Synthesis and anti-hepatitis B virus activity of 9-(2-Deoxy-2-fluoro- β-L-arabinofuranosyl)purine nuleosides

Article abstract of DOI:10.1021/jm970233+

Since the discovery of 2'-fluoro-5-methyl-β-L-arabhiofurunosyluracil (L-FMAU) as a potent antiHBV and anti-EBV agent, we have studied the structure-activity relationships of 2'-deoxy2'-fluoro-β-L- arabinofuranosylpyrimidine nucleosides as anti-HBV agents.

Full text of DOI:10.1021/jm970233+

2750
J . Med. Chem. 1997, 40, 2750-2754
Syn th esis a n d An ti-Hep a titis B Vir u s Activity of
9-(2-Deoxy-2-flu or o-â-L-a r a bin ofu r a n osyl)p u r in e Nu cleosid es
Tianwei Ma,† J u-Sheng Lin,‡ M. Gary Newton,§ Yung-Chi Cheng,‡ and Chung K. Chu*,†
Department of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry, The University of Georgia,
Athens, Georgia 30602, and Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut 06510
Received April 7, 1997X
Since the discovery of 2′-fluoro-5-methyl-â-L-arabinofuranosyluracil (L-FMAU) as a potent anti-
HBV and anti-EBV agent, we have studied the structure-activity relationships of 2′-deoxy-
2′-fluoro-â-L-arabinofuranosylpyrimidine nucleosides as anti-HBV agents. Therefore it is
rational to extend this study to the purine nucleosides. Thus, 3,5-di-O-benzoyl-2-deoxy-2-fluoro-
â-L-arabinofuranosyl bromide (1), which was prepared from L-xylose via a multistep procedure,
was coupled with several purines by the sodium salt method. From this general synthesis, 10
purine nucleosides containing the 2-deoxy-2-fluoro-â-L-arabinofuranosyl moiety have been
obtained. The anti-HBV activity and toxicity of the synthesized nucleosides were evaluated
in HepG2 2.2.15 cells. Among them, the adenine (10) and hypoxanthine (15) derivatives exhibit
good in vitro anti-HBV activity (EC50 ) 1.5 and 8 µM, respectively) without significant toxicity
up to 200 µM.
In tr od u ction
encouraging results prompted us to extend our studies
to the purine nucleosides.
As part of our continuing efforts to synthesize nu-
cleosides as antiviral agents, we have recently reported
2′-fluoro-5-methyl-â-L-arabinofuranosyluracil (L-FMAU)
as a potent antiviral agent against hepatitis B virus
(HBV) as well as Epstein-Barr virus (EBV).1-3 Com-
pared to the corresponding D-enantiomer, L-FMAU exhi-
bits more potent anti-HBV activity in vitro without any
significant cytotoxicity in a variety of cell lines such as
CEM, 2.2.15, H1, and bone marrow progenitor cells. Ad-
ditionally, L-FMAU does not interfere with the mito-
chondrial function, which has been the major concern
for some of the antiviral nucleosides such as FIAU and
DDC.4-6 In vitro studies with primary duck hepatocytes
indicated that L-FMAU also exhibits potent antiviral
Previously, several purine nucleosides containing the
2-deoxy-2-fluoro-â-D-arabinofuranosyl moiety have been
synthesized as potential antiviral and antileukemic
agents.10-12 One of the analogues, 9-(2-deoxy-2-fluoro-
â-D-arabinofuranosyl)guanine (2′-F-ara-G), was found to
be selectively toxic to human T-cell leukemia.11,12 More
recently, a purine analogue, 2,4-diamino-7-(2-deoxy-2-
fluoro-â-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine, has
been reported to exhibit good in vitro anti-HBV activ-
ity.13 It is well known that incorporation of a fluorine
atom at the 2′-position of purine nucleosides can in-
crease the stability of these compounds toward chemical
as well as metabolic degradation.14,15 Recent studies
with antiviral L-nucleosides suggest that different af-
finities toward anabolic and catabolic enzymes exist
between the biologically active L-nucleosides and their
D-counterparts, which could account for the enhanced
antiviral potency of the L-isomers.16,17 On the basis of
these observations, it is interesting to see whether the
incorporation of the 2-deoxy-2-fluoro-â-L-arabinofura-
nosyl moiety into purine nucleosides may influence the
biological activity. We wish to report herein the syn-
thesis and anti-HBV activity of 9-(2-deoxy-2-fluoro-â-L-
arabinofuranosyl)purine nucleosides.
activity against duck hepatitis B virus (DHBV; EC50
)
0.1 µM). Oral administration of L-FMAU (40 mg/kg/
day) for 5 days to Peking ducks congenitally infected
with chronic DHBV markedly reduced the viremia level
without any abnormalities.7 Furthermore, in vivo ef-
ficacy studies in woodchucks chronically infected with
woodchuck hepatitis virus indicated that L-FMAU ef-
fectively suppresses the virus during the drug treatment
at 10 mg/kg/day, and no significant viral rebound was
noted after discontinuation of the drug up to 24 weeks.8
L-FMAU is currently undergoing preclinical toxicology
studies.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of the purine nucleosides
reported in this paper was accomplished by the coupling
of glycosyl bromide 1 with the sodium salts18 of the
corresponding purines 2-4 followed by derivatization.
