Synthesis and antiviral evaluation of novel 4′-hydroxymethyl branched thioapiosyl nucleosides

Article abstract of DOI:10.1002/ardp.200500174

In this study, we synthesized novel 4′-hydroxymethyl branched thioapiosyl nucleosides. The thioapiosyl sugar moiety was constructed using sequential ozonolysis and reduction. The natural bases (uracil, thymine, cytosine, and adenine) were efficiently coup

Full text of DOI:10.1002/ardp.200500174

Arch. Pharm. Chem. Life Sci. 2005, 338, 577−581
DOI 10.1002/ardp.200500174
577
Synthesis and Antiviral Evaluation of Novel
4Ј-Hydroxymethyl Branched Thioapiosyl Nucleosides
Jin Woo Kim, Joon Hee Hong
College of Pharmacy, Chosun University, Kwangju, Republic of Korea
In this study, we synthesized novel 4Ј-hydroxymethyl branched thioapiosyl nucleosides. The thioapiosyl
sugar moiety was constructed using sequential ozonolysis and reduction. The natural bases (uracil,
thymine, cytosine, and adenine) were efficiently coupled using the Vorbrüggen glycosyl condensation
procedure (persilyated base and TMSOTf). The antiviral activities of the synthesized compounds were
evaluated against HIV-1, HSV-1, HSV-2, and HCMV. Compound 19 showed moderate anti-HIV ac-
tivity (EC50 ϭ 19.3 µg/mL) without exhibiting any cytotoxicity up to 100 µM.
Keywords: Keywords: Antiviral agents; Branched nucleoside; Thioapiosyl nucleoside; Ozonolysis;
Vorbrüggen condensation
Received: July 22, 2005; Accepted: September 09, 2005
Introduction
Apio dideoxynucleosides (apio-ddNs) are non-classical nu-
cleosides in which the 4Ј-hydroxymethyl group of the classi-
cal 2Ј,3Ј-dideoxynucleosides migrates to the C3Ј position
[1, 2]. Among this type of nucleoside, the adenine analogue
1 (apio-ddA) was reported to show anti-HIV activity that
was comparable to the parent 2Ј,3Ј-dideoxyadenosine [3].
Apio-ddA also appears to show metabolic resistance to
adenosine deaminase or stabilizes the glycosyl bond [4]. A
systemic structure-activity relationship study of apio dide-
oxynucleosides has been reported. Hence, there is a need to
search for new antiviral agents in this class of nucleosides.
The branched nucleosides (Figure 1), 4Ј-cyanothymidine 2
[5], 4Ј-zidothymidine 3 [6], and 4Ј-hydroxymethylthymidine
4 [7] are of particular interest because they are a new class
of compounds that have significant biological activity. How-
ever, the side effects as well as the emergence of drug-resist-
ant mutants continue to be a problem with these antiviral
agents [8, 9]. Therefore, as part of an ongoing study aimed
at discovering more potent and less toxic antiviral agents,
this study examined the effect of the isosteric replacement
of the 4Ј-oxygen by a sulfur atom. While there has been a
great deal of effort aimed at modifying the sugar and the
heterocyclic base of the natural nucleosides with the goal to
develop new antiviral agents, there has been no investigation
into 4Ј-hydroxymethyl branched thionucleosides. This paper
reports the synthesis and biological evaluation of the titled
nucleosides.
Figure 1. Structures of potent 4’-branched nucleosides, and
target nucleosides.
