Thiated derivatives of 2',3'-dideoxy-3'-fluorothymidine: Synthesis, in vitro anti-HIV-1 activity and interaction with recombinant drug resistant HIV-1 reverse transcriptase forms

Article abstract of DOI:10.1016/j.antiviral.2011.05.012

Various thiated analogues of thymine 2',3'-dideoxy-3'-fluoronucleoside (FLT) and their 5'-monophosphates and 5'-triphosphates were prepared with the use of modified multistep procedures. The thiated analogues of FLT and FLTMP were evaluated against the wi

Full text of DOI:10.1016/j.antiviral.2011.05.012

Antiviral Research 92 (2011) 57–63
Contents lists available at ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Thiated derivatives of 20,30-dideoxy-30-fluorothymidine: Synthesis, in vitro
anti-HIV-1 activity and interaction with recombinant drug resistant HIV-1
reverse transcriptase forms
Agnieszka Miazga a, François Hamy b, Séverine Louvel b, Thomas Klimkait b, Zofia Pietrusiewicz c,
c
c
a
a,
⇑
´
´
Anna Kurzynska-Kokorniak , Marek Figlerowicz , Patrycja Winska , Tadeusz Kulikowski
a Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warszawa, Poland
b InPheno AG, Vesalgasse 1, CH-4051 Basel, Switzerland
c Institute of Bioorganic Chemistry, Polish Academy of Sciences, 12/14 Noskowskiego St., 61-704 Poznan, Poland
a r t i c l e i n f o
a b s t r a c t
Various thiated analogues of thymine 20,30-dideoxy-30-fluoronucleoside (FLT) and their 50-mono-
phosphates and 50-triphosphates were prepared with the use of modified multistep procedures. The
thiated analogues of FLT and FLTMP were evaluated against the wild type and drug- and multidrug-
resistant strains of HIV-1, using the replicative phenotyping format of the deCIPhR assay, and showed
potent inhibition of drug-resistant HIV-1 strains at low cytotoxicity. Additionally, inhibition of
recombinant drug resistant forms of reverse transcriptase from single and multiple HIV-1 mutants by
the synthesized 50-triphosphates was investigated. The strongest inhibition was observed for K103N
Article history:
Received 11 March 2011
Revised 16 May 2011
Accepted 26 May 2011
Available online 6 June 2011
Keywords:
Anti HIV-1 agents
NRTI
and
D67 mutants and the most potent anti-HIV-1 activity against drug resistant strains and the lowest
cytotoxicity was exerted by S4FLTMP and FLTMP which may be regarded as potential anti-HIV/AIDS
HIV-1 mutants
agents.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
(Miazga et al., 2003; Poopeiko et al., 1995), potently inhibit
HIV-1 in vitro with low cytotoxicity. In the present study the
20,30-Dideoxy-30-fluorothymidine (FLT, alovudineÒ) belongs to
the most potent agents inhibiting the replication of the human
immunodeficiency virus type 1 (HIV-1) (Balzarini et al., 1988).
Some aspects of the mechanism of biological action of FLT have
been studied including its phosphorylation in a cell-free system
and in various cell lines, incorporation into DNA in a cell-free
system and in vitro termination of DNA synthesis as well as anti-
proliferative activity towards human cell lines (Langen et al.,
1972; Chidgeavadze et al., 1985; Matthes et al., 1988). FLT
50-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse trans-
criptase (RT) (Cheng et al., 1987; Matthes et al., 1987). In addition,
the development of HIV mutants resistant to FLT is slower than of
mutants resistant to other RT inhibitors. Various HIV isolates with
multidrug resistance-associated mutations showed no evidence of
resistance to FLT (Kim et al., 2001). Unfortunately, FLT exerts sub-
stantial haematologic toxicity in man. Lipophylic analogues of FLT,
20,30-dideoxy-30-fluoro-2-thiothymidine (S2FLT) and 20,30-dideoxy-
30-fluoro-4-thiothymidine (S4FLT), as already reported by us
inhibition of HIV-1 drug- and multidrug-resistant strains by newly
synthesized thioanalogues of FLT and FLTMP as well as inhibition
of HIV-1 RT mutants by thiated analogues of FLTTP were
investigated.
2. Materials and methods
2.1. Chemicals
[Methyl-3H] dTTP (45.9 Ci/mmol) was purchased from Moravek
Biochemicals Inc., Brea, CA; [a
32P] dTTP (5000 Ci/mmol) was ob-
tained from Hartmann Analytic GmbH; dNTPs were from Sigma;
DE81 (2.3 cm) circles were from Whatman (Maidstone, UK). Triton
X-100 and rotiszint eco plus LSC-universal cocktail were from
Roth (Karlsruhe, Germany); HIV PBS DNA template 50-TTTTAGT-
CAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTG-30
(50-nt) and Lys 21 primer 50-CAAGTCCCTGTTCGGGCGCCA-30
(21-nt) were synthesized in the Laboratory of DNA Sequencing
and Oligonucleotide Synthesis of the Institute of Biochemistry
and Biophysics of the Polish Academy of Science. All other reagents
used in this study were of analytical grade.
⇑
Corresponding author. Fax: +48 22 592 21 90.
E-mail address: tk@ibb.waw.pl (T. Kulikowski).
