Newly synthesized L-enantiomers of 3'-fluoro-modified β-2'- deoxyribonucleoside 5'-triphosphates inhibit hepatitis B DNA polymerases but not the five cellular DNA polymerases α, β, Γ, δ, and ε nor HIV-1 reverse transcriptase

Article abstract of DOI:10.1021/jm9704210

Novel β-L-2',3'-dideoxy-3'-fluoro nucleosides were synthesized and further converted to their 5'-triphosphates. Their inhibitory activities against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) DNA polymerases, α, ?, γ, δ and ε were investigat

Full text of DOI:10.1021/jm9704210

2040
J . Med. Chem. 1998, 41, 2040-2046
New ly Syn th esized L-En a n tiom er s of 3’-F lu or o-Mod ified
â-2’-Deoxyr ibon u cleosid e 5’-Tr ip h osp h a tes In h ibit Hep a titis B DNA P olym er a ses
Bu t Not th e F ive Cellu la r DNA P olym er a ses r, â, γ, δ, a n d E Nor HIV-1 Rever se
Tr a n scr ip ta se
Martin von J anta-Lipinski,† Burkhardt Costisella,‡ Hansueli Ochs,§ Ulrich Hu¨bscher,§ Peter Hafkemeyer,| and
Eckart Matthes*,†
Max-Delbru¨ck-Centrum fu¨r Molekulare Medizin, Robert-Ro¨ssle-Strasse 10, D-13125 Berlin-Buch, Germany,
Institut fu¨r Angewandte Chemie, Rudower Chaussee 5, D-12484 Berlin-Adlershof, Germany, Institut fu¨r Veterina¨rbiochemie,
Universita¨t Zu¨rich-Irchel, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland, and Medizinische Klinik,
Universita¨t Freiburg, Hugstetterstrasse 55, D-79106 Freiburg, Germany
Received J une 26, 1997
Novel â-L-2’,3’-dideoxy-3’-fluoro nucleosides were synthesized and further converted to their
5’-triphosphates. Their inhibitory activities against hepatitis B virus (HBV) and duck hepatitis
B virus (DHBV) DNA polymerases, human immunodeficiency virus (HIV) reverse transcriptase
(RT), and the cellular DNA polymerases R, â, γ, δ, and ꢀ were investigated and compared with
those of the corresponding 3’-fluoro-modified â-D-analogues. The 5’-triphosphates of 3’-deoxy-
3’-fluoro-â-L-thymidine (â-L-FTTP), 2’,3’-dideoxy-3’-fluoro-â-L-cytidine (â-L-FdCTP), and 2’,3’-
dideoxy-3’-fluoro-â-L-5-methylcytidine (â-L-FMetdCTP) emerged as effective inhibitors of HBV/
DHBV DNA polymerases (IC50 ) 0.25-10.4 µM). They were either equally (FTTP) or less
(FMetdCTP, FdCTP) effective than their â-D-counterparts. Also the 5’-triphosphate of â-L-
thymidine (â-L-TTP) was shown to be a strong inhibitor of these two viral enzymes (IC50
)
0.46/1.0 µM). However, all â-L-FdNTPs (also â-L-TTP) were inactive against HIV-RT, a result
which contrasts sharply with the high efficiency of the â-D- FdNTPs against this polymerase.
Between the cellular DNA polymerases only the â and γ enzymes displayed a critical
susceptibility to â-D-FdNTPs which is largely abolished by the â-L-enantiomers. These results
recommend â-L-FTdR, â-L-FCdR, and â-L-FMetCdR for further evaluation as selective inhibitors
of HBV replication at the cellular level.
In tr od u ction
triphosphates of such 3’-fluoro-modified â-L-pyrimidine
nucleosides whose enantiomeric analogues proved to be
extremely efficient inhibitors of HIV reverse tran-
scriptase (HIV-RT) (2’,3’-dideoxy-3’-fluoro-â-D-thymi-
dine-5’-triphosphate, D-FTTP; 2’,3’-dideoxy-3’-fluoro-â-
D-uridine-5’-triphosphate, D-FdUTP)8-10 and HBV DNA
polymerase (2’,3’-dideoxy-3’-fluoro-â-D-5-methylcytidine-
5’-triphosphate, D-FMetdCTP).11,12 In addition, as D-
nucleosides these 3’-fluoro-modified analogues have
been shown to be very strong inhibitors of HIV and/or
HBV replication at the cellular level.8,10,13-15 First
clinical applications of â-D- FLT as an anti-AIDS drug
produced an unexpectedly severe toxicity emphasizing
the need for compounds with higher selectivity.16
Here we report the synthesis of the 2’,3’-dideoxy-3’-
fluoro-â-L-analogues of thymidine, uridine, cytidine, and
5-methylcytidine and the inhibitory activity of their 5’-
triphosphates (â-L-FTTP, â-L-FdUTP, â-L-FdCTP, and
â-L-FMetdCTP) on the HIV reverse transcriptase, HBV
and duck hepatitis B virus (DHBV) DNA polymerases,
and the cellular DNA polymerases R, â, γ, δ, and ꢀ in
comparison to the corresponding D-enantiomers.
A series of modified nucleosides with the unnatural
L-configuration have been described recently as potent
inhibitors of hepatitis B virus (HBV) and human im-
munodeficiency virus (HIV) replication. These com-
pounds include 2’,3’-dideoxy-3’-thia-â-L-cytidine (3TC,
Lamivudine),1 2’,3’-dideoxy-3’-thia-â-L-5-fluorocytidine
(L-FTC),2 2’,3’-dideoxy-â-L-cytidine (L-ddC), 2’,3’-dideoxy-
â-L-5-fluorocytidine (L-FddC),3,4 2’-deoxy-2’-fluoro-5-
methyl-â-L-arabinofuranosyluracil (L-FMAU),5 2’,3’-
dideoxy-2’,3’-didehydro-â-L-cytidine, and 2’,3’-dideoxy-
2’,3’-didehydro-â-L-5-fluorocytidine.6 For the most part,
they display higher antiviral activity and lower cyto-
toxicity than their D-counterparts.
The approval of 3TC in combination with AZT for the
treatment of HIV infection and its first promising
results in the therapy of chronic HBV infection7 have
prompted the synthesis of new nucleosides with the
unnatural L-configuration. One of the key factors of this
enantioselectivity is the interaction of the 5’-triphos-
phates of the L-nucleosides with cellular and viral
polymerases.
To get direct insights into enantioselectivity at the
viral and cellular levels, we decided to prepare the 5’-
Ch em istr y
Methods developed for preparation of the D-nucleo-
sides and their derivatives were modified and used to
prepare the new L-analogues for this work, as sum-
marized in Scheme 1. The synthesis of 1-(2,3-dideoxy-
† Max-Delbru¨ck-Centrum fu¨r Molekulare Medizin.
‡ Institut fu¨r Angewandte Chemie.