Chemistry
As shown in Scheme 1, the γ,δ-unsaturated ester derivative
5, which was readily synthesized from 1,3-dihydroxyacetone
using a method reported elsewhere (Scheme 1) [10], was
selected as the starting compound for the synthesis of the
4Ј-hydroxymethyl branched thioapiosyl nucleosides. The
ester 5 was treated with ozone in methylene chloride at
Ϫ78°C, followed by the decomposition of the ozonide with
dimethylsulfide (DMS) to give the aldehyde 6. Compound
6 was reduced using DIBAL-H in toluene at Ϫ20°C to give
the diol 7. After treating compound 7 with mesyl chloride,
the resulting dimesylate 8 was reacted with sodium sulfide
in DMF leading to the intermediate 9 (Scheme 1). Com-
pound 9 was oxidized to the corresponding sulfoxide with
Correspondence: Joon Hee Hong, College of Pharmacy, Chosun
University, Kwangju 501-759, Republic of Korea. Phone: ϩ82 62
230-6378, Fax: ϩ82 62 222-5414, e-mail: hongjh@chosun.ac.kr
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

578
J. W. Kim, J. H. Hong
Arch. Pharm. Chem. Life Sci. 2005, 338, 577−581
mCPBA, which was then subjected to a Pummerer re-
arrangement [11] with Ac2O at 110°C to yield the desired
glycosyl donor 10. The uracil and thymine nucleoside anal-
ogs 11 and 12 were prepared by condensing compound 10
with the corresponding per-O-silylated bases using tri-
methylsilyl trifluoromethanesulfonates (TMSOTf) as the
catalyst in 1,2-dichloroethane (DCE) (Scheme 2). The cyto-
sine nucleoside 17 was prepared from the corresponding
N4-benzoyl cytosine nucleoside analogue 13, which was pre-
pared from the corresponding N4-benzoyl cytosine using a
similar method described for 11 and 12. The adenine
nucleoside was synthesized by the condensation of com-
pound 10 with silylated 6-chloropurine using TMSOTf as a
catalyst in DCE to give the protected 6-chloropurine deriva-
tive 14, which was treated with ammonia in methanol in
a steel bomb at 95-100°C leading to compound 18. The
deprotected nucleosides 15, 16, 19, and 20 were obtained
from the corresponding nucleoside derivatives 11, 12, 17,
and 18 by a treatment with tetrabutylammonium fluoride
(TBAF).
Scheme 1. Synthesis of thiophene structure 9.
Scheme 2. Synthesis of target thioapiosyl nucleosides.
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Arch. Pharm. Chem. Life Sci. 2005, 338, 577−581
Synthesis of Novel Branched Thioapiosyl Nucleosides
579
Table 1. The antiviral activities of the synthesized compounds.
Compound
HIV-1
EC50 [mM]
HSV-1
EC50 [mM]
HSV-2
EC50 [mM]
HCMV
EC50 [mM]
Cytotoxicity
CC50 [mM]
†
†
†
†
‡
15
16
19
20
>100
>100
>19.3
>100
0.0007
Ϫ
>100
>100
>100
>100
Ϫ
>100
>100
>100
>100
Ϫ
>100
>100
38.4
>100
Ϫ
Ϫ
10.0
>100
>100
>100
>100
2.1
>10
300.00
AZT
Ganciclovir
Ribavirin
2.2
Ϫ
2.2
Ϫ
Ϫ
Ϫ, Not determined.
† EC50 [mM], Concentration required to inhibit 50 % of virus induced cytopathicity.
‡ CC50 [mM], Concentration required to reduce cell viability by 50 %.
Co. (Newark, DE, USA). The dry THF was obtained by distillation
from Na and benzophenone when the solution became purple.
Results and discussion
The synthesized compounds 15, 16, 19, and 20 were tested
against several viruses such as the HIV (MT-4 cells),
HSV-1 (CCL81 cells), HSV-2 (CCL-81 cells), and HCMV
(AD-169, Davis cells). Almost all the synthesized com-
pounds showed no antiviral activity or cytotoxicity when
tested up to 100 µg/mL. However, the cytosine analogue 19
showed moderate antiviral activity against the HIV-1
(Table 1).
Syntheses
Ethyl-3,3Ј-bis(tert-butyldimethylsilyloxymethyl)-4-oxo-
butyrate 6
A solution of compound 5 (10 g, 23.9 mmol) in anhydrous CH2Cl2
(200 mL) was cooled to Ϫ78°C and ozone gas was then bubbled
into the reaction mixture until a blue color persisted for 5 min. The
reaction mixture was then degassed with nitrogen, and methyl sulf-
ide (8.7 mL, 119 mmol) was added slowly at Ϫ78°C.
In summary, this study developed a novel synthetic method
for 4Ј-branched apiosyl nucleosides from a simple 1,3-di-
hydroxyacetone derivative. When the synthesized com-
pounds were tested against several viruses such as the HIV-1,
HSV-1, HSV-2, and HCMV, the cytosine analogue 19
showed weak antiviral activity against the HIV-1. The lack
of antiviral activity of these racemic compounds is possibly
due to their unfavorable conformations for phosphoryl-
ation, which occurred during the nucleotide activation pro-
cess. However, it is expected that the information obtained
in this study will be useful for developing novel apiosyl
nucleosides.