0166-3542/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2011.05.012

58
A. Miazga et al. / Antiviral Research 92 (2011) 57–63
2.2. General methods
2.3.2. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-2-ethoxythymine
(3)
Melting points (uncorrected) were measured on a Buchi Melting
Point B-540 apparatus, UV spectra were recorded on a Cary 300
instrument, using 10 mm path length cuvettes. Mass spectra were
recorded on an AMD-604 spectrometer or a Q-TOF MICROMASS
spectrometer. High resolution 1H NMR spectra were recorded on
a Varian 500 MHz in D2O, with DSS as an internal standard or in
CDCl3, with tetramethylsilane as an internal standard. 31P NMR
spectra were recorded on a Varian 200 MHz in D2O, with 85%
H3PO4 as an external standard. Thin-layer chromatography (TLC)
was run on Merck silica gel F254 glass plates developed with the
following solvents (v/v): (A) CHCl3-MeOH, 90:10; (B) CHCl3-MeOH,
95:5.
1.6 g (4 mmol) of 2 was suspended in 90 mL of absolute EtOH
and 2.6 mL (17.5 mmol) of DBU (1,8-diazabicyclo[5.4.0]undec-7-
ene) was added. The mixture was refluxed for 3 h, cooled and trea-
ted with 20 mL of Dowex 50 WX8 (pyridine form) in 30 mL of
water. The reaction mixture was stirred for 5 min, filtered and
evaporated in vacuo. The residual oil was dissolved in 50 mL of
CHCl3, washed with water, dried with Na2SO4 and concentrated
in vacuo. The crude product was purified on a silica gel column
using CHCl3-MeOH (0–5%) as eluent to yield 3: 950 mg (88%);
mp. 144–145 °C; UV kmax (pH 0) 264 nm (
e
8.3 ꢀ 103); kmax (pH
1) 258.5 nm (
e
9.4 ꢀ 103); kmax (pH 2) 256.5 nm (
e
10.8 ꢀ 103); kmax
(pH 7) 255.5 nm
(
e
10.5 ꢀ 103); kmax (pH 12) 256 nm
(e
10.1 ꢀ 103); TLC (silica gel) Rf (A) 0.47; 1H NMR d [ppm] (D2O)
7.48 (1H, d, H6), 6.44 (1H, dd, H10) 5.37 (1H, dd, H30), 4.59–4.52
(3H, m, H40, CH2CH3), 3.85 (2H, d, H50, H500), 2.84–2.75 (1H, m,
H200), 2.50–2.36 (1H, m, H20), 1.97 (3H, s, CH3), 1.43 (3H, t, CH2CH3);
ESI-MS m/z 273 (M+H)+.
2.3. Syntheses
2.3.1. 1-(2,3-Dideoxy-3-fluoro-5-O-tosyl-b-D-pentofuranosyl)-
thymine (2) (Scheme 1)
3.6 g (9.08 mmol) of 1-(2-deoxy-5-O-tosyl-b-D-threo-pentofur-
anosyl)-thymine (1) was suspended in 30 mL of CH2Cl2 and 2 mL
of DAST ((diethylamino)sulphur trifluoride) was added at 0 °C.
The mixture was stirred for 1 h at 0 °C, diluted with 100 mL of
CHCl3 and 50 mL of saturated NaHCO3 was added. The organic
layer was separated and extracted with CHCl3. The combined or-
ganic layers were washed with water, dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The residual oil
was purified by column chromatography on silica gel using
CHCl3-MeOH (0–3%) as eluent. Fractions containing 2 were com-
bined, evaporated and crystallized from EtOH to yield 1.95 g
(54%) of white crystals: mp. 148–149 °C dec.; UV kmax (pH 0)
2.3.3. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-2-thiothymine (6)
900 mg (3.3 mmol) of 3, 70 mL of acetic anhydride and 7 mg of
DMAP (4-(dimethylamino)pyridine) were stirred for 2 h at room
temperature. The mixture was evaporated to dryness with toluene
and EtOH. The residual oil was dissolved in 75 mL of CHCl3 and
washed twice with water. The organic layer was dried over anhy-
drous Na2SO4, and evaporated under reduced pressure to oil of 1-
(2,3-dideoxy-3-fluoro-b-D-pentofuranosyl)-2-ethoxythymine (4)
which was directly used in the next reaction step. Hydrogen sul-
phide gas was bubbled into a stirred solution of 4 and 4.15 mL
(33.06 mmol) of tetramethylguanidine in 20 mL of dry pyridine
at 0 °C for 1 h. The stirred reactants were allowed to warm up to
room temperature. After 12 h the mixture was diluted with
75 mL of CHCl3 and argon was bubbled through the solution
(1 h). Then, 70 mL of 1 N HCl were added, the organic layer was
separated, washed twice with water, dried over Na2SO4 and evap-
orated in vacuo. The product was purified by silica gel column
chromatography (CHCl3) and crystallized from EtOH to yield
630 mg (65%) of 1-(5-O-acetyl-2,3-dideoxy-3-fluoro-b-D-pentofur-
anosyl)-2-thiothymine (5). 600 mg (1.98 mmol) of 5 was dissolved
in 20 mL of MeOH saturated with ammonia. The mixture was stir-
265 nm
265 nm
270 nm
273 nm (
(
(
e
e
10.5 ꢀ 103), 222 nm
(
e
(e
17.6 ꢀ 103); kmax (pH 1)
19 ꢀ 103); kmax (pH 7)
10.8 ꢀ 103), 223.5 nm
(
e
10.4 ꢀ 103), 224 nm
(
e
18.2 ꢀ 103); kmax (pH 12)
e
8.3 ꢀ 103), 224.5 nm (
e
22 ꢀ 103); TLC (silica gel) Rf (B)
0.63; 1H NMR d [ppm] (CDCl3) 8.04 (1H, br s, NH), 7.79 (2H, d,
Ph), 7.41–7.39 (3H, m, H6, Ph), 6.43 (1H, dd, H10), 5.21 (1H, dd,
H30), 4.39 (1H, d, H40), 4.29 (1H, dd, H50), 4.23 (1H, dd, H500),
2.63–2.58 (1H, m, H200), 2.48 (3H, s, CH3Ph), 2.24–2.11 (1H, m,
H20), 1.96 (3H, s, CH3); MS m/z 421.0867 (M+Na)+.