§ Universita¨t Zu¨rich-Irchel.
| Universita¨t Freiburg.
S0022-2623(97)00421-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/15/1998

Novel â-L-2′,3′-Dideoxy-3′-fluoro Nucleosides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2041
Sch em e 1
Sch em e 2
4-chlorophenyl phosphorodichloridate and 1,2,4-triazole
in anhydrous pyridine followed by a solution of am-
monium hydroxide and dioxane (3:1, v/v) yielded the
corresponding cytidine derivatives 13 and 14.
Nucleoside 5’-triphosphates of the L-nucleosides were
synthesized via the monophosphates24 as described
earlier for the corresponding D-counterparts8,11,12 ac-
cording to the method of Hoard.25
Bioch em ica l Eva lu a tion
In h ibition of Vir a l P olym er a ses by th e Tr ip h os-
p h a tes of â-L- a n d â-D-3’-F lu or o-Mod ified P yr im i-
d in e Nu cleosid es a n d of â-L-Th ym id in e (â-L-TTP ).
The 5’-triphosphates of â-D- and â-L-enantiomers of 3’-
fluoro-modified thymidine and 2’-deoxycytidine deriva-
tives were evaluated as inhibitors of HIV-RT and the
endogenous DNA polymerases of HBV and DHBV.
Earlier, the â-D-enantiomers of FTTP, FdUTP, and
FMetdCTP were shown to be extremely efficient inhibi-
tors of HIV-RT (FTTP, FdUTP: IC50 ) 0.05 and 0.07
µM)8,10 and HBV DNA polymerase (FTTP, FMetdCTP:
IC50 ) 0.15 and 0.03 µM).11,12 In contrast, â-L-FTTP (16)
and â-L-FdUTP (17) synthesized here (Scheme 2) lacked
any inhibitory effects against HIV-RT (see Table 1).
Moreover â-L-FdCTP (18) and â-L-FMetdCTP (19) lost
all inhibitory effects possessed by their enantiomeric
counterparts (â-D-FdCTP, â-D-FMetdCTP: IC50 ) 0.5
and 0.6 µM). In our hands also â-L-TTP (15) did not
interfere with the activity of the HIV-RT in contrast to
what has been reported recently.26 We find, however,
that this compound is a strong inhibitor of the HBV and
DHBV DNA polymerases (IC50 ) 0.46 and 1.0 µM).
Further differences in the stereospecific inhibition of
the hepatitis B virus DNA polymerases in comparison
to HIV-RT were revealed by the evaluation of â-L- and
â-D-enantiomers of the 3’-fluoro-modified dTTP and
dCTP analogues. â-L- and â-D-FTTP displayed similar
inhibitory effects on HBV DNA polymerase (IC50 ) 0.25
versus 0.15 µM) as well as on DHBV DNA polymerase
(IC50 ) 0.48 versus 0.17 µM). The same was true for
the less effective compounds, â-L- and â-D-FdUTP (see
Table 1), whereas hepatitis B virus DNA polymerases
proved to be more susceptible to the â-D-enantiomers
of FdCTP and FMetdCTP than to their L-enantiomers
(Table 1).
3-fluoro-â-L-ribofuranosyl)thymine was recently de-
scribed without experimental details, using a fluoro-1-
thiopentofuranoside as the starting synthon17 in
Vorbru¨ggen-type18 condensation reactions with silylated
thymine. Coupling by this method preferentially results
in the R-anomer. Because we were interested in the
synthesis of â-L-nucleoside 5’-triphosphates, a more
general synthetic approach was chosen.
L-5-Methyluridine19 was deoxygenated using the
method developed by Robins et al.20 to give compound
1. 2’-Deoxy-â-L-uridine (2) was synthesized from L-
arabinose according to the methodology of Holy.21
Protection of the 5’-hydroxyl function with a trityl group,
followed by treatment with methanesulfonyl chloride,
gave compounds 3 and 4, respectively. The configura-
tion in the 3’-position was easily inverted by the action
of sodium hydroxide in ethanolic solution to yield the
1-(2-deoxy-5-O-trityl-â-L-threo-pentofuranosyl)nucleo-
sides 5 and 6. Treatment of compounds 5 and 6,
respectively, with 2 equiv of (diethylamino)sulfur tri-
fluoride (DAST)22 in dichloroethane at room tempera-
ture overnight produced the desired fluoro compounds
7 and 8 without detectable amounts of elimination
products in contrast to results obtained in the R-L-
series.23 Detritylation gave the 1-(2,3-dideoxy-3-fluoro-
â-L-erythro-pentofuranosyl)nucleosides 9 and 10. Treat-
ment of the acetylated compounds 11 and 12 with

2042 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
von J anta-Lipinski et al.
Ta ble 1. Effects of â-L-TTP and â-L- and â-D-Enantiomers of 3’-Fluoro-Modified TTP and dCTP Analogues on Viral and Cellular
Polymerases
IC50 (µM)a
viral polymerases
HBV
cellular DNA polymerases
compound
HIV-RT
DHBV
R
â
γ
δ
ꢀ
â-L-TTP (15)
â-L-FTTP (16)
â-D-FTTP
â-L-FdUTP (17)
â-D-FdUTP
â-L-FdCTP (18)
â-D-FdCTP
â-L-FMetdCTP (19)
â-D-FMetdCTP
>200
0.46 ( 0.08
0.25 ( 0.06
0.15 ( 0.02d
35 ( 8
1.0 ( 0.25
0.48 ( 0.1
0.17 ( 0.04
>40
>100
>200
>200b
>100
>200c
>100
>200
>200
>200e
>200
>200
80 ( 15
>100
>200
>200
>200
>200
>100
>200
>100
>200
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
0.05 ( 0.005b
>200
2.20 ( 0.45b
>100
1.5 ( 0.26
>100
0.07 ( 0.01c
>100
25 ( 6d
>40
3.0 ( 0.65c
>100
3.5 ( 0.9
>50
7.0 ( 1.6
10.4 ( 1.9
0.8 ( 0.17
2.0 ( 0.45
0.015 ( 0.005
0.5 ( 0.08
>100
0.6 ( 0.07
0.5 ( 0.1
1.0 ( 0.22
>200
2.0 ( 0.34
>50
1.9 ( 0.42
0.03 ( 0.007e
1.0 ( 0.29e
0.5 ( 0.09
a
Concentrations required for a 50% inhibition of the enzymes. Poly(A)‚oligo(dT)15, poly(dA)‚oligo(dT)15, or activated DNA and the TTP
or dCTP concentrations were used as described in the Experimental Section. For estimation of endogenous HBV/DHBV DNA polymerase
activities, the TTP or dCTP concentrations were 0.4 µM. Means of three experiments are given. For DNA polymerase â and γ and the
b
d
viral polymerases, standard deviations are included. Data from ref 8. c Data from ref 10. Data from ref 11. e Data from ref 12.