The mixture was stirred for 2 h at rt. (room temperature) under an
N2 atmosphere. The mixture was concentrated under reduced press-
ure and the residue was purified by silica gel column chromatogra-
phy (EtOAc/hexane, 1:30) to give compound 6 (8.7 g, 87%) as a
colorless oil; 1H NMR (CDCl3, 300 MHz) d 9.66 (s, 1H), 4.05 (q,
J ϭ 7.2 Hz, 2H), 3.84-3.64 (m, 4H), 2.36 (s, 2H), 1.19 (t, J ϭ 7.2
Hz, 3H), 0.83 (s, 18H), 0.06 (s, 6H), 0.03 (s, 6H); 13C NMR (CDCl3,
75 MHz) d 204.41, 171.06, 65.40, 60.22, 55.11, 25.48, 18.22,
14.10, -5.72.
2,2Ј-Bis(tert-butyldimethylsilanyloxymethyl)butane-1,4-diol 7
To a solution of compound 6 (4.5 g, 10.7 mmol) in anhydrous meth-
ylene chloride (150 mL), a 1.0 M solution of DIBAL-H (32.1 mL,
32.1 mmol) in hexane was added dropwise at Ϫ20°C under nitrogen
with constant stirring. The mixture was then stirred for 1 h at the
same temperature and quenched with MeOH (30 mL) which was
followed by increasing the temperature to rt. After stirring at rt. for
2 h, the resulting solid was removed by Celite filtration, and the
filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (EtOAc/hexane, 1:2)
to give compound 7 (3.52 g, 87%) as a colorless oil; 1H NMR
(CDCl3, 300 MHz) d 3.65-3.47 (m, 8H), 3.36 (t, J ϭ 5.4 Hz, 1H),
3.02 (t, J ϭ 6.0 Hz, 1H), 1.52 (t, J ϭ 5.4 Hz, 2H), 0.80 (s, 18H),
0.03 (s, 12H); 13C NMR (CDCl3, 75 MHz) δ 66.63, 65.75, 58.38,
43.65, 34.39, 25.81, 18.16, -5.66; Anal. calc. for C18H42O4Si2: C,
57.09; H, 11.18. Found: C, 57.24; H, 10.89.
Experimental
General
All the chemicals were of reagent grade and were used as purchased.
All the moisture-sensitive reactions were performed in an inert at-
mosphere of either N2 or Ar using distilled dry solvents. The melt-
ing points were determined using a Mel-temp II laboratory device
(Laboratory Devices, Holliston , MA, USA) and were uncorrected.
The NMR spectra were recorded on a JEOL JNM-LA 300 MHz-
NMR spectrometer (Jeol, Tokyo, Japan). The chemical shifts are
reported in parts per million (d) and the signals are quoted as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and dd
(doublet of doublets). The UV spectra were obtained using a
Beckman DU-7 spectrophotometer (Beckman, South Pasadena,
CA, USA). The elemental analyses were performed using an
Elemental Analyzer System (Leco-932, St. Joseph, MI, USA). TLC
was performed on Uniplates (silica gel) purchased from Analtech
Methanesulfonic acid 2,2-bis(tert-butyldimethylsilanyloxy-
methyl)-4-methanesulfonyloxy-butylester 8
To a solution of compound 7 (4.2 g, 11.09 mmol) in anhydrous
methylene chloride (100 mL) and triethylamine (7.1 mL, 50 mmol),
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J. W. Kim, J. H. Hong
Arch. Pharm. Chem. Life Sci. 2005, 338, 577−581
methanesulfonyl chloride (571 mg, 4.99 mmol) was added slowly at
0°C and the mixture was stirred 3 h at the same temperature under
N2 atmosphere. The solvent was evaporated under reduced pressure.
The residue was extracted with EtOAc/H2O, dried over MgSO4, and
filtered. The filtrate was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (EtOAc/
hexane, 1:1) to give compound 8 (4.74 g, 80%) as a colorless oil;
1H NMR (CDCl3, 300 MHz) d 4.33 (t, J ϭ 7.2 Hz, 1H), 4.08 (s,
2H), 3.61 (s, 2H), 3.41 (s, 4H), 2.95 (s, 6H), 1.79 (t, J ϭ 6.9 Hz,
2H), 0.82 (s, 18H), 0.05 (s, 12H); 13C NMR (CDCl3, 75 MHz)
δ64.78, 60.48, 57.34, 34.89, 26.64, 25.62, 18.42, -5.71; Anal. calc.
for C20H46O8S2Si2: C, 44.91; H, 8.67. Found: C, 45.12; H, 8.50.