O
N
O
N
O
NH
NH
N
O
O
OEt
N
ii
iii
i
TsO
TsO
HO
O
OH
O
O
F
F
1
2
3
O
O
N
O
HN
N
HN
S
OEt
S
N
N
iv
v
AcO
AcO
HO
O
O
O
F
F
F
6
5
4
Scheme 1. Synthesis of 30-fluoro-2-thiothymidine (6). (i) DAST, 0 °C, 1 h; (ii) DBU, EtOH, reflux, 3 h; (iii) Ac2O, DMAP, r.t., 2 h; (iv) H2S, TMG, Py, 0 °C, 1 h; (v) NH3/MeOH, r.t.,
2 h.

A. Miazga et al. / Antiviral Research 92 (2011) 57–63
59
m, H200, J1 ,2 = 5.5 Hz), 2.46–2.33 (1H, m, H20, J1 ,2 = 8.5 Hz), 2.09
(3H, s, CH3); ESI-MS m/z 261 (M+H)+.
0
00
0
0
red at room temperature for 2 h, concentrated under reduced pres-
sure and crystallized from EtOH to yield white crystals: 500 mg
(97%), mp. 164-165 °C dec.; UV kmax (pH 0) 278.5 nm
(
(
e
e
18 ꢀ 103), 221 nm
(
e
15.5 ꢀ 103); kmax (pH 1) 277.5 nm
2.3.7. General procedure for the synthesis of nucleoside 50-
monophosphates (Scheme 3)
To an ice-cooled solution of dried 1,2,4-triazole (314 mg,
4.4 mmol) in 8 mL of dry 1,4-dioxane was added POCl3 (150 lL,
18.2 ꢀ 103), 220 nm
(
e
16 ꢀ 103); kmax (pH 2) 277.5 nm
(e
18.4 ꢀ 103), 219 nm (
e
16 ꢀ 103); kmax (pH 7) 278 nm (
e
e
17.8 ꢀ 103), 220.5 nm (
e
15.6 ꢀ 103); kmax (pH 12) 241.5 nm (
24.7 ꢀ 103); TLC (silica gel) Rf (A) 0.57; 1H NMR d [ppm] (D2O)
7.89 (1H, d, H6), 7.08 (1H, dd, H10) 5.35 (1H, dd, H30), 4.45 (1H,
m, H40), 3.87 (2H, d, H50, H500), 2.94–2.85 (1H, m, H200), 2.31–2.18
(1H, m, H20), 1.95 (3H, s, CH3); ESI-MS m/z 261 (M+H)+.
1.6 mmol) and a solution of triethylamine (0.62 mL, 4.44 mmol)
in 2 mL of 1,4-dioxane. After stirring for 1 h at room temperature
the reaction mixture was directly filtered into a flask containing
1 mmol of nucleoside. Phosphorylation of nucleoside was com-
pleted in 15 min and then 0.5 mL of H2O was added, and left to
stand overnight at room temperature. The solvent was removed
in vacuo and the product was purified by chromatography on a
DEAE-Sephadex A-25 column eluted with a linear gradient of TEAB
(triethylammonium bicarbonate buffer) buffer (1 M, pH 7.5) (0–
0.4 M). The fractions containing pure monophosphate were pooled
and lyophilised to remove excess of buffer to yield nucleoside 50-
phosphate. The product was dissolved in a small amount of water
and 5% solution of NaI in acetone was added dropwise; the formed
precipitate was filtered off, washed several times with acetone and
dried.
2.3.4. General procedure for the synthesis of 4-thiated nucleosides
(Scheme 2)
To the solution of 0.5 mmol of acetylated nucleoside analogue
(5), (7) (Huang et al., 1991) in 5 mL of anhydrous 1,4-dioxane
300 mg (0.75 mmol) of Lawesson Reagent was added. The mixture
was refluxed for 3 h. The mixture was cooled, 1 mL of H2O was
added and the total was evaporated to dryness. The residue was
dissolved in CHCl3, extracted with NaHCO3 and brine. The organic
layer was dried over Na2SO4 and evaporated to dryness. The result-
ing oil was dissolved in 10 mL of MeOH saturated with ammonia
and the mixture was stirred at room temperature for 2 h. The mix-
ture was than evaporated in vacuo and purified on a silica gel col-
umn using CHCl3 as eluent.