Kinetic studies were performed with 15 and 16, the
most efficient inhibitors of HBV DNA polymerase. The
Lineweaver-Burk plots (not shown) indicate a competi-
tive type of inhibition for HBV DNA polymerase by both
15 and 16 with regard to TTP. The Km value of TTP
was estimated to be 0.4 µM, and the Ki values were 0.30
µM for 15 and 0.2 µM for 16. These results are in
contrast to earlier findings demonstrating that HBV
DNA polymerase displays some degree of selectivity for
the L-enantiomers.27 Thus L-ddTTP and L-ddCTP de-
rivatives proved to be more active inhibitors of HBV
DNA polymerase than their D-counterparts.28
Surprisingly, this preference for â-L-enantiomers is
abolished by the replacement of the 3’-hydrogen by the
bulkier and more electronegative fluoro substituent in
these nucleotides. Moreover, FTTP and FdCTP in their
L-configurations have even lost all activity as inhibitors
of HIV-RT as in the case of ddTTP and ddCTP.27
F igu r e 1. Incorporation of â-D-FTTP and â-L-FTTP (16) by
HIV-RT into poly(A)‚oligo(dT)15 in comparison to â-L-TTP (15).
[5’-32P]-(dT)15 (350 nM) was hybridized with poly(A) (3.5 µM)
and incubated with 10 µM â-D-FTTP or 50 µM 16 or 15 and
0.1 unit of HIV-RT as described in the Experimental Section
for different times. The standard assay was incubated with
10 µM TTP. The products were separated on 20% polyacryla-
mide-8 M urea gels. The gels were dried and phosphorimaged.
In h ibition of Cellu la r P olym er a ses by â-L- a n d
â-D-3’-F lu or o-Mod ified P yr im id in e Nu cleosid e Tr i-
p h osp h a tes. We tested the â-L- and â-D-enantiomers
for their inhibition of the cellular DNA polymerases R,
â, γ, δ, and ꢀ. Table 1 shows that none of the L- or
D-enantiomers displayed a significant inhibitory activity
against DNA polymerases R, δ or ꢀ. In contrast, DNA
polymerases â and γ proved to be very susceptible to
the fluoro-modified TTP and dCTP analogues but only
against their â-D-enantiomers, whereas the correspond-
ing L-enantiomers did not show inhibitory effects against
both of these polymerases. These results are in agree-
ment with recent findings demonstrating that the
cellular DNA polymerases â and γ are less sensitive
against the triphosphates of L-ddC, L-FddC, 3TC, and
carbovir compared with their D-counterparts.29-31 As
a consequence, a lower cytotoxicity of 3’-fluoro-modified
â-L-analogues might be expected. Experiments are
under way to estimate both the antiproliferative activity
and the efficiency of the 2’,3’-dideoxy-3’-fluoro-â-L-
derivatives of thymidine (9), 5-methylcytidine (13), and
cytidine (14) to inhibit the HBV replication in Hep G2
2.2.15 cells.
â-L-F TTP (16) a n d â-D-F TTP a s Su b st r a t es for
HIV-RT. A terminating incorporation of â-D-FTTP into
growing oligonucleotide chains by various DNA poly-
merases was described earlier.33,34 Here we compared
the ability of HIV-RT to incorporate â-L-FTTP (16) or
â-D-FTTP and â-D-TTP or â-L-TTP (15) into poly(A)‚
oligo(dT)15. As can be seen from Figure 1, HIV-RT
incorporated significant amounts of 16 but not 15 into
poly(A)‚oligo(dT)15, when given at 50 µM, whereas it
used 10 µM â-D-FTTP more efficiently as an alternative
substrate. About 30% of the primer was extended by
10 µM â-D-FTTP during 30 min (Figure 1). Such chain-
terminating incorporation of L-enantiomers by HIV-RT
has been described earlier for the triphosphates of 2’,3’-
dideoxy-â-L-thymidine, 2’,3’-didehydro-2’,3’-dideoxy-â-L-
thymidine, 3TC, and FTC.19,35,36 However, these com-
pounds are strong inhibitors of the viral enzyme (Ki
values: 0.11, 0.015, 10.6, and 1.6 µM),7,35,37 whereas the
Ki value for 16 was estimated to be 45 µM.
Also â-L-TTP (15) did not inhibit any cellular DNA
polymerases (Table 1), an observation reported recently
to be restricted to the DNA polymerases R, â, δ, and ꢀ,
whereas a high affinity to the DNA polymerase γ has
been reported.32
In contrast to the incorporation of some modified â-L-
dNTPs by HIV-RT, this enzyme and the cellular DNA
polymerases R, â and ꢀ proved to be unable to utilize
unmodified â-L-dNTPs as substrates.38

Novel â-L-2′,3′-Dideoxy-3′-fluoro Nucleosides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2043
sera11 (>100 pg of HBV (DHBV) DNA/mL) and suspended in
1/40 of the original volume in PBS; 20 µL of the virus
suspension was lysed in 2.6 µL of 6% 2-mercaptoethanol in
10% Nonidet P-40, and the endogenous DNA polymerase
activities were assayed at 37 °C for 60 min in 50 µL containing
50 mM Tris-HCl, pH 8.1, 60 mM KCl, 40 mM MgCl2, 100 µM
concentrations of the three dNTPs, and, on the basis of the
inhibitor tested (TTP or dCTP analogues), 0.4 µM [3H]TTP or
[3H]dCTP (8500 cpm/pmol).12
Con clu sion s
The newly synthesized â-L-enantiomers of 3’-fluoro-
modified pyrimidine nucleoside 5’-triphosphates emerged
as effective inhibitors of HBV/DHBV DNA polymerases.
They proved to be equally or less effective than their
â-D-counterparts. However, the â-L-enantiomers were
completely inactive against HIV-RT, whereas the cor-
responding â-D-enantiomers have been described earlier
as very efficient inhibitors of both HIV-RT and HBV
DNA polymerase.8-12
Inhibition of cellular DNA polymerases â and γ by
â-D-FdNTPs, which seems to be mainly responsible for
their toxicity at the cellular level, is largely abolished
by the â-L-FdNTPs. Therefore, a lower antiproliferative
activity of 3’-fluoro-modified â-L-nucleosides can be
expected than that described earlier for the correspond-
ing â-D-nucleosides. Indeed, preliminary results show
that â-L-FTdR displayed much less antiproliferative
activity against MT-4 cells (CD50 about 500 µM) than
â-D-FTdR (CD50 about 1 µM13). It remains to be seen
whether â-L-FTdR, â-L-FMetCdR, and â-L-FCdR share
some of the favorable metabolic properties of other â-L-
ddC analogues39 and of L-FMAU,40 respectively, causing
potent activity against HBV replication at the cellular
level.
Ch em istr y. Reactions were monitored by TLC using
precoated silica gel 60-F254 sheets, and compounds were
detected either with UV light or with a 10% sulfuric acid spray,
followed by heating on a hot plate. Elemental analyses were
performed by the Institut fu¨r Angewandte Chemie, Berlin.