The sugar intermediate 1 was prepared from L-xylose,
via a multistep procedure as we reported earlier,9 with
the adaption of the new fluorination method reported
by Chou et al.19 Thus, the fully blocked 6-chloropurine
derivative 5 was obtained in 65% isolated yield by the
condensation of 1 with the sodium salt of 2. Am-
monolysis by an appropriate amine with concomittant
or subsequent deacylation in saturated NH3/CH3OH
gave the 6-substituted amino analogues 10 and 12-14
in good yields (66-82%). The 6-unsubstituted purine
Recently, we have also reported the structure-activ-
ity relationships of 2′-fluoro-â-L-arabinofuranosyl pyri-
midines as anti-HBV agents,9 from which we found that
L-FMAU exhibits the most potent anti-HBV activity
among these pyrimidine nucleosides, whereas two other
derivatives, namely, 2′-deoxy-2′-fluoro-â-L-arabinofura-
nosylcytosine (L-FAC) and its 5-iodocytosine derivative
(L-FIAC), show good in vitro anti-HBV activity. These
* Address correspondence to this author. Tel: 706-542-5379. Fax:
706-542-5381. E-mail: dchu@rx.uga.edu.
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
§ Department of Chemistry.
X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0022-2623(97)00233-1 CCC: $14.00 © 1997 American Chemical Society

Synthesis of Anti-HBV Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2751
Ta ble 1. Anti-HBV Activity and Toxicity of
9-(2-Deoxy-2-fluoro-â-L-arabinofuranosyl)purine Nucleosides
toxicities,
IC50 (µM)
HBV (2.2.15)
F igu r e 1. ORTEP drawing of 9-(2-deoxy-2-fluoro-â-L-arabino-
furanosyl)-9H-purine (11).
no.
X
Y
EC50 (µM)
2.2.15 CEM
11
10
12
13
14
15
16
21
22
23
H
H
H
H
H
H
H
H
Cl
NH2
NH2
>10
1.5
>10
>10
>10
8
>10
>10
>10
>10
>200
>200
>200
>200
>200
>200
>200
>200
180
NDa
90
ND
ND
ND
>100
ND
ND
ND
NH2
NHMe
nucleoside 11 was obtained in 86% yield by dehaloge-
nation of 5 with 10% Pd-C in EtOAc in the presence of
Et3N followed by deacylation with saturated NH3/CH3-
OH. Treatment of 5 with mercaptoethanol in the
presence of NaOCH3 in refluxing methanol gave the
hypoxanthine derivative 15 in 64% yield. Our attempt
to synthesize the hypoxanthine derivative 15 by the
deamination of 10 with adenosine deaminase was
unsuccessful. This suggests that 10 is not as good a
substrate of this enzyme as the corresponding D-enan-
tiomer, which can be degraded at a comparable rate as
adenosine.15 The 6-mercaptopurine derivative 16 was
obtained in 65% yield by the treatment of 5 with
thiourea in refluxing ethanol followed by deacylation
with saturated NH3/CH3OH.
Coupling of the sodium salt of 2,6-dichloropurine in
acetonitrile with 1 gave the fully blocked nucleoside 17
in 29% yield, from which the 2-chloroadenine derivative
21 was obtained in 81% yield by direct ammonolysis in
saturated NH3/CH3OH at 90 °C. Treatment of 17 with
LiN3 in refluxing EtOH followed by hydrogenation on
5% Pd-C and subsequent deacylation gave the 2,6-
diaminopurine analogue 22 in 40% yield. Although the
coupling reaction of the sodium salt of 2-amino-6-
chloropurine with 1 in DMF gave a complex mixture,
the desired product 20 was isolated in 9% yield and was
converted to the guanine derivative 23 in 70% yield by
reaction with mercaptoethanol in refluxing methanol in
the presence of NaOCH3.
cyclopropylamino
NMe2
OH
SH
NH2
NH2
OH
>200
>100
a
ND: not determined.
Further exploration of the structure modification re-
vealed that the free 6-NH2 group is preferred for the
anti-HBV activity, since either attachment of a small
alkyl group (methyl, cyclopropyl, dimethyl) to or sub-
stitution of an NH2 group with a hydrogen atom results
in the loss of anti-HBV activity. Similar findings have
been observed in the study of L-nucleosides with other
sugar moieties.22 Although the hypoxanthine derivative
15 exhibits moderate antiviral activity, the 6-mercapto
analogue 16 does not show any significant activity up
to 10 µM. Substitutions with either a chlorine atom (21)
or an NH2 group (22) at the 2-position of compound 10
also decrease the anti-HBV activity.
In summary, we have synthesized a number of purine
nucleosides containing the 2-deoxy-2-fluoro-â-L-arabino-
furanosyl moiety. From the structure-activity relation-
ships studies, we have found that the adenine and
hypoxanthine derivatives (10 and 15) exhibit moder-
ately potent in vitro anti-HBV activity without signifi-
cant cytotoxicity.
All the nucleosides obtained were characterized by
microanalysis and spectroscopic methods, and the physi-
cal data are consistent with those of the known D-
isomers. In addition to the presence of a doublet for
H-8 in the 1H NMR spectra, which results from the
coupling with the 2′-arabino fluorine atom (J F-H8 ) 1.6-
3.0 Hz), the â-configuration was also confirmed by the
Exp er im en ta l Section
Melting points were determined on a Mel-temp II apparatus
and are uncorrected. NMR spectra were recorded on a Bruker
400 AMX spectrometer at 400 MHz for H NMR and 100 MHz
1
for 13C NMR with Me4Si as internal standard. Chemical shifts
(δ) are reported in parts per million (ppm), and signals are
reported as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or bs (broad singlet). IR spectra were recorded
on a Nicolet 510P FT-IR spectrometer. Optical rotations were
measured on a J asco DIP-370 digital polarimeter. Mass
spectra were recorded on a Micromass Autospec high-resolu-
tion mass spectrometer. TLC were performed on Uniplates
(silica gel) purchased from Analtech Co. Column chromatog-
raphy was performed using either silica gel-60 (220-440 mesh)
for flash chromatography or silica gel G (TLC grade, >440
mesh) for vacuum flash column chromatography. UV spectra
were obtained on a Beckman DU 650 spectrophotometer.
Elemental analyses were performed by Atlantic Microlab, Inc.,
Norcross, GA, or Galbraith Laboratories, Inc., Knoxville, TN.