2.3.8. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-2-thiothymine
50-monophosphate disodium salt (11)
Yield 276 mg (75%); 1H NMR d [ppm] (D2O) 8.03 (1H, psd, H6),
7.13–7.10 (1H, dd, H10), 5.51–5.39 (1H, dd, H30, J3 ,F = 52.5 Hz),
0
4.61–4.55 (1H, m, 40, J4 ,F = 27.5 Hz), 4.13–4.05 (2H, m, H50, H500),
0
2.3.5. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-2,4-
dithiothymine (8)
2.87–2.79 (1H, m, H200), 2.36–2.22 (1H, m, H20), 1.97 (3H, s, CH3);
Crystallized from Et2O to yield yellow crystals: 125 mg (90%),
31P NMR d [ppm] (D2O) 1.66; ESI-MS m/z 339 (MꢁH)ꢁ.
mp. 155–157 °C dec.; UV kmax (pH 0) 285 nm (
e
20.6 ꢀ 103); kmax
(pH 1) 284 nm (
e
20.3 ꢀ 103); kmax (pH 7) 279 nm (
e
18.7 ꢀ 103);
2.3.9. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-4-thiothymine
50-monophosphate disodium salt (12)
kmax (pH 12) 319 nm
(
e
22.8 ꢀ 103), 273 nm
(
e
17 ꢀ 103),
222.5 nm (
e
6.8 ꢀ 103); TLC (silica gel) Rf (A) 0.75; 1H NMR d
Yield 250 mg (65%); 1H NMR d [ppm] (D2O) 7.92 (1H, s, H6),
6.37 (1H, dd, H10) 5.51–5.36 (1H, dd, H30, J3 ,F = 53 Hz), 4.56–4.49
0
[ppm] (DMSO) 13.88 (1H, br s, NH), 8.02 (1H, d, H6), 6.84 (1H,
dd, H10) 5.32 (1H, dd, H30), 4.32 (1H, m, H40), 3.70 (2H, t, H50,
H500), 2.77–2.62 (1H, m, H200), 2.31–2.14 (1H, m, H20), 2.01 (3H, s,
CH3); ESI-MS m/z 277 (M+H)+.
(1H, m, H40, J4 ,F = 27 Hz), 4.07–3.96 (2H, d, H50, H500), 2.71–2.6
0
(1H, m, H200), 2.49–2.3 (1H, m, H20), 2.1 (3H, s, CH3); 31P NMR d
[ppm] (D2O) 3.95; ESI-MS m/z 339 (MꢁH)ꢁ.
2.3.10. 1-(2,3-dideoxy-3-fluoro-b-D-pentofuranosyl)-2,4-
dithiothymine 50-monophosphate disodium salt (13)
2.3.6. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-4-thiothymine (9)
Crystallized from Et2O to yield yellow crystals: 98 mg (75%),
Yield 200 mg (50%); 1H NMR d [ppm] (D2O) 8.07 (1H, s, H6), 7.03
mp. 131–132 °C; UV kmax (pH 0) 278.5 nm (
e
18 ꢀ 103), 221 nm
(1H, dd, H10) 5.52–5.38 (1H, dd, H30, J3 ,F = 53.1 Hz), 4.62–4.55 (1H,
0
(
e
15.5 ꢀ 103); kmax (pH 1) 277.5 nm (
e
18.2 ꢀ 103), 220 nm (
e
e
e
m, H40, J4 ,F = 27.1 Hz), 4.08 (2H, m, H50, H500), 2.94–2.83 (1H, m,
0
16 ꢀ 103); kmax (pH 2) 277.5 nm
16 ꢀ 103); kmax (pH 7) 278 nm
(
e
e
18.4 ꢀ 103), 219 nm
(
(
H200), 2.41–2.24 (1H, m, H20), 2.15 (3H, s, CH3); 31P NMR d [ppm]
(D2O) 4.45; ESI-MS m/z 356 (MꢁH)ꢁ.
(
e
17.8 ꢀ 103), 220.5 nm
15.6 ꢀ 103); kmax (pH 12) 241.5 nm (
24.7 ꢀ 103); TLC (silica gel)
Rf (A) 0.69; 1H NMR d [ppm] (D2O) 7.76 (1H, s, H6), 6.32 (1H, dd,
2.3.11. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-thymine 50-
monophosphate disodium salt (14)
H10) 5.41–5.29 (1H, dd, H30, J3 ,F = 53.3 Hz), 4.45–4.38 (1H, m, H40,
0
J4 ,F = 27 Hz), 3.82 (2H, d, H50, H500, J4 ,5 = 4.5 Hz), 2.75–2.67 (1H,
0
0
0
Yield 173 mg (47%); 1H NMR d [ppm] (D2O) 7.92 (1H, s, H6),
6.53 (1H, dd, H10) 5.63–5.48 (1H, dd, H30, J3 ,F = 52.7 Hz), 4.67–4.6
0
(1H, m, H40, J4 ,F = 27.3 Hz), 4.23–4.14 (2H, m, H50, H500), 2.79–
0
O
N
S
N
2.68 (1H, m, H200), 2.58–2.41 (1H, m, H20), 2.03 (3H, s, CH3); 31P
NMR d [ppm] (D2O) 0.75.