MgSO4, filtered, and concentrated under reduced pressure. The resi-
due was purified by silica gel column chromatography (EtOAc/hex-
ane, 2:1) to give compound 11 (446 mg, 69%); 1H NMR (CDCl3,
300 MHz) d 8.11 (br s, 1H), 7.72 (d, J ϭ 8.1 Hz, 1H), 6.04 (s, 1H),
5.71 (dd, J ϭ 8.4, 2.4 Hz, 1H), 3.63 (dd, J ϭ 17.7, 10.8 Hz, 2H),
3.57 (d, J ϭ 9.9 Hz, 1H), 3.50 (d, J ϭ 9.9 Hz, 1H), 3.21 (m, 1H),
2.88 (m, 1H), 2.16 (dd, J ϭ 13.5, 6.9 Hz, 1H), 1.96 (dd, J ϭ 12.8,
8.8 Hz, 1H), 0.85 (s, 18 H), 0.02 (s, 12H); 13C NMR (CDCl3, 75
MHz) d 165.62, 153.62, 143.96, 101.32, 63.00, 62.44, 59.39, 32.05,
29.92, 25.74, 18.13, -5.62; Anal. calc. for C22H42N2O4SSi2: C, 54.28;
H, 8.70; N, 5.75. Found: C, 54.50; H, 8.72; N, 5.85.
(
)-1-[3,3Ј-C-Bis(tert-butyldimethylsilyloxymethyl)tetra-
3,3Ј-Bis(tert-butyldimethylsilanyloxymethyl)tetrahydrothio-
phene 9
hydrothiophen-2-yl] thymine 12
Compound 12 was prepared from thymine using the similar method
as described for synthesizing compound 11; yield 63%; 1H NMR
(CDCl3, 300 MHz) d 8.23 (brs, 1H), 7.45 (s, 1H), 6.02 (s, 1H), 3.71
(dd, J ϭ 13.4, 10.0 Hz, 2H), 3.58 (d, J ϭ 8.8 Hz, 1H), 3.51 (d, J ϭ
8.8 Hz, 1H), 3.24 (m, 1H), 2.85 (m, 1H), 2.16 (dd, J ϭ 13.6, 6.4 Hz,
1H), 1.90 (dd, J ϭ 13.8, 5.8 Hz, 1H), 1.30 (s, 3H), 0.87 (s, 18 H),
0.03 (s, 12H); 13C NMR (CDCl3, 75 MHz) d 165.04, 152.25, 136.45,
105.77, 64.23, 61.76, 57.56, 33.23, 29.76, 25.65, 18.28, 12.98, -5.54;
Anal. calc. for C23H44N2O4SSi2: C, 55.16; H, 8.85; N, 5.59. Found:
C, 55.02; H, 8.70; N, 5.38.
To a solution of compound 8 (2.4 g, 4.48 mmol) in DMF (10 mL),
Na2S (524 mg, 6.72 mmol) was added, and the mixture was stirred
at 0°C overnight under nitrogen. The mixture was quenched by
adding water and extracted with diethyl ether. The organic layer
was washed in brine, dried over anhydrous MgSO4, and evaporated.
The residue was purified by silica gel column chromatography
(EtOAc/hexane, 1:40) to give compound 9 (1.0 g, 60%) as a colorless
oil; 1H NMR (CDCl3, 300 MHz) d 3.49 (dd, J ϭ 12.0, 9.3 Hz, 4H),
2.79 (t, J ϭ 6.9 Hz, 2H), 2.58 (s, 2H), 1.79 (t, J ϭ 6.6 Hz, 2H), 0.91
(s, 18H), 0.03 (s, 12H); 13C NMR (CDCl3, 75 MHz) d 63.80, 54.49,
34.60, 30.07, 29.70, 25.56, 18.52, -5.47; Anal. calc. for
C18H40O2SSi2: C, 57.38; H, 10.70. Found: C, 57.21; H, 10.61.