9-(3,5-Di-O-ben zoyl-2-d eoxy-2-flu or o-â-L-a r a bin ofu r a -
n osyl)-9H-6-ch lor op u r in e (5). A mixture of 6-chloropurine
(2; 0.19 g, 1.2 mmol) and NaH (60 mg, 1.5 mmol, 60% in oil)
in anhydrous CH3CN (5 mL) was stirred under Ar at room
C1′-F coupling constant (J C1′-F ) 15-18 Hz) in the 13
C
NMR spectra of the intermediates 5, 17, and 21.20
Furthermore, the configuration of compound 11 has
been unambiguously determined by single-crystal X-ray
crystallography,21 and the ORTEP diagram is shown in
Figure 1.
An ti-HBV Activities. Anti-HBV activity and cyto-
toxicity of the synthesized nucleosides were evaluated
in 2.2.15 cells as described before,1 and the results are
summarized in Table 1. Initially, we synthesized three
compounds, namely, the adenine 10, hypoxanthine 15,
and guanine 23 derivatives. We found 10 and 15 exhibit
significant in vitro anti-HBV activity (EC50 ) 1.5 and 8
µM) without significant cytotoxicity up to 200 µM, while
23 does not show any anti-HBV activity up to 200 µM.

2752 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Ma et al.
temperature for 30 min, to which 1 (0.42 g, 1.0 mmol) in CH3-
CN (10 mL) was added. The resulting mixture was stirred at
room temperature for 4 h, filtered, and washed with CH2Cl2.
The combined filtrate was evaporated to dryness to give a
mixture that was separated on a silica gel column (100:1
CHCl3:CH3OH). The major product was collected and recrys-
tallized from EtOH to give 5 as a white solid (270 mg, 65.6%):
mp 101-105 °C; UV (EtOH) λmax 235.0, 264.5 nm; [R]25D +29.84
CHCl3:CH3OH) and crystallization from MeOH gave 13 as
white needles (45 mg, 66%): mp 131-133 °C; UV (H2O) λmax
265.0 (ꢀ 21 609) (pH 2), 268.5 (ꢀ 19 202) (pH 7), 268.5 nm (ꢀ
20 138) (pH 11); [R]25D -42.47 (c 0.11, H2O); 1H NMR (DMSO-
d6) δ 8.24 (bs, 2H, H-8, H-2), 8.02 (bs, 1H, NH, D2O exchange-
able), 6.40 (dd, 1H, H-1′, J 1′,2′ ) 4.6 Hz, J 1′,F ) 14.1 Hz), 5.96
(d, 1H, 3′-OH, D2O exchangeable), 5.18 (dt, 1H, H-2′, J 2′,F
)
52.5 Hz), 5.12 (t, 1H, 5′-OH, D2O exchangeable), 4.42 (dt, 1H,
H-3′, J 3′,F ) 18.8 Hz), 3.85 (m, 1H, H-4′), 3.64 (m, 2H, H-5′ab),
3.15 (m, 1H, CH(CH2)2), 0.72 (d, 4H, CH(CH2)2); FABMS m/z
310 (M + 1)+. Anal. (C13H16FN5O3‚H2O) C, H, N.
1
(c 0.22, CHCl3); H NMR (CDCl3) δ 8.78 (s, 1H, H-2), 8.42 (d,
1H, H-8, J ) 2.9 Hz), 8.12-7.44 (m, 10H, benzoyl), 6.71 (dd,
1H, H-1′, J 1′,2′ ) 2.6 Hz, J 1′,F ) 21.9 Hz), 5.80 (dd, 1H, H-3′,
J 3′,F ) 21.9 Hz), 5.40 (dd, 1H, H-2′, J 2′,F ) 50.0 Hz), 4.84 (m,
2H, H-5′a,b), 4.64 (m, 1H, H-4′); 13C NMR (CDCl3) 152.64,
144.90 (CO), 134.71, 134.59, 133.92, 130.39, 130.24, 130.16,
129.91, 129.21, 129.00 (Ar), 92.93 (d, J ) 192.96 Hz, C-2′),
84.29 (d, J ) 17.01 Hz, C-1′), 81.95 (C-4′), 76.89 (C-3′), 63.52
(C-5′). Anal. (C24H18ClFN4O5‚C2H5OH) C, H, N.
9-(2-De oxy-2-flu or o-â-L-a r a b in ofu r a n osyl)-N 6,N6-d i-
m eth yl-9H-p u r in e (14). A mixture of 5 (0.2 g, 0.4 mmol) and
N,N-dimethylamine (0.3 mL, 40% in H2O) in 1,4-dioxane (20
mL) was stirred at room temperature for 1 h and then
evaporated to dryness to give 7 as a syrup: UV (MeOH) λmax
273.0 nm. The crude 7 was treated with saturated NH3/CH3-
OH at room temperature for 16 h; removal of solvent and
purification by silica gel column chromatography (10:1 CHCl3:
CH3OH) gave 14 as a foam (80 mg, 67%); an analytical sample
was obtained by crystallization from 2-propanol to give 14 as
a white solid: mp 152-154 °C; UV (H2O) λmax 267.0 (ꢀ 15 912)
(pH 2), 274.0 (ꢀ 15 121) (pH 7), 273.5 nm (ꢀ 16 547) (pH 11);
[R]25D -47.88 (c 0.11, H2O); 1H NMR (DMSO-d6) δ 8.25 (d, 1H,
9-(2-Deoxy-2-flu or o-â-L-a r a bin ofu r a n osyl)a d en in e (10).