Melting points were determed with a Boetius microscope hot
stage and are uncorrected. Optical rotations were measured
on a Perkin-Elmer model 141 polarimeter and are given in
units of 10-1 deg cm2 g-1 1H NMR spectra were recorded on
.
a Varian Unity 500 NMR spectrometer at 300 MHz using
DMSO-d6 as solvent and tetramethylsilane as internal stan-
dard; 13C NMR spectra were obtained at 75 MHz on the same
spectrometer. All chemical shifts (δ) are reported in parts per
million (ppm), and the coupling constants (J ) are quoted in
Hz. Electron impact mass spectra were measured at 70 eV
on the GC/MS-Datensystem HP 5985 B. MALDI time-of-flight
(TOF) mass spectra of the triphosphates were recorded on a
Bruker Reflex (UV laser) instrument using dihydroxybenzoic
acid as the matrix. UV spectra were recorded on a Shimadzu
UV-161A spectrometer. For column chromatography Merck
silica gel 60 (230-400 mesh) was used. Solvents and reagents
were purified and dried by standard procedures.
Exp er im en ta l Section
1-(2-Deoxy-3-O-(m eth ylsu lfon yl)-5-O-(tr iph en ylm eth yl)-
â-L-r ibofu r a n osyl)th ym in e (3). A mixture of L-thymidine
(1) (1.81 g, 7.47 mmol) and triphenylmethyl chloride (2.5 g,
8.97 mmol) in anhydrous pyridine (40 mL) was refluxed for
2.5 h. After the mixture cooled to room temperature, meth-
anesulfonyl chloride (1.8 mL, 22.5 mmol) was added to the ice-
cooled solution. The reaction mixture was kept at room
temperature overnight. Water (1 mL) was added, and the
solution was dropped into 1.2 L of vigorously stirred ice-water.
The resulting amorphous precipitate was collected by filtration,
washed with water, and dried in vacuo. Compound 2 so
obtained (3.9 g, 7 mmol, 93%) was homogeneous by TLC
analysis and was used without further purification in the next
reaction step. An analytical sample was obtained by prepara-
tive TLC. Elution with EtOAc/n-hexane (3/1) afforded pure 3
as a foam: [R]20D +8.74° (c ) 0.9, CHCl3); 1H NMR (Me2SO-d6)
δ 1.43 (s, 3H, CH3) (2H-2’ and CH3SO2 under Me2SO), 3.19-
3.23 (m, 2H, H-5’, H-5’’), 4.14 (m, 1H, H-4’), 5.31 (m, 1H, H-3’),
6.15 (dd, 1H, H-1’), 7.14-7.29 (m, trityl), 7.43 (s, 1H, H-6),
11.34 (br s, 1H, NH); 13C NMR (Me2SO-d6) δ 11.64 (CH3), 36.55
(CH3SO2), 37.71 (C-2’), 62.98 (C-5’), 80.05 (C-3’), 82.45 (C-4’),
83.55 (C-1’), 86.63 (quaternary trityl), 109.78 (C-5), 127.17-
127.91 (aromatic trityl), 135.50 (C-6), 147.66 (CH3SO2), 150.22
(C-2), 163.48 (C-4). Anal. (C30H30N2O7S) C, H, N.
DNA polymerase R (0.4 unit) from human placenta (pro-
vided by Dr. M. Kukhanova, Engelhardt Institute of Molecular
Biology, Russian Academy of Sciences, Moscow, Russia) was
assayed at 37 °C for 15 min in 20 µL containing 50 mM Tris-
HCl, pH 7.5, 10 mM KCl, 2 mM MgCl2, 2 mM â-mercapto-
ethanol, 100 µg/mL BSA, 0.35 µM poly(dA)‚oligo(dT)15, and 10
µM [3H]TTP (5000 cpm/pmol) for testing of the TTP-derived
inhibitors or 1 µg of activated calf thymus DNA, 5 µM [3H]-
dCTP (20 000 cpm/pmol), and 50 µM each of the three other
dNTPs and the dCTP-derived inhibitors.
DNA polymerase â (0.25 unit) purified from HeLa cells
(MoBiTec, Go¨ttingen) was assayed at 37 °C for 15 min in 20
µL containing 50 mM Tris-HCl, pH 8.7, 100 mM KCl, 10 mM
MgCl2, 1 mM DTT, 0.4 mg/mL BSA, poly(dA)‚oligo(dT)15, and
[3H]TTP or activated DNA and the three dNTPs, [3H]dCTP,
and inhibitors as described above.
DNA polymerase γ (0.5 unit; provided by Dr. H. Ko¨nig, Paul
Ehrlich Institut, Langen, Germany) was assayed at 37 °C for
20 min in 20 µL containing 50 mM Tris-HCl, pH 8.2, 150 mM
KCl, 5 mM Mg-acetate, 1 mM DTT, 100 µg/mL BSA, 1 µg of
activated DNA, 50 µM concentrations each of the three dNTPs,
and, on the basis of the inhibitor tested (TTP or dCTP
analogues), a 5 µM concentration of the fourth one as a labeled
triphosphate ([3H]TTP, 5000 cpm/pmol; [3H]dCTP, 20 000 cpm/
pmol).
DNA polymerases δ and ꢀ (0.05 unit), each purified from
calf thymus according to Weiser et al.,41 were assayed at 37
°C for 30 min in 25 µL containing 50 mM Bis-Tris, pH 6.5, 6
mM MgCl2, 1 mM DTT, 250 µg BSA/mL, 3.5 µM poly(dA)‚
oligo(dT)15, and 10 µM [3H]TTP (5000 cpm/pmol) or activated
DNA, substrates, and the dCTP analogues as described above.
Recombinant PCNA (10 µg/mL)42,43 was added to each DNA
polymerase δ assay.
1-(2-Deoxy-5-O-(tr ip h en ylm eth yl)-â-L-xylofu r a n osyl)-
th ym in e (5). Compound 3 (4.4 g, 7.8 mmol) was dissolved in
ethanol (60 mL) and heated at reflux. Sodium hydroxide (0.36
g, 9 mmol) was added, and refluxing was continued for 1.5 h.
After cooling to room temperature the reaction mixture was
concentrated to 20 mL and poured into water (500 mL). The
turbid solution was extracted with chloroform (3 × 75 mL).