NH
NH
2.3.12. General procedure for the synthesis of nucleoside 50-
triphosphates
R
R
i,ii
AcO
HO
O
O
To the ice-cold suspension of 0.5 mmol of appropriate nucleo-
side analogue (6, 9, 10) in 1 mL of trimethylphosphate, 148 lL
(1.6 mmol) of POCl3 were added and the mixture was stirred at
0 °C. After 6 h 1.48 mL of tri-n-butylamine and a solution of 1.5 g
of bis (tri-n-butylammonium) pyrophosphate in 2 mL of DMF were
added. The mixture was stirred in an ice-bath for 30 min and TEAB
buffer was added, so as to adjust the pH to about 7. The solution
was extracted with diethyl ether and the aqueous layer evaporated
to dryness. The residue was purified on a DEAE-Sephadex A-25
F
F
5: R = S
7: R = O
8: R = S
9: R = O
Scheme 2. Synthesis of 30-fluoro-2,4-dithiothymidine (8) and 30-fluoro-4-thiot-
hymidine (9). (i) Lawesson Reagent, 1,4-dioxane, reflux, 3 h; (ii) NH3/MeOH, r.t., 2 h.

60
A. Miazga et al. / Antiviral Research 92 (2011) 57–63
R2
NH
6: R1 = S, R2 = O
R1
N
8: R1 = O, R2 = S
9: R1 = S, R2 = S
10: R1 = R2 = O
HO
O
F
i
ii,iii
R2
N
R2
N
NH
NH
R1
O
O
P
O
O
R1
P O
O
O
P O
O
O
O
O
P O
O
O
O
F
F
11: R1 = S, R2 = O
12: R1 = O, R2 = S
13: R1 = R2 = S
15: R1 = S, R2 = O
16: R1 = O, R2 = S
17: R1 = R2 = O
14: R1 = R2 = O
Scheme 3. Synthesis of nucleoside 50-monophosphates (11–14) and 50-triphosphates (15–17). (i) 1,2,4-triazole, POCl3, 1,4-dioxane, H2O; (ii) POCl3, TMP, 0 °C, 6 h; (iii) bis(tri-
n-butylammonium) pyrophosphate, n-Bu3N, DMF, 30 min 0 °C, TEAB.
column using linear gradient of TEAB (0.1–0.6 M) as eluent. Frac-
tions containing 50-triphosphate were collected, lyophilised, and
the product was dissolved in a minimum of water and 100 mg of
NaI in 10 mL of acetone was added. The precipitated sodium salt
was collected by centrifugation, washed with acetone and dried
over P2O5.
2.3.15. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-thymine 50-
triphosphate sodium salt (17)
21 mg (21%). 1H NMR d [ppm] (D2O) 7.80 (1H, d, H6), 6.44–6.41
(1H, dd, H10), 5.60–5.49 (1H, dd, H30), 4.57–4.51 (1H, m, H40), 4.31–
4.15 (2H, m, H50, H500), 2.65–2.57 (1H, m, H200), 2.48–2.34 (1H, m,
H20), 1.93 (3H, s, CH3); 31P NMR d [ppm] (D2O) ꢁ8.80 (
c P),
ꢁ11.24 (d,
a
P), ꢁ21.02 (b P); ESI-MS m/z 483 (MꢁH)ꢁ.
2.3.13. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-2-thiothymine
50-triphosphate sodium salt (15)
2.4. Generation of HIV-1 resistant mutants
55 mg (20%). 1H NMR d [ppm] (D2O) 8.05 (1H, psd, H6), 7.15–
7.13 (1H, dd, H10), 5.54–5.42 (1H, dd, H30), 4.63–4.58 (1H, m, 40),
4.15–4.06 (2H, m, H50, H500), 2.90–2.82 (1H, m, H200), 2.38–2.25
(1H, m, H20), 1.97 (3H, s, CH3); 31P NMR d [ppm] (D2O) ꢁ7.11
HIV-1 mutants displaying high resistance to drugs while main-
taining sufficient replication capacity were selected (Table 1).
Mutations in the reference strain pNL4-3 (GenBank accession num-
ber #AF3244939) were inserted using the QuickChange mutagen-
esis kit (Stratagene) according to Manufacturer0s instructions. The
presence of mutations in the engineered proviral plasmids was
ascertained by sequencing them on ABI 310 prism sequencer.
(c
P), ꢁ11.00 (d,
a
P, J = 19.86), ꢁ21.27 (b P); ESI-MS m/z 499
(MꢁH)ꢁ.
2.3.14. 1-(2,3-Dideoxy-3-fluoro-b-D-pentofuranosyl)-4-thiothymine
50-triphosphate sodium salt (16)
2.5. Determination of antiviral activity and cytotoxicity
49 mg (18%). 1H NMR d [ppm] (D2O) 7.85 (1H, d, H6), 6.39 (1H,
dd, H10) 5.58–5.48 (1H, d, H30), 4.61–4.56 (1H, m, H40), 4.46–4.18
(2H, m, H50, H500), 2.79–2.64 (1H, m, H200), 2.45–2.36 (1H, m, H20),
Anti-HIV properties of thiated thymine 20,30-dideoxy-30-fluoro-
nucleosides and their 50-monophosphates were evaluated against
wild-type and multidrug-resistant HIV-1 strains using the replica-
tive phenotyping assay deCIPhR as described previously (Louvel
et al., 2008; Opravil et al., 2010; Fehr et al., 2011). For determining
2.12 (3H, s, CH3); 31P NMR d [ppm] (D2O) ꢁ7.12 (
P), ꢁ10.73 (d,
c
a
P), ꢁ20.94 (b P); ESI-MS m/z 499 (MꢁH)ꢁ.