A solution of 5 (0.15 g, 0.30 mmol) in saturated NH3/CH3OH
(20 mL) was sealed in a stainless steel bomb and heated at 90
°C for 16 h. The solvent was evaporated, and the residue was
purified by preparative TLC (7:1 CHCl3:CH3OH) and recrys-
tallized from MeOH to give 10 as white crystals (60 mg,
74%): mp 231-233 °C; UV (H2O) λmax 256.0 (ꢀ 18 171) (pH 2),
258.5 (ꢀ 17 679) (pH 7), 258.0 nm (ꢀ 18 674) (pH 11); [R]25
H-8, J ) 1.8 Hz), 8.22 (s, 1H, H-2), 6.42 (dd, 1H, H-1′, J 1′,2′ )
D
-44.55 (c 0.11, H2O); 1H NMR (DMSO-d6) δ 8.23 (d, 1H, H-8,
4.6 Hz, J 1′,F ) 14.2 Hz), 5.96 (d, 1H, 3′-OH, D2O exchangeable),
5.18 (dt, 1H, H-2′, J 2′,F ) 52.7 Hz), 5.12 (t, 1H, 5′-OH, D2O
exchangeable), 4.43 (dt, 1H, H-3′, J 3′,F ) 18.9 Hz), 3.84 (m,
1H, H-4′), 3.64 (m, 2H, H-5′ab), 3.16 (s, 6H, NH(CH3)2);
FABMS m/z 298 (M + 1)+. Anal. (C12H16FN5O3‚0.8H2O) C,
H, N.
J
) 1.9 Hz), 8.15 (s, 1H, H-2), 7.34 (bs, 2H, NH2, D2O
exchangeable), 6.40 (dd, 1H, H-1′, J 1′,2′ ) 4.6 Hz, J 1′,F ) 14.4
Hz), 5.94 (d, 1H, 3′-OH, D2O exchangeable), 5.18 (dt, 1H, H-2′,
J 2′,F ) 52.7 Hz), 5.12 (t, 1H, 5′-OH, D2O exchangeable), 4.44
(dt, 1H, H-3′, J 3′,F ) 19.8 Hz), 3.83 (m, 1H, H-4′), 3.64 (m, 2H,
H-5′a,b); FABMS m/z 270 (M + 1)+. Anal. (C10H12FN5O3) C,
H, N.
9-(2-De oxy-2-flu or o-â-L-a r a b in ofu r a n osyl)h yp oxa n -
th in e (15). To a solution of 5 (0.2 g, 0.4 mmol) in methanol
(20 mL) was added NaOCH3 (91 mg, 1.6 mmol) followed by
2-mercaptoethanol (0.11 mL, 1.6 mmol). The mixture was
refluxed under Ar for 4 h. The solvent was evaporated, and
the residue was dissolved in 50% MeOH/H2O, neutralized with
Dowex 50w × 8 (H+) resin, and then filtered and washed with
50% MeOH/H2O. Removal of solvent gave a syrup that was
purified on preparative TLC (4:1 CH2Cl2:CH3OH) and then
loaded on a short silica gel column. Elution with EtOAc gave
15 as a white solid (70 mg, 64%): mp >118 °C dec; UV (H2O)
λmax 248.0 (ꢀ 8097) (pH 2), 248.5 (ꢀ 6068) (pH 7), 252.5 nm (ꢀ
8680) (pH 11); [R]25D -45.13 (c 0.12, MeOH); 1H NMR (DMSO-
d6) δ 8.20 (d, 1H, H-8, J ) 1.9 Hz), 8.08 (s, 1H, H-2), 6.35 (dd,
1H, H-1′, J 1′,2′ ) 4.7 Hz, J 1′,F ) 13.3 Hz), 5.90 (d, 1H, 3′-OH,
D2O exchangeable), 5.21 (dt, 1H, H-2′, J 2′,F ) 52.7 Hz), 5.10
9-(2-Deoxy-2-flu or o-â-L-a r a b in ofu r a n osyl)-9H -p u r in e
(11). A mixture of 5 (0.25 g, 0.50 mmol), 10% Pd-C (150 mg)
in EtOAc (30 mL), and Et3N (5 mL) was subjected to hydro-
genolysis at 40 psi for 5 h. After filtration through a Celite
pad and washing with EtOAc, the combined filtrate was
evaporated to dryness to give 6 as a white solid: UV (MeOH)
λmax 230.5, 262.0 nm. It was then stirred in saturated NH3/
CH3OH at room temperature for 16 h. The solvent was
evaporated, and the residue was purified by silica gel column
chromatography (10:1 CHCl3:CH3OH). Recrystallization from
MeOH gave 11 as white crystals (0.11 g, 86%): mp 174-176
°C; UV (H2O) λmax 261.5 (ꢀ 6673) (pH 2), 261.5 (ꢀ 6343) (pH 7),
262.0 nm (ꢀ 6330) (pH 11); [R]25D -61.49 (c 0.10, H2O); 1H NMR
(DMSO-d6) δ 9.24 (s, 1H, H-6), 9.01 (s, 1H, H-2), 8.78 (d, 1H,
H-8, J ) 1.8 Hz), 6.60 (dd, 1H, H-1′, J 1′,2′ ) 4.7 Hz, J 1′,F ) 13.1
Hz), 6.05 (d, 1H, 3′-OH, D2O exchangeable), 5.33 (dt, 1H, H-2′,
J 2′,F ) 52.6 Hz), 5.16 (t, 1H, 5′-OH, D2O exchangeable), 4.50
(dm, 1H, H-3′, J 3′,F ) 19.0 Hz), 3.91 (m, 1H, H-4′), 3.67 (m,
2H, H-5′a,b); FABMS m/z 255 (M + 1)+. Anal. (C10H11FN4O3)
C, H, N.
(t, 1H, 5′-OH, D2O exchangeable), 4.42 (dt, 1H, H-3′, J 3′,F
)
18.9 Hz), 3.84 (m, 1H, H-4′), 3.63 (dm, 2H, H-5′a,b); FABMS
m/z 271 (M + 1)+. Anal. (C10H11FN4O4‚EtOAc) C, H, N.