(
)-N4-Benzoyl-1-[3,3Ј-C-bis(tert-butyldimethylsilyloxy-
methyl)tetrahydrothiophen-2-yl] cytosine 13
Compound 13 was prepared from N4-benzoyl cytosine using the
method described for synthesizing compound 11: yield 66%; 1H
NMR (CDCl3, 300 MHz) d 7.95-7.50 (m, 7H), 6.01 (s, 1H), 3.80
(dd, J ϭ 12.5, 6.0 Hz, 2H), 3.72 (dd, J ϭ 13.8, 6.2 Hz, 2H), 3.21 (t,
J ϭ 7.2 Hz, 1H), 2.83 (m, 1H), 2.15 (m, 1H), 0.99 (dd, J ϭ 12.4,
5.2 Hz, 1H), 0.84 (s, 18H), 0.02 (s, 12H); 13C NMR (CDCl3, 75
MHz) d 190.87, 162.45, 132.12, 127.65, 125.49, 64.72, 62.73, 58.78,
32.85, 30.10, 25.72, 18.65, -5.62; Anal. calc. for C29H47N3O4SSi2:
C, 59.04; H, 8.03; N, 7.12. Found: C, 58.89; H, 7.90; N, 7.26.
( )-Acetic
tetrahydrothiophen-2-yl ester 10
acid
4,4Ј-bis(tert-butyl-dimethyl-silanyloxymethyl)-
To a solution of compound 9 (1.4 g, 3.71 mmol) in CH2Cl2 (15 mL)
at Ϫ78°C, 80% mCPBA (797 mg, 3.71 mmol) in methylene chloride
(13 mL) was added. The mixture was stirred at the same tempera-
ture for 1 h, quenched with saturated NaHCO3 and extracted with
saturated CH2Cl2 two times. The organic layer was washed with a
10% sodium sulfite solution, saturated NaHCO3 and brine, dried
over MgSO4, and filtered. The filtrate was concentrated and the
residue was dissolved in Ac2O (12 mL). The mixture was stirred at
110°C for 2 h and concentrated under reduced pressure. The residue
was extracted with EtOAc/H2O, and the organic layer was washed
with saturated NaHCO3 and brine, dried over anhydrous MgSO4,
and filtered. The residue was purified by silica gel column chroma-
tography (EtOAc/hexane, 1:20) to give compound 10 (871 mg, 54%)
(
)-6-Chloro-9-[3,3Ј-C-bis(tert-butyldimethylsilyloxymethyl)-
tetrahydrothiophen-2-yl] purine 14
Compound 14 was prepared from 6-chloropurine using a similar
method described for synthesizing compound 11: yield 64%; 1H
NMR (CDCl3, 300 MHz) d 8.48 (s, 1H), 8.10 (s, 1H), 6.02 (s, 1H),
3.69 (dd, J ϭ 12.6, 6.2 Hz, 2H), 3.34 (d, J ϭ 10.5Hz, 1H), 3.22 (d,
J ϭ 10.5 Hz, 1H), 3.06 (dd, J ϭ 10.6, 4.8 Hz, 1H), 2.83 (m, 1H),
2.10 (m, 1H), 1.93 (m, 1H), 0.84 (s, 18H), 0.03 (s, 12H); 13C NMR
(CDCl3, 75 MHz) d 155.76, 152.84, 148.26, 147.71, 130.92, 63.66,
62.34, 58.42, 33.49, 29.55, 25.73, 18.81, 18.76, -5.54; Anal. calc. for
1
as a colorless oil; H NMR (CDCl3, 300 MHz) d 5.57 (s, 1H), 3.82
(dd, J ϭ 12.4, 10.2 Hz, 2H), 3.70 (dd, J ϭ 12.0, 8.8 Hz, 2H), 2.74
(dd, J ϭ 10.8, 8.8 Hz, 2H), 2.18 (dd, J ϭ 10.2, 8.2 Hz, 2H), 2.01 (s,
3H), 0.89 (s, 18H), 0.02 (s, 12H); 13C NMR (CDCl3, 75 MHz)
δ170.74, 82.45, 63.81, 62.54, 58.72, 45.82, 32.60, 29.07, 25.44, 18.62,
16.4, -5.57; Anal. calc. for C20H42O4SSi2: C, 55.25; H, 9.74. Found:
C, 55.02; H, 9.59.
C
23H41ClN4O2SSi2: C, 52.19; H, 7.81; N, 10.59. Found: C, 51.98;
H, 8.06; N, 10.37.