A. Miazga et al. / Antiviral Research 92 (2011) 57–63
61
Table 1
HIV-1 drug resistant mutants.
cooled at room temperature. The mixtures were subsequently
stored at ꢁ20 °C.
Mutant Number of Resistance to drug
mutations
2.7.2. Assay of HIV-1 reverse transcriptase activity
M184V
K103N
K65R
1
1
1
4
7
Lamivudine,
Efavirenz,
Tenofovir,
Emtricitabine
Nevirapine
Abacavir
The activities of HIV-1 RTs were assayed by DE81 filter isotopic
method (Reardon and Miller, 1990) as was described previously
´
(Winska et al., 2010). Reaction mixtures contained 75 mM KCl,
M4a
The class of thymidine analogues
All classes of nucleosidic and non-nucleosidic RT
inhibitors
67b
5 mM NaCl, 50 mM Tris–HCl, pH 8.3, 6 mM MgCl2, 0.01% Triton
D
X-100, 5 mM DTT, 50
DPM/pmol), 15 g/ml HIV PBS DNA and one of the following:
10 ng WT (1 g/
70 ng K103N (0.2
l
M dATP, dCTP, dGTP, [3H]dTTP (200–600
SQc
28
l
a
b
c
D67N, T69D, T215Y, K219Q.
Deletion of D67, substitutions: T69G, K70R, L74I, K103N, T215F, K219Q.
E29Q, M41L, E44D, K49R, K64H, T69D, L74V, V90I, K103N, V108I, F116S, V118I,
l
l
l
l), 10 ng
D
67 (1
l
g/
l
l), 30 ng M4 (0.3
l
g/
l
l
l),
g/
g/ll) or 132 ng multi-mutant SQ (0.265
E169K, M184V, D192N, I195T, G196E, E203K, L210Y, R211S, T215Y, K223E, L228R,
M230L, S251N, L283I, R284K, E297K.
ll). All the reactions were initiated with enzyme in a total volume
of 20 l and incubated at 37 °C for 30 min.
l
2.7.3. Inhibition studies
IC50 values for the studied compounds were determined at
3.5 mM ATP with minimum seven concentrations of each inhibitor
tested in the range of 0.01–1000
K103N and WT, and 50 M dTTP for multi-mutant SQ and calcu-
the effective concentration of 50% (EC50), test compounds were as-
sayed across a range of concentrations and data were modelled
using a curve fitting software (XLfit, idbs) yielding a dose–response
curve from which EC50 was extrapolated.
In addition, the Alamar blue cell viability assay (Invitrogen) was
used to determine the cytotoxic dose of 50% (CD50) for each ana-
logue. Data were modelled with the help of a curve fitting software
(XLfit, idbs) yielding a dose–response curve from which CD50 was
extrapolated.
lM at 10 lM dTTP for D67, M4,
l
lated using the equation in GOSA fit (Global Optimisation by Sim-
ulated Annealing) Bio-Log software:
ðmax ꢁ minÞ
Signal ¼ min þ
1 þ 10ðlog Xꢁlog IC50Þ
where IC50 is the concentration of unlabelled ligand that inhibits
50% of the binding of a fixed concentration of the radioligand. X is
the log of the concentration of the unlabelled ligand.
2.6. Generation of reverse transcriptase mutants
Proviral plasmids containing cDNAs of genomic RNA of the HIV-
1 variants resistant to commonly used anti-HIV-1 RT drugs were
provided by InPheno. In order to obtain a collection of recombinant
RTs, an HIV-1 RT expression vector, generously provided by S.H.
Hughes, was applied (Clark et al., 1995). Originally, this vector
was used to produce the wild type HIV-1 RT (HIV-1 RT WT) in Esch-
erichia coli. In the first stage, fragments of the proviral plasmids
encoding the drug-resistant RTs were amplified by PCR. Next, the
wild type RT coding sequence present in the expression vector
was replaced with the PCR generated products. The structures of
the newly created plasmids were determined by sequencing of at
least three individual clones for each prepared construct. The
expression, isolation and purification of recombinant RTs were car-
ried out according to the protocol previously described for HIV-1
RT WT (Clark et al., 1995). The expression of each recombinant
2.8. Steady-state kinetic assays
Kampp values for
variable concentrations of [3H]dTTP (200–600 DPM/pmol) in the
range of 0.5–192 M, and for SQ at variable concentrations of
D67, M4, K103N and WT were determined at
l
[
a
32P] dTTP (1000 CPM/pmol) in the range of 10–360
l
M with
app
20 lg/ml HIV PBS DNA. V
and Kampp values were calculated using
max
nonlinear regression by fitting of the experimental data to Michae-
lis–Menten equation in GOSA fit (Global Optimisation by Simulated
Annealing) Bio-Log software. Kai pp values were calculated with the
use of Cheng and Prusoff (1973) equation: Ki = IC50/(1 + [S]/Km).