9-(2-Deoxy-2-flu or o-â-L-a r a bin ofu r a n osyl)-9H-p u r in e-
6-th iol (16). A mixture of 5 (0.4 g, 0.8 mmol) and thiourea
(76 mg, 1.0 mmol) in EtOH (25 mL) was stirred at reflux for
2 h and then cooled in an ice-water bath. The white
precipitate formed was filtered and washed with EtOH (5 mL)
to give 9 (0.33 g, 82%): UV (MeOH) λmax 323.0, 227.5 nm
(shoulder). Compound 9 was treated with saturated NH3/CH3-
OH at room temperature for 15 h and then evaporated to
dryness. The residue was purified by silica gel column
chromatography (10:1 CHCl3:CH3OH) to give 16 as a white
solid (0.14 g, 81%): mp 224-226 °C; UV (H2O) λmax 321.0 (ꢀ
9-(2-Deoxy-2-flu or o-â-L-a r a bin ofu r a n osyl)-N6-m eth yl-
9H-p u r in e (12). A solution of 5 (0.16 g, 0.32 mmol) in MeOH
(10 mL) and methylamine (10 mL, 40% in H2O) in a sealed
stainless steel bomb was heated at 85 °C for 12 h. Removal
of solvent and purification by silica gel column chromatography
(10:1 CHCl3:CH3OH) gave 12 as a pale white solid (75 mg,
82%): mp 91-94 °C; UV (H2O) λmax 261.5 (ꢀ 12 601) (pH 2),
265.0 (ꢀ 10 985) (pH 7), 265.0 nm (ꢀ 11 647) (pH 11); [R]25
D
-37.43 (c 0.17, H2O); 1H NMR (DMSO-d6) δ 8.25 (bs, 2H, H-8,
H-2), 7.94 (bs, 1H, NH, D2O exchangeable), 6.42 (dd, 1H, H-1′,
J 1′,2′ ) 4.6 Hz, J 1′,F ) 14.2 Hz), 5.99 (d, 1H, 3′-OH, D2O
exchangeable), 5.20 (dt, 1H, H-2′, J 2′,F ) 52.7 Hz), 5.15 (t, 1H,
5′-OH, D2O exchangeable), 4.46 (dt, 1H, H-3′, J 3′,F ) 19.0 Hz),
3.86 (m, 1H, H-4′), 3.67 (m, 2H, H-5′ab), 2.96 (s, 3H, NHCH3);
FABMS m/z 284 (M + 1)+. Anal. (C11H14FN5O3) C, H, N.
N6-Cyclop r op yl-9-(2-d eoxy-2-flu or o-â-L-a r a bin ofu r a n o-
syl)-9H-p u r in e (13). A solution of 5 (0.11 g, 0.22 mmol) in
THF (20 mL) and cyclopropylamine (1 mL) was heated in a
sealed steel bomb at 90 °C for 4 h and then evaporated to
dryness to give 8 as a syrup: UV (MeOH) λmax 230.0, 268.5
nm. The crude product thus obtained was treated with
saturated NH3/CH3OH at room temperature for 16 h. Removal
of solvent followed by purification on preparative TLC (9:1
16 697) (pH 2), 315.5 (ꢀ 8796) (pH 7), 309.5 nm (ꢀ 16 246) (pH
1
11); [R]25 -37.39 (c 0.10, H2O); H NMR (DMSO-d6) δ 13.86
D
(s, 1H, NH, D2O exchangeable), 8.42 (d, 1H, H-8, J ) 1.6 Hz),
8.22 (s, 1H, H-2), 6.38 (dd, 1H, H-1′, J 1′,2′ ) 4.8 Hz, J 1′,F ) 12.8
Hz), 5.98 (d, 1H, 3′-OH, D2O exchangeable), 5.22 (dt, 1H, H-2′,
J 2′,F ) 52.4 Hz), 5.14 (t, 1H, 5′-OH, D2O exchangeable), 4.42
(dt, 1H, H-3′, J 3′,F ) 18.6 Hz), 3.86 (m, 1H, H-4′), 3.66 (dm,
2H, H-5′a,b); FABMS m/z 287 (M + 1)+. Anal. (C10H11FN4O3S)
C, H, N.
9-(3,5-Di-O-b en zoyl-2-d eoxy-2-flu or o-â-L-a r a b in ofu r -
a n osyl)-2,6-d ich lor o-9H-p u r in e (17). A mixture of 2,6-
dichloropurine (3; 0.75 g, 4.0 mmol) and NaH (95%, 0.15 g,
6.0 mmol) in CH3CN (20 mL) was stirred under Ar at room
temperature for 30 min. To this was added 1 (0.84 g, 0.20
mmol), and the mixture was stirred at room temperature for

Synthesis of Anti-HBV Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2753
3 h and then washed with CH2Cl2. The combined filtrate was
evaporated to dryness to give a mixture that was purified by
silica gel column chromatography (4:1 hexanes:EtOAc). The
major product was collected to give 17 as a white foam (0.31
g, 29%); an analytical pure sample was obtained by crystal-
NMR (CDCl3) 165.20, 162.13 (CO), 148.15, 141.70, 134.62,
133.86, 130.37, 130.06, 129.18, 128.97, 113.74 (Ar), 92.95 (d,
J ) 193.39 Hz, C-2′), 83.78 (d, J ) 17.20, C-1′), 81.42 (C-4′),
76.84 (C-4′), 63.62 (C-5′). Anal. (C24H19ClFN5O5‚1.5H2O) C,
H, N.
lization from MeOH: mp 153-154 °C; UV (MeOH) λmax 273.5
9-(2-Deoxy-2-flu or o-â-L-a r a bin ofu r a n osyl)gu a n in e (23).
