘‘capturing’’ virus from supernatants on anti-HBsAg coated plates,
washing, denaturing to release HBV DNA, performing PCR with biotinylated primers,
streptavidin capture of biotinylated PCR products with concomitant probe hybridiza-
tion, addition of substrate, and reading optical densities of the colorimetric reac-
tion. Dilutions of a standardized HBV-containing supernatant were included on every
plate, and HBV DNA concentrations of test wells were calculated from this HBV
standard curve.
HIV
Anti-HIV-1 (3B) activity and cytotoxicity in MT-4 lymphocytes was determined as
previously described by Averett et al.b
REFERENCES
1. Herdewijn, P.; Balzarini, J.; DeClercq, E.; Pauwels, R.; Baba, M.; Broder, S.;
Vanderhaeghe, H. J. Med. Chem. 1987, 30, 1270–1278.
2. Huryn, D.M.; Okabe, M. Chem. Rev. 1992, 92, 1745–1768.
3. Mansour, T.S.; Storer, R. Curr. Pharm. Des. 1997, 3, 227–264.
4. Seela, F.; Rosemeyer, H. Helv. Chim. Acta 1989, 72, 1084–1089.
5. Krawczyk, S.H.; Townsend, L.B. Nucleosides Nucleotides 1989, 8, 97–115.
6. Pauwels, R.; Baba, M.; Balzarinin, J.; Herdewijn, P.; Desmyter, J.; Robins, M.J.;
Zou, R.; Madej, D.; De Clercq, E. Biochem. Pharmacol. 1988, 37, 1317–1325.
aCompounds were tested for anti-HBV activity according to the method described by: From
Ref. [23].
bCompounds were assayed for HIV activity in MT-4 cells according to the method described by:
[From Ref. 22].

2’,3’-dideoxy-2’,3’-didehydro-7-deazaadenosine
1937
7. McGuigan, C.; Wedgwood, O.M.; De Clercq, E.; Balzarini, J. Bioorg. Med. Chem.
Lett. 1996, 6, 2359–2362.
8. Balzarini, J.; Kruining, J.; Wedgwood, O.; Pannecouque, C.; Aquaro, S.; Perno, C.-
F.; Naesens, L.; Witvrouw, M.; Heijtink, R.; De Clercq, E.; McGuigan, C. FEBS
Lett. 1997, 410, 324–328.
9. Siddiqui, A.Q.; McGuigan, C.; Ballatore, C.; Wedgwood, O.; De Clercq, E.;
Balzarini, J. Bioorg. Med. Chem. Lett. 1999, 9, 2555–2560.
10. McGuigan, C.; Cahard, D.; Sheeka, H.M.; De Clercq, E.; Balzarini, J.J. J. Med.
Chem. 1996, 39, 1748.
11. McGuigan, C.; Tsang, H.-W.; Sutton, P.W.; De Clercq, E.; Balzarini, J. Antivir.
Chem. Chemother. 1998, 9, 109–115.
12. McGuigan, C.; Velazquez, S.; De Clercq, E.; Balazrini, J. Antivir. Chem.
Chemother. 1997, 8, 519–527.
13. Siddiqui, A.; McGuigan, C.; Ballatore, C.; Zucotto, F.; Gilbert, I.H.; De Clercq,
E.; Balzarini, J. J. Med. Chem. 1999, 42, 4122–4128.
14. Siddiqui, A.; Ballatore, C.; McGuigan, C.; De Clercq, E.; Balzarini, J. J. Med.
Chem. 1999, 42, 393–399.
15. Meier, C.; Knispel, T.; De Clercq, E.; Balzarini, J. J. Med. Chem. 1999, 42, 1604–
1614.
16. Perigaud, C.; Gosselin, G.; Imbach, J.-L. Antivir. Ther. 1996, 1, 39–46.
17. Valette, G.; Pompon, A.; Girardet, J.-L.; Cappellacci, L.; Franchetti, P.; Grifantini,
M.; La Colla, P.; Loi, A.G.; Perigaud, C.; Gosselin, G.; Imbach, J.-L. J. Med.
Chem 1996, 39, 1981–1990.
18. Gudmundsson, K.S.; Daluge, S.M.; Johnson, L.C.; Jansen, R.; Hazen, R.;
Condreay, L.D.; McGuigan, C. Nucleosides Nucleotides Nucleic Acids 2003, 22,
1953–1963.
19. Lagraverend, M.; Aubertin, A.M.; Obert, G.; Huel, C.; Bisagni, E. Nucleosides
Nucleotides 1994, 13, 915–923.
20. Daluge, S.M.; Gudmundsson, K. Preparation of Carbocyclic Nucleoside (1R,cis)-
4-(4-Amino-7H-Pyrrolo[2,3-d]Pyrimidine-7-yl)-2-Cyclopentene-1-Methanol
Derivatives as Antivirals. WO2001042255, 2001.
21. Montgomery, J.A.; Hewson, K. J. Med. Chem. 1967, 10, 665–667.
22. Averett, D.R. J. Virol. Methods 1989, 23, 263–276.
23. Jansen, R.W.; Johnson, L.C.; Averett, D.R. Antimicrob. Agents Chemother. 1993,
37, 441–447.
24. Sells, M.A.; Chen, M.-L.; Acs, G. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 1005–
1009.
Received March 22, 2004
Accepted September 8, 2004