3. Results and discussion
RT: WT, M184V, K103N, K65R, M4,
D67 and SQ was carried out
3.1. Anti-HIV activity
in the BL21(DE3)pLysS E. coli strain. In each case, the total protein
fraction was isolated and subjected to a two-step purification: (i)
affinity purification on Ni-column, and (ii) ion-exchange column
purification. Western blot analysis and MALDI-TOF mass spec-
trometry analysis were applied to confirm that the expected re-
combinant HIV-1 RTs were indeed produced in bacteria. The
experiments undertaken did not allow to obtain sufficient amounts
of homogenous preparations of M184V and K65R mutants. Thus,
Anti-HIV properties of thioanalogues of FLT and FLTMP were
evaluated against HIV-1 wild type as well as drug and multidrug
resistant HIV-1 strains, using the replicative phenotyping format
of the deCIPhR assay (Table 2).
Most of the thiated nucleoside analogues are not more active
than the parent unthiated compounds. However, S4FLTMP (12) is
the exception, as its activity against the HIV-1 WT strain is similar
to that of FLTMP (14) (and even better in the case of K103N mutant
strain) as well as to that of the reference compound FLT (10) with-
out increase of cytotoxicity. It should be underlined that S4FLTMP
(12) exerts at least equipotent antiviral activity against the multi-
the collection of five recombinant RTs, WT, K103N, M4,
D67 and
SQ, were used for further studies. The polymerase activity of all ob-
tained HIV-1 RT mutants was confirmed in the primer extension
reactions involving either a DNA or RNA template annealed to
the 50 end-labelled DNA primer.
drug resistant HIV-1
D67 (EC50 98 nM) and SQ (EC50 103 nM)
strains as the reference compound FLT. Both S4FLTMP (12) and
FLTMP (14) may be regarded as a potential agent against HIV-1
drug and multidrug resistant HIV-1 strains.
2.7. Assays of HIV-1 reverse transcriptase activity and inhibition
studies
The results obtained for the thiated FLTMP analogues 11, 12 and
13 point to a more important role of 4-thio- than 2-thio- or 2,4-
dithio-substituents in the thymine moiety of FLTMP in enhancing
the antiviral activity without increase in cytotoxicity in the deC-
IPhR assay (Table 2). This may be related to the closer steric anal-
2.7.1. Preparation of template-primer
21-nt primer Lys21 and 50-nt HIV PBS DNA were mixed at mo-
lar ratio of 3:1 with 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 6 mM
MgCl2 and 10 mM DTT, heated at 95 °C for 3 min and then slowly

62
A. Miazga et al. / Antiviral Research 92 (2011) 57–63
Table 2
Anti-HIV-1 activity of synthesized analogues of FLT in deCIPhR™ assay.
Compound
EC50
WT
(l
M)a
CD50 (l
M)b
M184V
K103N
K65R
M4
D
67
SQ
S2FLT (6)
1.75
0.61
NAc
0.22
1.56
0.51
NAc
0.14
0.043
NAc
1.12
0.27
NAc
0.11
0.011
NAc
3.02
0.51
NAc
0.25
0.48
NAc
NAc
NAc
11.7
NCd
NCd
NCd
NCd
NCd
NCd
20.2
S4FLT (9)
NAc
NAc
S2,4FLT (8)
S2FLTMP (11)
S4FLTMP (12)
S2,4FLTMP (13)
FLTMP (14)
FLT (10)
NAc
NAc
0.5
0.4
NAc
NAc
0.024
0.095
0.031
0.098
0.103
NAc
NAc
NAc
NAc
NAc
0.029
0.021
0.02
0.027
0.03
0.018
0.086
0.032
0.042
0.016
0.139
0.131
0.159
0.069
a
b
c
Effective dose of compound achieving 50% inhibition of HIV-1 replication.
Cytotoxic dose of compound required to reduce the viability of normal uninfected cells by 50%.
Not active at the maximal tested dose of 10 M.
Not cytotoxic at the maximal tested dose of 10
l
d
lM, or extrapolated.
Table 3
Steady-state constants for HIV-1 RT variants:
D
67, M4, K103N, SQ and WT.
app
max
Kampp for dTTP SDa
(lM)
V
SDa pmol/min/
lg
Kai pp SD (
AZTTP
lM)
HIV-1 RT variant
FLTTP
S4FLTTP
S2FLTTP
WT
8.58 0.90
7.38 0.43
7.54 0.83
2.66 0.35
44.08 3.15
123.58 33.10
82.66 5.04
37.27 6.33
14.72 2.03
7.31 0.83
1.26 0.09
0.78 0.14
1.48 0.23
0.44 0.13
294.27 0.17
0.90 0.08
0.49 0.07
0.73 0.24
0.09 0.03
104.77 7.66
7.27 0.26
3.76 0.99
7.28 0.42
1.10 0.15
116.56 5.21
0.85 0.04
0.53 0.08
1.06 0.16
0.14 0.08
58.71 10.06
D
67
M4
K103N
SQ
a
SD calculated from a minimum of three independent experiments.
ogy to FLTMP of S4FLTMP (12) (conformation anti) than those of
S2FLTMP (11) and S2,4FLTMP (13) (conformations syn) congeners
RT variants (WT and mutants with TAMs, M4 and D67) only M4
demonstrated a very small increase in ATP-phosphorolytic activity
in comparison to WT (between 0% and 15%, with the highest differ-
´
(Winska et al., 2010).