The extracts were combined and washed successively with a
saturated aqueous solution of NaHCO3 and water. The
organic phase was dried (MgSO4), filtered, and evaporated. The
resulting residue was purified by column chromatography on
silica gel with chloroform (1% methanol, 0.1% triethylamine)
as eluant to give 5 (2.64 g, 5.45 mmol, 69.7%), which crystal-
HIV-RT (0.05 unit; Boehringer, Mannheim) was assayed at
37 °C for 60 min in 20 µL containing 50 mM Tris-HCl, pH 8.0,
150 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, 5 mM DTT, 0.3
mM GSH, 0.35 µM poly(A)‚oligo(dT)15, and 10 µM [3H]TTP
(5000 cpm/pmol) for testing of the TTP-derived inhibitors or 1
µg of activated calf thymus DNA, 5 µM [3H]dCTP (10 000 cpm/
pmol), and 50 µM each of the three other dNTPs and the dCTP-
derived inhibitors.8
lized from acetone: mp 236-239 °C dec; [R]20 =12.51° (c )
D
1, CHCl3); 1H NMR (Me2SO-d6) δ 1.59 (s, 3H, CH3) (2H-2’ under
Me2SO), 3.15-3.34 (m, 2H, H-5’, H-5’’), 4.04 (m, 1H, H-4’), 4.13
(m, 1H, H-3’), 5.16 (s, 1H, 3’-OH), 6.07 (dd, 1H, H-1’), 7.21-
7.38 (m, trityl), 7.55 (s, 1H, H-6), 11.23 (br s, 1H, NH); 13C
NMR (Me2SO) δ 12.38 (CH3), 40.68 (C-2’), 62.94 (C-5’), 68.83
(C-3’), 83.14 (C-4’), 84.04 (C-1’), 85.99 (quaternary trityl),
For estimation of HBV and DHBV DNA polymerase activi-
ties, virus particles were pelleted from HBV- or DHBV-positive

2044 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
von J anta-Lipinski et al.
108.20 (C-5), 126.90-128.19 (aromatic trityl), 136.64 (C-6),
150.39 (C-2), 163.71 (C-4). Anal. (C29H28N2O5) C, H, N.
λmin 253 nm (ꢀ 4320); UV (H2O, pH 1) λmax 286 nm (ꢀ 11 360),
1
λmin 244 nm (ꢀ 800); [R]D -31.91° (c ) 1.0, MeOH); H NMR
(Me2SO-d6) δ 1.85 (s, 3H, CH3), 2.11-2.17 (m, 1H, H-2’), 2.20-
2.25 (m, 1H, 2-H’’), 3.59-3.52 (m, 2H, H-5’, H-5’’), 4.13 (m,
1H, J 4’ - F ) 27.4, H-4’), 5.18 (d, 1H, OH-5’), 5.22 (m, 1H, J 3’ -
1-(2,3-Did eoxy-3-flu or o-5-O-(tr ip h en ylm eth yl)-â-L-r ibo-
fu r a n osyl)th ym in e (7). To a solution of 5 (2.55 g, 5.9 mmol)
in dichloromethane (60 mL) was added DAST (1.4 mL), and
the reaction mixture was stirred at room temperature for 1 h.
The mixture was diluted with chloroform (60 mL) and poured
into saturated sodium bicarbonate solution (200 mL). The
organic phase was separated, washed with water (20 mL),
dried (Na2SO4), filtrated, and evaporated in vacuo. The
residue was purified by chromatography on a silica gel column
using chloroform (1% methanol, 0.1% triethylamine) as eluant.
7 was isolated as a foam (1.45 g, 50.5%): [R]20D -23.20° (c ) 1,
) 53.7, H-3’), 6.13 (dd, 1H, J 1’ - 2’ ) 5.4, J 1’ - 2’’ ) 5.9, H-1’),
F
7.79 (s, 1H, H-6), 8.09 (br s, 1H, NH2), 8.23 (br s, 1H, NH2);
13C NMR (Me2SO-d6) δ 30.59 (CH3), 42.88 (C-2’, J F - 2’ ) 20.3),
65.86 (C-5’), 90.17 (C-1’), 90.50 (C-4’), 99.97 (C-3’, J F
174.2), 107.30 (C-5), 144.81 (C-6), 156.17 (C-2), 167.46 (C-4).
)
-
3’
Anal. (C10H14N3O3F‚HCl) C, H, N.
1-(2-Deoxy-3-O-(m eth ylsu lfon yl)-5-O-(tr iph en ylm eth yl)-
â-L-r ibofu r a n osyl)u r a cil (4). 2’-Deoxy-â-L-uridine (11.41 g,
50 mmol) was dissolved in pyridine (250 mL), and triphenyl-
methyl chloride (16.7 g, 60 mmol) was added. The mixture
was heated to 100 °C for 1 h. The solution was chilled to 0 °C
followed by the addition of methanesulfonyl chloride (12 mL,
60 mmol). After workup similar to that for 2, precipitation in
water yielded 4 as a solid (21.4 g, 39 mmol, 78%): 1H NMR
(Me2SO-d6) δ (2H-2’, 3H (mesyl group) under Me2SO), 3.32-
3.35 (m, 2H, H-5’, H-5’’), 4.17 (t, 1H, H-4’), 5.30 (d, 1H, H-3’),
1
CHCl3); H NMR (Me2SO-d6) δ 1.36 (s, 3H, CH3), 3.04-3.18
(m, 2H, H-2’, H-2’’), 3.30-3.62 (m, 2H, H-5’, H-5’’), 4.17 (dt,
1H, J 4’ - F ) 27.6, H-4’), 5.36 (dd, 1H, J 3’ - F ) 53.7, H-3’), 6.17
(dd, 1H, J ) 5.6 and 8.9, H-1’), 7.27-7.35 (m, trityl), 7.44 (s,
1H, H-6), 11.34 (br s, 1H, NH); 13C NMR (Me2SO-d6) δ 12.27
(CH3), 37.36 (C-2’), 63.37 (d, C-5’, J F - C5’ ) 9.8), 82.86 (d, C-4’,
J F C4’ ) 25.7), 86.67 (quaternary trityl), 94.20 (d, C-3’, J F
-
-
) 174.7), 109.79 (C-5), 127.18-128.13 (aromatic trityl),
C3’
135.32 (C-6), 150.27 (C-2), 163.45 (C-4). Anal. (C29H27N2O4F)
C, H, N.
5.46 (d, 1H, H-5, J 5
(m, 15H, trityl), 7.64 (d, 1H, H-6, J 6
) 8.1), 6.15 (dd, 1H, H-1’), 7.32-7.38
-
6
) 8.1), 11.41 (s, 1H,
-
5
NH); 13C NMR (Me2SO) δ 36.49 (mesyl), 37.62 (C-2’), 62.70
(C-5’), 79.54 (C-3’), 82.42 (C-4’), 84.16 (C-1’), 86.56 (quaternary
trityl), 101.74 (C-5), 127.89-128.17 (aromatic trityl), 140.42
(C-6), 150.15 (C-2), 163.38 (C-4). Anal. (C29H28N2O7S) C, H,
N.
1-(2,3-Did eoxy-3-flu or o-â-L-r ibofu r a n osyl)th ym in e (9).