A suspension of 20 (80.0 mg, 0.156 mmol), 2-mercaptoethanol
(45 µL, 0.63 mmol), and NaOCH3 (36 mg, 0.63 mmol) in MeOH
(10 mL) was stirred at reflux under Ar for 6 h. The solvent
was evaporated, and the residue was dissolved in 50% MeOH/
H2O, neutralized with Dowex 50w × 8 (H+) resin, filtered, and
washed with 50% MeOH/H2O. The filtrate was evaporated
to dryness and then triturated with acetone. The residue was
further triturated with ether to leave a white solid which was
recrystallized from water to give a white solid (31 mg, 70%):
mp 249-251 °C; UV (H2O) λmax 254.0 (ꢀ 11 786) (pH 2), 254.0
1
nm; [R]25 +26.80 (c 0.1, CHCl3); H NMR (CDCl3) δ 8.40 (d,
D
1H, H-8, J ) 3.0 Hz), 8.10-7.44 (m, 10H, benzoyl), 6.65 (dd,
1H, H-1′, J 1′,2′ ) 2.7 Hz, J 1′,F ) 21.8 Hz), 5.78 (dd, 1H, H-3′,
J 3′,F ) 16.9 Hz), 5.42 (dd, 1H, H-2′, J 2′,F ) 49.8 Hz), 4.84 (m,
2H, H-5′a,b), 4.64 (m, 1H, H-4′); 13C NMR (CDCl3) 166.15,
165.14 (CO), 153.25, 152.44, 152.12, 145.18, 145.12, 134.32,
133.53, 130.57, 129.99, 129.72, 129.55, 129.21, 128.80, 128.68,
128.60, 127.97 (Ar), 92.57 (d, J ) 193.00 Hz, C-2′), 83.91 (d, J
) 17.0 Hz, C-1′), 81.73 (C-4′), 76.46 (C-3′), 63.08 (C-5′). Anal.
(C24H17Cl2FN4O5) C, H, N.
2-Ch lor o-9-(2-d eoxy-2-flu or o-â-L-a r a bin ofu r a n osyl)a d -
en in e (21). Compound 17 (130 mg, 0.245 mmol) was treated
with saturated NH3/CH3OH (20 mL) in a sealed bomb at 90
°C for 7 h. Removal of solvent followed by purification on a
silica gel column (10:1-7:1 CHCl3:CH3OH) gave 21 as a foam
(60 mg, 81%); an analytical sample was obtained by crystal-
lization from 2-propanol to give 21 as a white solid: mp 226-
228 °C; UV (H2O) λmax 263.0 (ꢀ 14 858) (pH 2), 263.0 (ꢀ 13 438)
(pH 7), 263.0 nm (ꢀ 13 073) (pH 11); [R]25D -31.86 (c 0.10, H2O);
1H NMR (DMSO-d6) δ 8.27 (d, 1H, H-8, J ) 1.9 Hz), 7.89 (bs,
2H, NH2, D2O exchangeable), 6.31 (dd, 1H, H-1′, J 1′,2′ ) 4.7
Hz, J 1′,F ) 13.7 Hz), 5.95 (d, 1H, 3′-OH, D2O exchangeable),
5.22 (dt, 1H, H-2′, J 2′,F ) 52.5 Hz), 5.08 (t, 1H, 5′-OH, D2O
exchangeable), 4.41 (dt, 1H, H-3′, J 3′,F ) 18.7 Hz), 3.83 (m,
1H, H-4′), 3.65 (dm, 2H, H-5′a,b); FABMS m/z 304 (M + 1)+.
Anal. (C10H11ClFN5O3‚0.15IPr-OH) C, H, N.
(ꢀ 10 786) (pH 7), 254.5 nm (ꢀ 11 214) (pH 11); [R]25 -24.96
D
(c 0.10, H2O); 1H NMR (DMSO-d6) δ 7.80 (d, 1H, H-8, J ) 2.1
Hz), 6.14 (dd, 1H, H-1′, J 1′,2′ ) 4.2 Hz, J 1′,F ) 16.0 Hz), 5.92 (d,
1H, 3′-OH, D2O exchangeable), 5.12 (dt, 1H, H-2′, J 2′,F ) 52.4
Hz), 5.12 (t, 1H, 5′-OH, D2O exchangeable), 4.36 (dt, 1H, H-3′,
J 3′,F ) 17.9 Hz), 3.82 (m, 1H, H-4′), 3.62 (dm, 2H, H-5′a,b);
FABMS m/z 286 (M + 1)+. Anal. (C10H12FN5O4) C, H, N.
Ack n ow led gm en t. This research was supported by
Grant AI 33655 from the National Institutes of Health.
We thank Dr. Michael G. Bartlett for performing the
mass spectroscopy.
Refer en ces
2,6-Dia m in o-9-(2-d eoxy-2-flu or o-â-L-a r a bin ofu r a n osyl)-
9H-p u r in e (22). A mixture of 17 (130 mg, 0.245 mmol) and
LiN3 (48 mg, 1.0 mmol) in 95% EtOH was stirred at reflux for
40 min and then evaporated to dryness. The residue was
extracted with CHCl3, washed with water, dried (MgSO4).