Antiviral activity of FLTMP (14) is similar or several times less
(some mutant strains) than that of FLT. On the contrary, S2- and
S4-thiated nucleoside 50-monophosphates (11, 12) exert increased
antiviral activity against WT and nearly all HIV-1 mutant strains in
comparison to their parent nucleos(t)ides (6, 9). The latter in vitro
effect is rather difficult to explain. Nevertheless it should be added
that there would be an advantage to the use in patients of a drug in
the form of nucleotide instead of nucleoside as the former is much
more water soluble and allows intravenous administration in a
small volume. Moreover, nucleoside analogue 50-monophosphates
may act as extracellular prodrugs of nucleoside analogues gradu-
ally formed by hydrolytic cleavage of the 50-monophosphate moi-
ety by membrane-bound enzymes, such as phosphomonoesterase
(LePage et al., 1972, 1975). Such a slow conversion of a prodrug
to a drug provides sustained blood plasma level of the latter.
ence at 4 mM and the lowest at 8 mM ATP), whereas D67 un-
blocked the AZTMP terminated primer with even lower efficiency
than WT (data not shown). Consequently, M4 mutant demon-
strated slightly higher IC50 for AZTTP (3.44 0.54 lM), than the
WT (2.74 0.19 lM). However, with regard to the slightly lower
Kampp for dTTP in comparison to WT, the difference in IC50 values
does not reflect the difference in Kai pp values (almost the same val-
ues for WT and M4, see Table 3). Similarly to AZTTP, all the com-
pounds tested were ATP-mediated chain-terminated primers
excised from DNA by the examined RTs (WT, M4,
D67 and
K103N), with the order of excision: S4FLTTP > S2FLTTP > FLTTP
(data not shown).
3.2.2.2. Steady-state inhibition results. Amongst the tested com-
pounds the strongest inhibition was observed for K103N and
D
67 RT mutants, with Kai pp value for the latter one about 2-fold
3.2. Reverse transcriptase inhibition
lower than those for WT and M4 (Table 3).
Amongst the two studied thioanalogues, S2FLTTP was a better
inhibitor of all the examined HIV-1 RT variants than S4FLTTP, with
lower Kai pp values as compared to the latter, namely about 7–8-fold
3.2.1. Kinetics of dTMP incorporation
The rates of dTMP incorporation into HIV PBS DNA by HIV-1 RTs
were linear with respect to time and enzyme concentration with
lower for WT,
D67, M4 and K103N and about 2-fold lower for the
app
and Kampp values presented in Table 3. Steady state kinetic
SQ mutant (Table 3). The activities of S2FLTTP were comparable
(and for SQ RT even almost twice higher) than those obtained for
FLTTP.
V
max
parameters of HIV-1 SQ RT with Kampp for dTTP of 44.08 3.15
lM
app
max
and V
of 7.31 0.83 pmol/min/lg differed strongly from those
obtained for other studied HIV-1 RT variants. Kampp value for dTTP
Anti-HIV-1 inhibitory activities only partially correlate with the
steady-state inhibition values, showing FLTTP and S2FLTTP to be
better inhibitors of drug resistant HIV-1-RT variants than S4FLTTP.
Obviously, a precise correlation between enzyme inhibition and
the effects on cells cannot be expected, in spite of using a physio-
logical template. Many processes of intracellular metabolism of
nucleoside analogues, like reduction, oxidation, glycosylation and
especially phosphorylation of nucleosides in cells, can influence
app
max
for this mutant was about 5-fold higher than for WT, with a V
that was almost 17-fold lower.
3.2.2. Inhibition of HIV-1 RT variants by nucleotide analogues
3.2.2.1. Primer unblocking studies. The primer unblocking control
studies at variable concentration of ATP (2–8 mM) and fixed con-
centration of AZTTP (10 lM) showed that amongst all the tested

A. Miazga et al. / Antiviral Research 92 (2011) 57–63
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HIV-1 RT variants with TAMs, with the exception of the SQ mutant,
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of physiological concentrations of ATP.
4. Conclusions
Langen, P., Kowollik, G., Etzold, G., Venner, H., Reinert, H., 1972. The
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LePage, G.A., Naik, S.R., Katakkar, S.B., Khaliq, A., 1975. 9-b-D-arabinofuranosyl-
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On the basis of the antiviral profile, inhibition of drug resistant
HIV-1 strains and low cytotoxicity, the most effective inhibitors
were S4FLTMP (12) and FLTMP (14) (anti-HIV-1 K103N activity
EC50 = 11 nM and 30 nM, respectively, no cytotoxicity up to
10
lM).
The best thiated inhibitor S4FLTMP (12) exerts potent antiviral
activity (EC50 103 nM) against the multiresistant SQ mutant, RT
of which is 200-fold more resistant to AZTTP than that of the WT
enzyme (Table 3). S4FLTMP (12) and FLTMP (14) may therefore
be regarded as a selective potential agent against HIV-1 drug-
and multidrug-resistant strains.
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Acknowledgements
We thank Dr. Vincent Vidal for critical review and discussion of
the manuscript and Mrs. Carla Garcia Llansó for excellent technical
assistance.
This work was supported by the EC Grant LSHP-CT-2007-
037760.
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