A solution of 7 (1.45 g, 2.98 mmol) in aqueous acetic acid (80%,
50 mL) was heated to 90 °C for 1 h. After cooling the solvent
was removed under reduced pressure, and the residue was
purified chromatographically on silica gel with chloroform
(1.5% methanol) as eluant. Evaporation of the appropriate
fractions afforded the title compound (360 mg, 49.5%) which
crystallized from ethyl acetate: mp 176-177 °C (methanol);
UV (H2O, pH 7) λmax 265 nm (ꢀ 7620), λmin 234 nm (ꢀ 2070);
1-(2-Deoxy-5-O-(tr ip h en ylm eth yl)-â-L-xylofu r a n osyl)-
u r a cil (6). A mixture of 4 (28.0 g, 51 mmol), sodium hydroxide
(3.4 g, 85 mmol), and ethanol (400 mL) was refluxed for 2 h.
After workup similar to that for 3, purification by silica gel
column chromatography (chloroform-methanol-triethylamine,
98:2:0.1) gave 6 (11.2 g, 23.8 mmol, 46.6%) which was crystal-
MS m/z 244 (M+), 126 (base + H), 119 (C5H5O2F, sugar moiety);
1
[R]20 +1.44° (c ) 0.9, methanol); H NMR (Me2SO-d6) δ 1.85
D
(s, 3H, CH3), 2.19-2.26 (m, 2H, H-2’, H-2’’), 3.54-3.75 (m, 2H,
lized from methanol: mp 224-226 °C; [R]20 -6.37° (c ) 1,
D
H-5’, H-5’’), 4.08 (dt, 1H, H-4’, J 4’ - ) 27.9), 5.17 (br s, 1H,
CHCl3); 1H NMR (Me2SO-d6) δ (2H-2’ under Me2SO), 3.20-
3.34 (m, 1H, H-5’), 3.37-3.40 (m, 1H, H-5’’), 4.08-4.12 (m, 1H,
H-4’), 4.18 (m, 1H, H-3’), 5.27 (d, 1H, OH-3’), 5.55 (d, 1H, H-5,
F
5’-OH), 5.24 (dd, 1H, J ) 4.4, J 3’ - ) 54.2, H-3’), 6.15 (dd,
F
1H, J ) 5.4 and 9.1, H-1’), 7.63 (s, 1H, H-6), 11.35 (br s, 1H,
NH); 13C NMR (Me2SO-d6) δ 12.16 (CH3), 36.78 (C-2’, J ) 20.1),
60.75 (C-5’, J ) 11.1), 83.58 (C-1’), 84.67 (C-4’, J ) 22.4), 94.75
J 5
) 8.1), 6.12 (dd, 1H, H-1’), 7.36-7.44 (m, 15H, trityl),
-
6
7.73 (d, 1H, H-6, J 6
) 8.1), 11.28 (s, 1H, NH); 13C NMR
-
5
(C-3’, J F C3’ ) 173.8), 109.66 (C-5), 135.64 (C-6), 150.37 (C-
(Me2SO) δ 40.79 (C-2’), 60.79 (C-5’), 68.73 (C-3’), 83.40 (C-4’),
84.28 (C-1’), 85.98 (quaternary trityl), 100.88 (C-5), 126.92-
128.19 (aromatic trityl), 140.95 (C-6), 150.41 (C-2), 163.14 (C-
4). Anal. (C28H26N2O5) C, H, N.
-
2), 163.54 (C-4). Anal. (C10H13N2O4F) C, H, N.
1-(5-O-Acetyl-2,3-d id eoxy-3-flu or o-â-L-r ibofu r a n osyl)-
th ym in e (11). A mixture of 9 (240 mg, 0.98 mmol) and acetic
anhydride (1.5 mL, mmol) in pyridine (6 mL) was kept at room
temperature overnight. After removal of the solvent under
reduced pressure, the residue was purified by silica gel column
chromatography with chloroform (1% methanol) as eluant to
give 11 as a white foam (262 mg, 93%): [R]20D -8.74° (c ) 0.9,
1-(2,3-Did eoxy-3-flu or o-5-O-(tr ip h en ylm eth yl)-â-L-r ibo-
fu r a n osyl)u r a cil (8). Compound 6 (10.3 g, 21.8 mmol) was
suspended in dichloromethane (250 mL), and DAST (3.3 mL,
25 mmol) was added. Treatment according to the procedure
for 7 gave 8 as a solid material (4.9 g, 10.4 mmol, 47.7%): [R]20
D
1
CHCl3); H NMR (Me2SO-d6) δ 1.73 (s, 3H, CH3), 2.02 (s, 3H,
-28.51° (c ) 0.9, CHCl3); 1H NMR (Me2SO-d6) δ 2.33-2.38
CH3, acetyl), 2.33-2.43 (m, 2H, H-2’, H-2’’), 4.13-4.19 (m, 2H,
(m, 1H, H-2’), 2.41-2.48 (m, 1H, H-2’’), 3.20-3.24 (m, 1H,
H-5’, H-5’’), 4.26 (dt, 1H, H-4’, J 4’
) 26.8), 5.28 (dd, 1H,
H-5’), 3.32-3.38 (m, 1H, H-5’’), 4.26 (dt, 1H, H-4’, J F
- 4’
)
-
F
H-3’, J 3’ - F ) 54.7), 6.24 (dd, 1H, H-1’, J 1’ - 2’ ) 8.3, J 1’ - 2’’
6.8), 7.42 (d, 1H, H6), 11.32 (br s, 1H, H-N3); 13C NMR (Me2-
SO-d6) δ 11.70 (CH3), 20.14 (CH3, acetyl), 35.70 (C-2’, J F
)
26.2), 5.35 (dd, 1H, H-3’, J F - 3’ ) 54.3), 5.43 (d, 1H, H-5, J 5
-
) 8.2), 6.16 (m, 1H, H-1’), 7.35-7.39 (m, 15 H, trityl), 7.59
6
)
(d, 1H, H-6, J 6 - 5 ) 8.2), 11.38 (br s, 1H, NH); 13C NMR (Me2-
- 2’
20.5), 62.77 (C-5’), 81.09 (C-4’, J F - 4’ ) 24.1), 83.77 (C-1’), 93.01
(C-3’, J F - 3’ ) 175.5), 109.46 (C-5), 135.24 (C-6), 149.98 (C-2),
163.15 (C-4), 169.63 (CdO, acetyl). Anal. (C12H15N2O5F) C,
H, N.
SO) δ 36.91 (C-2’, J F
82.98 (C-4’, J F
) 20.9), 63.16 (C-5’, J F
- 5’
) 9.8),
-
2’
) 25.2), 84.26 (C-1’), 86.60 (quaternary
-
4’
trityl), 93.37 (C-3’, J F 3’ ) 175.1), 127.94-128.14 (aromatic
-
trityl), 140.14 (C-6), 150.18 (C-2), 162.78 (C-4). Anal.
(C28H25N2O7F) C, H, N.