Removal of solvent gave 18 as a syrup: UV (MeOH) λmax 295.5
nm. It was redissolved in EtOH (20 mL) and stirred with 10%
Pd-C (20 mg) under H2 at 1 atm for 2 h and then filtered and
washed with EtOAc. The combined filtrate was evaporated
to dryness and purified by silica gel column chromatography
(10:1 CHCl3:CH3OH) to give 19 as a syrup (0.11 g, 91%): UV
(MeOH) λmax 255.0, 279.0 nm. 19 was treated with saturated
NH3/CH3OH (15 mL) at room temperature for 16 h and then
evaporated to dryness. The residue was triturated with
acetone, and the solid was recrystallized from MeOH to give
22 as a white solid (28 mg, 40% total yield from 17): mp 220-
223 °C; UV (H2O) λmax 251.5 (ꢀ 9680), 289.5 (ꢀ 8196) (pH 2),
255.5 (ꢀ 6751), 278.5 (ꢀ 7302) (pH 7), 255.0 (ꢀ 7941), 278.0 nm
(ꢀ 8279) (pH 11); [R]25D -44.24 (c 0.12, H2O); 1H NMR (DMSO-
d6) δ 7.79 (d, 1H, H-8, J ) 2.0 Hz), 6.80 (bs, 2H, NH2, D2O
exchangeable), 6.17 (dd, 1H, H-1′, J 1′,2′ ) 4.2 Hz, J 1′,F ) 16.0
Hz), 5.90 (bs, 3H, 3′-OH, NH2, D2O exchangeable), 5.08 (t, 1H,
5′-OH, D2O exchangeable), 5.07 (dt, 1H, H-2′, J 2′,F ) 52.4 Hz),
4.37 (dt, 1H, H-3′, J 3′,F ) 18.2 Hz), 3.80 (m, 1H, H-4′), 3.61 (m,
2H, H-5′a,b); FABMS m/z 285 (M + 1)+. Anal. (C10H13FN6O3)
C, H, N.
2-Am in o-9-(3,5-d i-O-ben zoyl-2-d eoxy-2-flu or o-â-L-a r a -
bin ofu r a n osyl)-6-ch lor o-9H-p u r in e (20). A mixture of
2-amino-6-chloropurine (4; 0.44 g, 2.6 mmol) and NaH (80 mg,
3.0 mmol, 95% in mineral oil) in anhydrous DMF (10 mL) was
stirred under Ar at room temperature for 15 min. To this was
added 1 (0.87 g, 2.0 mmol) in DMF (20 mL), and the mixture
was stirred at room temperature for 30 h. It was evaporated
to dryness, and the residue was purified on a silica gel column
(100:1 CHCl3:CH3OH) to give a mixture of mainly two products
(N-9 and N-7). Separation on preparative TLC (3:2 hexane:
EtOAc) followed by recrystallization in MeOH gave the desired
product 20 (fast moving band) as a white solid (95 mg, 9.3%):
(1) Chu, C. K.; Ma, T.; Shanmuganathan, K.; Wang, C.; Xiang, Y.;
Pai, S. B.; Yao, G.-Q.; Sommadossi, J .-P.; Cheng, Y.-C. Use of
2′-fluoro-5-methyl-â-L-arabinofuranosyluracil as a novel antiviral
agent for hepatitis B virus and Epstein-Barr virus. Antimicrob.
Agents Chemother. 1995, 39, 979-981.
(2) Pai, S. B.; Liu, S.-H.; Zhu, Y.-L.; Chu, C. K.; Cheng, Y.-C.
Inhibition of hepatitis B virus by a novel L-nucleoside, 2′-fluoro-
5-methyl-â-L-arabinofuranosyluracil. Antimicrob. Agents Chemo-
ther. 1996, 40, 380-386.
(3) Yao, G.-Q.; Liu, S.-H.; Chou, E.; Kukhanova, M.; Chu, C. K.;
Cheng, Y.-C. Inhibition of Epstein-Barr virus replication by a
novel L-nucleoside, 2′-fluoro-5-methyl-â-L-arabinofuranosyluracil.
Biochem. Pharmacol. 1996, 51, 941-947.
(4) Chen, C.-H.; Vazquez-Padua, M.; Cheng, Y.-C. Effect of anti-
human immunodeficiency virus nucleoside analogs on mitocho-
drial DNA and its implication for delayed toxicity. Mol. Phar-
macol. 1991, 39, 625-628.
(5) Parker, W. B.; Cheng, Y.-C. Mitochondrial toxicity of antiviral
nucleoside analogs. J . NIH Res. 1994, 6, 57-61.
(6) Cui, L.; Yoon, S.; Schinazi, R. F.; Sommadossi, J .-P. Cellular and
molecular events leading to mitochondrial toxicity of 1-(2-deoxy-
2-fluoro-1-â-D-arabinofuranosyl)-5-iodouracil in human. J . Clin.
Invest. 1994, 95, 555-563.
(7) Zoulim, F.; Aguesse, S.; Borel, C.; Trepo, C.; Cheng, Y.-C. 2′-
Fluoro-5-methyl-â-L-arabinofuranosyluracil, a novel L-nucleoside
analog, inhibits duck hepatitis B virus replication in primary
hepatocytes and in vivo. Antiviral Res. 1996, 30, A24.
(8) Tennant, B.; J acob, J .; Graham, L. A.; Peek, S.; Du, J .; Chu, C.
K. Pharmacokinetic and pharmacodynamic studies of 1-(2-fluoro-
5-methyl-â-L-arabinofuranosyl)uracil (L-FMAU) in the wood-
chuck model of hepatitis B virus (HBV) infection. Antiviral Res.
1997, 34, A52, 36.
(9) Ma, T. W.; Pai, S. B.; Zhu, Y.-L.; Kim, H.-B.; Wang, C.-G.;
Newton, M. G.; Shanmuganathan, K.; Cheng, Y.-C.; Chu, C. K.
Structure-activity relationship of 1-(2-deoxy-2-fluoro-â-L-ara-
binofuranosyl)pyrimidine nucleosides as anti-hepatitis B virus
agents. J . Med. Chem. 1996, 39, 2835-2843.
(10) Montgomery, J . A.; Shortnacy, A. T.; Carson, D. A.; Secrist, J .
A., III. 9-(2-Deoxy-2-fluoro-â-D-arabinofuranosyl)guanine:
A
metabolically stable cytotoxic analogue of 2′- deoxyguanosine.
J . Med. Chem. 1986, 29, 2389-2392.