1-(2,3-Did eoxy-3-flu or o-â-L-r ibofu r a n osyl)-5-m eth ylcy-
tosin e (13). A mixture of 1-(5-O-acetyl-2,3-dideoxy-3-fluoro-
â-L-ribofuranosyl)thymine (286 mg, 1 mmol), 1,2,4-triazole (136
mg, 2 mmol), and 4-chlorophenyl dichlorophosphate (0.244 mL,
1.53 mmol) in pyridine (6 mL) was kept for 3 days at room
temperature. Ammonium hydroxide (40 mL) was added, and
the solution was allowed to stand overnight. The solvent was
removed in vacuo. The resulting residue was dissolved in
water (50 mL) and applied on a column of DOWEX W ×8 (H+-
form). Elution with water (800 mL) and with ammonia (5%,
300 mL) gave 13 as crude product. This material was purified
by chromatography (silica gel, eluant: chloroform/15% metha-
nol) to give 13 which was isolated as the hydrochloride from
methanol/HCl (314 mg, 41%): mp 139-142 °C (2-propanol);
MS m/z 243 (M+ - HCl); UV (H2O, pH 7) λmax 278 nm (ꢀ 8350),
1-(2,3-Did eoxy-3-flu or o-â-L-r ibofu r a n osyl)u r a cil (10).
Compound 8 (4.9 g, 10.4 mmol) was treated according to the
procedure for 7 to give 10 (2.1 g, 9.1 mmol, 87.7%) as a white
solid which crystallized from methanol: mp 187-188 °C; [R]20
D
-11.27° (c ) 1, H2O); UV (H2O, pH 7) λmax 261 nm (ꢀ 10 100),
λmin 230 nm (ꢀ 2400); 1H NMR (Me2SO-d6) δ 2.18-2.23 (m, 1H,
H-2’), 2.32-2.39 (m, 1H, H-2’’), 3.61 (m, 2H, H-5’, H-5’’), 4.16
(dt, 1H, H-4’, J 4’ - F ) 27.6), 5.20 (d, 1H, OH-5’), 5.28 (dd, 1H,
H-3’, J 3’ - ) 54.3), 5.67 (d, 1H, H-5, J 5
) 8.1), 6.19 (m,
F
- 6
1H, H-1’), 7.85 (d, 1H, H-6, J 6 - 5 ) 8.1), 11.36 (br s, 1H, NH);
13C NMR (Me2SO) δ 37.14 (C-2’, J F - 2’ ) 20.1), 60.73 (C-5’, J F
5’ ) 11.3), 83.99 (C-1’), 84.88 (C-4’, J F 4’ ) 22.9), 94.79 (C-
-
-
3’, J F
) 173.7), 102.04 (C-5), 140.12 (C-6), 150.33 (C-2),
-
3’
162.91 (C-4). Anal. (C9H11N2O4F) C, H, N.

Novel â-L-2′,3′-Dideoxy-3′-fluoro Nucleosides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2045
Ta ble 2. MALDI TOF Mass Spectral Analyses of the
3’-Fluoro-Modified â-L-Nucleoside Triphosphates
Ackn owledgm en t. The authors thank Dr. P. Franke,
Institute of Biochemistry, Free University Berlin, for
performing the mass spectra of the triphosphates.
compound
calcd for (M - H)-
found (M - H)-
16 (L-FTTP)
C10H15N2O13FP3: 483.16
C9H13N2O13FP3: 469.13
C9H14N3O12FP3: 468.14
C10H16N3O12FP3: 482.18
482.9
469.0
468.4
482.2
17 (L-FdUTP)
18 (L-FdCTP)
19 (L-FMedCTP)
Refer en ces
(1) Beach, J . W.; J eong, L. S.; Alves, A. J .; Pohl, D.; Kim, H. O.;
Chang, C. N.; Doong, S. L.; Schinazi, R. F.; Cheng, Y.-C.; Chu,
C. K. Synthesis of Enantiomerically Pure (2’R,5’S)-(-)-1-[2-
(Hydroxymethyl)-1,3- oxathiolan-5-yl]cytosine as Potent Anti-
1-(5-O-Acetyl-2,3-d id eoxy-3-flu or o-â-L-r ibofu r a n osyl)-
u r a cil (12). Compound 10 (1 g, 4.34 mmol) was treated with
acetic anhydride (8 mmol) in pyridine (10 mL) for 10 h at room
temperature, and the solvent was evaporated to dryness under
reduced pressure. The residue was purified by silica gel
column chromatography (chloroform-methanol, 4:1) to give
viral Agent Against Hepatitis
B Virus (HBV) and Human
Immunodeficiency Virus (HIV). J . Org. Chem. 1992, 57, 2217-
2219.
(2) 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.;
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-ox-
athiolan-5-yl]cytosine. Antimicrob. Agents Chemother. 1992, 36,
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(3) Lin, T. S.; Luo, M. Z.; Liu, M. C.; Pai, S. B.; Dutschman, G. E.;
Cheng, Y.-C. Antiviral Activity of 2’,3’-Dideoxy-â-L-5-fluorocy-
tidine (â-L-FddC) and 2’,3’-Dideoxy-â-L-cytidine (â-L-ddC) against
Hepatitis B Virus and Human Immunodeficiency Virus Type 1
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(4) Gosselin, G.; Schinazi, R. F.; Sommadossi, J .-P.; Mathe, C.;
Bergogne, M.-C.; Aubertin, A.-M.; Kirn, A.; Imbach, J .-L. Anti-
Human Immunodeficiency Virus Activities of the â-L-Enanti-
omer of 2’,3’-Dideoxycytidine and Its 5-Fluoro Derivative in
Vitro. Antimicrob. Agents Chemother. 1994, 38, 1292-1297.
(5) 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-arabinosyluracil as Novel Antiviral Agent
for Hepatitis B Virus and Epstein-Barr Virus. Antimicrob.
Agents Chemother. 1995, 39, 979-981.
(6) Lin, T. S.; Luo, M. Z.; Liu, M. C.; Zhu, Y. L.; Gullen, E.;
Dutschman, G. E.; Cheng, Y.-C. Design and Synthesis of 2’,3’-
Dideoxy-2’,3’-didehydro-â-L-cytidine (â-L-d4C) and 2’,3’-Dideoxy-
2’,3’-didehydro-â-L-5-fluorocytidine (â-L-Fd4C), Two Exception-
ally Potent Inhibitors of Human Hepatitis B Virus (HBV) and
Potent Inhibitors of Human Immunodeficiency Virus (HIV) in
Vitro. J . Med. Chem. 1996, 39, 1757-1759.
(7) Dienstag, J . L.; Perrillo, R. P.; Schiff, E. R.; Bartholomew, M.;
Vicary, C.; Rubin, M. A Preliminary Trial of Lamivudine for
Chronic Hepatitis B Infection. N. Engl. J . Med. 1995, 333, 1657-
1661.
(8) Matthes, E.; Lehmann, C.; Scholz, D.; von J anta-Lipinski, M.;
Gaertner, G.; Rosenthal, A.; Langen, P. Inhibition of HIV-
Associated Reverse Transcriptase by Sugar-Modified Derivatives
of Thymidine 5’-Triphosphates in Comparison to Cellular Poly-
merases R and â. Biochem. Biophys. Res. Commun. 1987, 148,
78-85.
(9) Cheng, Y.-C.; Dutschman, G. E.; Bastow, K. F.; Sarngadharan,
M. G.; Ting, R. Y. Human Immunodeficiency Virus Reverse
Transcriptase and Its Interactions With Nucleoside Triphos-
phate Analogues. J . Biol Chem. 1987, 262, 2187-2189.
(10) Matthes, E.; Lehmann, C.; von J anta-Lipinski, M.; Scholz, D.
Inhibition of HIV-Replication by 3’-Fluoro-Modified Nucleosides
with Low Toxicity. Biochem. Biophys. Res. Commun. 1989, 165,
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(11) Meisel, H.; Reimer, K.; von J anta-Lipinski, M.; Ba¨rwolff, D.;
Matthes, E. Inhibition of Hepatitis B Virus DNA Polymerase
by 3’-Fluorothymidine Triphosphate and other Modified Nucleo-
side Triphosphate Analogues. J . Med. Virol. 1990, 30, 137-141.
(12) Matthes, E.; Reimer, K.; von J anta-Lipinski, M.; Meisel, H.;
Lehmann, C. Comparative Inhibition of Hepatitis B Virus DNA
Polymerase and Cellular DNA Polymerases by Triphosphates
of Sugar-Modified 5-Methyldeoxycytidines and of Other Nucleo-
side Analogues. Antimicrob. Agents Chemother. 1991, 35, 1254-
1257.
12 (1.02 g, 3.75 mmol, 87.2%) which crystallized from metha-
1
nol: mp 114-115 °C; [R]20 -7.08° (c ) 1, CHCl3); H NMR
D
(Me2SO-d6) δ 2.05 (s, 3H, CH3), 2.34-2.38 (m, 1H, H-2’), 2.43-
2.49 (m, 1H, H-2’’), 4.19-4.22 (m, 2H, H-5’, H-5’’), 4.36 (dt,
1H, H-4’, J 4’ - F ) 27.6), 5.33 (dd, 1H, H-3’, J 3’ - F ) 53.9), 5.70
(d, H-5, J 5
) 8.1), 6.18 (m, 1H, H-1’), 7.65 (d, 1H, H-6, J 6
-
- 6
5 ) 8.1), 11.40 (br s, 1H, NH); 13C NMR (Me2SO) δ 20.56 (CH3),
36.41 (C2’, J F ) 20.4), 63.23 (C-5’, J F ) 10.7), 81.75
-
2’
- 5’
(C-4’, J F 4’ ) 25.9), 84.69 (C-1’), 94.0 (C-3’, J F 3’ ) 175.5),
-
-
102.22 (CO, acetyl), 140.26 (C-5), 150.36 (C-6), 162.96 (C-2),
170.08 (C-4). Anal. (C11H13N2O5F) C, H, N.
1-(2,3-Dideoxy-3-flu or o-â-L-r ibofu r an osyl)cytosin e (14).
A solution of compound 12 (2.6 g, 9.85 mmol), 1,2,4-triazole
(1.38 g, 20 mmol), and 4-chlorophenyl dichlorophosphate (2.44
mL, 15 mmol) in pyridine (60 mL) was kept for 3 days under
an argon atmosphere at room temperature and worked up
according to the procedure for 13. Compound 14 was crystal-
lized from methanol yielding 1.38 g (6.02 mmol, 61%): mp
217-219 °C; UV (H2O, pH 7) λmax 270 nm (ꢀ 10 860), λmin 249
nm (ꢀ 1700); UV (H2O, pH 1) λmax 280 nm (ꢀ 12 700), λmin 250
1
nm (ꢀ 5650); [R]20 -35.34° (c ) 1.0, H2O); H NMR (Me2SO-
D
d6) δ 2.09-2.20 (m, 1H, H-2’), 2.39-247 (m, 1H, H-2’’), 3.52-
3.57 (m, 1H, H-5’), 3.60-3.64 (m, 1H, H-5’’), 4.14 (dt, 1H, H-4’,
J F 4’ ) 27), 5.13 (t, 1H, 5’-OH), 5.27 (dd, 1H, H-3’, J F
)
-
- 3’
53.7), 5.43 (d, 1H, H-5, J 5
) 7.3), 6.21 (m, 1H, H-1’), 7.20
-
6
(br d, 2H, NH2), 7.77 (d, 1H, H-6, J 6 - 5 ) 7.3); 13C NMR (Me2-
SO) δ 37.71 (C-2’, J F ) 20.0), 60.88 (C-5’, J F ) 9.1),
-
2’
- 5’
84.73 (C-4’, J F - 4’ ) 23.0), 84.96 (C-1’), 94.25 (C-5), 95.0 (C-3’,
J F - 3’ ) 173.4), 140.22 (C-6), 154.89 (C-2), 165.48 (C-2). Anal.
(C9H12N3O3F) C, H, N.
5′-Tr ip h osp h a tes. The 5′-triphosphates were isolated by
anion-exchange chromatography on a column (2 × 60 cm) of
Fractogel TSK DEAE-650 (M) (Merck) using a linear gradient
(0.05-0.3 M) of triethylammonium bicarbonate buffer (pH
7-8) as eluant. The triphosphates were eluted at buffer
concentrations of 0.2-0.26 M. The fractions containing the
triphosphate were pooled and concentrated under diminished
pressure. Residual triethylammonium bicarbonate was re-
moved by repeated coevaporation with deionized water. Purity
of the triphosphate solutions was controlled by anion-exchange
high-performance liquid chromatography on a WAX column
(DuPont, Wilmington) using the buffer system: (A) K2HPO4/
KH2PO4 (pH 7.0, 20 mM) and (B) K2HPO4/KH2PO4 (pH 7.0, 1
M); linear gradient, 0-50% B in 35 min; flow rate, 1.5 mL/
min. The retention times for L-FTTP (16), L-FdUTP (17),
L-FdCTP (18), and L-FMedCTP (19) were in the range between
25.0 and 25.60 min. Reverse-phase HPLC was conducted
using a Kontron liquid chromatograph. Analyses were per-
formed on a Spherisorb 50DS2 column using the following
buffer system: (A) aqueous triethylammonium acetate (0.1 M,
pH 7) and (B) acetonitrile/water (95/5, v/v); linear gradient,
100-90% buffer A in 30 min; flow rate, 1 mL/min. The
retention times for compounds 16, 17, 18, and 19 were 15.01,
12.75, 11.08, and 22.91 min, respectively. The triphosphates
used for biochemical evaluation were separated from possible
contaminations by this HPLC system. The new L-dNTPs were
characterized by MALDI TOF mass spectrometry (Table 2).
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