(11) Chu, C. K.; Matulic-Adamic, J .; Huang, J .-T.; Chou, T.-C.;
Burchenal, J . H.; Fox, J . J .; Watanabe, K. A. Nucleosides.
CXXXV. Synthesis of some 9-(2-deoxy-2-fluoro-â-D-arabinofur-
anosyl)-9H-purines and their biological activities. Chem. Pharm.
Bull. 1989, 37, 336-339.
mp 101-103 °C; UV (MeOH) λmax 232.5, 307.5 nm; [R]25
D
+42.46 (c 0.12, CHCl3); 1H NMR (CDCl3) δ 8.11, 7.43 (m, 10H,
benzoyl), 8.04 (d, 1H, H-8, J ) 3.0 Hz), 6.45 (dd, 1H, H-1′, J 1′,2′
) 2.7 Hz, J 1′,F ) 22.2 Hz), 5.77 (dd, 1H, H-3′, J 3′,F ) 16.6 Hz),
5.32 (dd, 1H, H-2′, J 2′,F ) 50.1 Hz), 5.13 (bs, 2H, NH2, D2O
exchangeable), 4.81 (m, 2H, H-5′a,b), 4.58 (m, 1H, H-4′); 13C
(12) Montgomery, J . A.; Shortnacy-Fowler, A. T.; Clayton, S. D.;
Riordan, J . M.; Secrist, J . A., III. Synthesis and biologic activity
of 2′-fluoro-2-halo derivatives of 9-â-D-arabinofuranosyladenine.
J . Med. Chem. 1992, 35, 397-401.

2754 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Ma et al.
(13) Ojwang, J . O.; Bhattacharya, B. K.; Marshall, H. B.; Korba, B.
E.; Revankar, G. R.; Rando, R. F. Inhibition of episomal hepatitis
B virus DNA in vitro by 2,4-diamino-7-(2-deoxy-2-fluoro-â-D-
arabinofuranosyl)pyrrolo[2,3-d]pyrimidine. Antimicrob. Agents
Chemother. 1995, 39, 2570-2573.
(14) Marquez, V. E.; Tseng, C. K.-H.; Mitsuya, H.; Aoki, S.; Kelley,
J . A.; Ford, H., J r.; Roth, J . S.; Broder, S.; J ohns, D. G.; Driscoll,
J . S. Acid-stable 2′-fluoro purine dideoxynucleosides as active
agents against HIV. J . Med. Chem. 1990, 33, 978-985.
(15) Stoeckler, J . D.; Bell, C. A.; Parks, R. E., J r.; Chu, C. K.; Fox, J .
J .; Ikehara, M. C(2′)- substituted purine nucleoside analogs:
Interactions with adenosine deaminase and purine nucleoside
phosphorylase and formation of analog nucleotides. Biochem.
Pharmacol. 1982, 31, 1723-1728.
(16) Schinazi, R. F.; Chu, C. K.; Peck, A.; McMillan, A.; Mathis, R.;
Cannon, D.; J eong, L.- S.; Beach, J . W.; Choi, W.-B.; Yeola, S.;
Liotta, D. C. Activities of the four optical isomers of 2′, 3′-dideoxy-
3′-thiacytidine (BCH-189) against human immunodeficiency
virus type-1 in human lymphocytes. Antimicrob. Agents Chemoth-
er. 1992, 39, 979-981.
(17) Furman, P. A.; Davis, M.; Liotta, D. C.; Paff, M.; Frick, L. W.;
Nelson, D. J .; Dornsife, R. E.; Wurster, J . A.; Wilson, L. J .; Fyfe,
J . A.; Tuttle, J . V.; Miller, W. H.; Condreay, L.; Averett, D. R.;
(18) Revankar, G. R.; Robins, R. K. Use of the sodium salt glycosy-
lation method in nucleoside synthesis. Nucleosides Nucleotides
1989, 8, 709-724.
(19) Chou, T. S.; Becke, L. M.; O’Toole, J . C.; Carr, M. A.; Parker, B.
E. Triethylamine poly(hydrogen fluorides) in the synthesis of a
fluorinated nucleoside glycon. Tetrahedron Lett. 1996, 37, 17-
20.
(20) Mansuri, M. M.; Krishnan, B.; Martin, J . C. A potentially simple
13C NMR method to assign regio- and stereochemistry of (2′-
deoxy-2′-fluoroarabinofuranosyl)nucleosides. Tetrahedron Lett.
1991, 32, 1287-1290.
(21) X-ray data for compound 11: crystal dimensions, 0.50 × 0.20 ×
0.30 mm; crystal color and habbit, colorless needle; empirical
formula, C10H11FN4O3; formula weight, 254.22; crystal system,
orthorhombic; lattice parameters, a ) 8.054(1), b ) 11.510(1), c
) 11.988(2) Å; space group, P212121 (No. 19), Z ) 4; pseudoro-
tation phase angle P ) -29.5°, νmax ) 40.2°, γ ) 176.7°, κ )
-158.3°.
(22) Bolon, P. J .; Wang, P.; Chu, C. K.; Gosselin, G.; Boudou, V.;
Pierra, C.; Mathe´, C.; Imbach, J .-L.; Faraj, A.; el Alaoui, M. A.;
Sommadossi, J .-P.; Pai, S. P.; Zhu, Y.-L.; Lin, J .-S.; Cheng, Y.-
C.; Schinazi, R. F. Anti-human immunodeficiency and anti-
hepatitis B virus activities of â-L-2′,3′-dideoxy purine nucleosides.
Bioorg. Med. Chem. Lett. 1996, 6, 1657-1662.
Schinazi, R. F.; Painter, G. R. The anti-hepatitis
B virus
activities, cytotoxicities, and anabolic profiles of the (-) and (+)
enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-
5-yl]cytosine. Antimicrob. Agents Chemother. 1992, 36, 2686-
2692.
J M970233+