(
)-1-[3,3Ј-C-Bis(tert-butyldimethylsilyloxymethyl)tetra-
( )-1-[3,3Ј-C-Bis(hydroxymethyl)tetrahydrothiophen-2-yl]
hydrothiophen-2-yl] uracil 11
uracil 15
Uracil (150 mg, 1.33 mmol), anhydrous HMDS (10 mL), and a
catalytic amount of ammonium sulfate were heated under reflux
until the solution turned clear and the solvent was distilled under
anhydrous conditions. The residue was dissolved in anhydrous 1,2-
dichloroethane (DCE). To this mixture, a solution of compound 10
(289 mg, 0.667 mmol) in dry DCE (6 mL) and TMSOTf (0.24 mL,
1.33 mmol) was added and the resulting mixture was stirred at rt.
for 4 h. The reaction mixture was quenched with 3 mL of saturated
NaHCO3 and stirred for 10 min. The resulting solid was filtered
through a Celite pad, and the filtrate was extracted twice with
CH2Cl2. The combined organic layers were dried over anhydrous
To a solution of compound 11 (92.98 mg, 0.191 mmol) in tetra-
hydrofuran (5 mL), tetrabutylammonium fluoride (0.573 mL, 1.0 M
solution in THF) was added at 0°C. The mixture was stirred over-
night at rt., and concentrated. The residue was purified by silica gel
column chromatography (MeOH/CH2Cl2, 1:7) to give compound
15 (37.5 mg, 76%): mp. 162-165°C; UV (H2O) lmax 261.5 nm; IR
(KBr) cm-1 3340, 3080, 2960, 2790, 1670; 1H NMR (DMSO-d6, 300
MHz) d 11.20 (brs, 1H), 7.58 (d, J ϭ 7.2 Hz, 1H), 6.03 (s, 1H), 5.55
(d, J ϭ 7.2 Hz, 1H) 4.99 (t, J ϭ 5.4 Hz, 1H), 4.78 (t, J ϭ 5.6 Hz,
1H), 3.80 (d, J ϭ 9.3 Hz, 1H), 3.67 (d, J ϭ 9.3 Hz, 1H), 3.42 (m,
2H), 3.21 (dd, J ϭ 12.4, 5.8 Hz, 1H), 2.88 (m, 1H), 2.03 (m, 2H);
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Arch. Pharm. Chem. Life Sci. 2005, 338, 577−581
Synthesis of Novel Branched Thioapiosyl Nucleosides
581
13C NMR (DMSO-d6, 75 MHz) d 165.65, 152.81, 141.67, 101.36,
64.12, 62.83, 58.43, 33.21, 30.65; Anal. calc. for C10H14N2O4S: C,
46.50; H, 5.46; N, 10.85. Found: C, 46.29; H, 5.71; N, 11.00.
(
)-9-[3,3Ј-C-Bis(hydroxymethyl)tetrahydrothiophen-2-yl]
adenine 20
Compound 20 was synthesized from 18 using a similar procedure
described for synthesizing compound 15, yield 60%; mp. 174-
176°C; UV (H2O) lmax 261.0 nm; IR (KBr) cm-1 3310, 3410, 2960,
2890, 1590; 1H NMR (DMSO-d6, 300 MHz) d 8.48 (s, 1H), 8.22 (s,
1H), 7.30 (brs, 2H), 5.99 (s, 1H), 5.00 (t, J ϭ 5.0 Hz, 1H), 4.79 (t,
J ϭ 5.2 Hz, 1H), 3.61 (d, J ϭ 9.2 Hz, 1H), 3.31 (d, J ϭ 9.2 Hz,
1H), 3.13 (d, J ϭ 10.2 Hz, 1H), 2.89 (d, J ϭ 10.2 Hz, 1H), 2.11 (m,
2H); 13C NMR (DMSO-d6, 75 MHz) d 155.78, 153.31, 148.53,
141.75, 120.36, 64.76, 62.60, 58.67, 33.21, 29.86; Anal. calc. for
C11H15N5O2S: C, 46.96; H, 5.37; N, 24.89. Found: C, 46.70; H,
5.55; N, 25.09.
(
)-1-[3,3Ј-C-Bis(hydroxymethyl)tetrahydrothiophen-2-yl]
thymine 16
Compound 16 was synthesized from 12 using a similar procedure
described for synthesizing compound 15, yield 70%; mp. 165-
168°C; UV (H2O) lmax 266.5 nm; IR (KBr) cm-1 3360, 3160, 2960,
2810, 1665, 1650; 1H NMR (DMSO-d6, 300 MHz) d 11.26 (brs,
1H), 7.31 (s, 1H), 6.00 (s, 1H), 3.80 (d, J ϭ 6.8 Hz, 1H), 3.63 (d,
J ϭ 6.8, 1H), 3.51 (d, J ϭ 8.6 Hz, 1H), 3.40 (d, J ϭ 8.6 Hz, 1H),
3.25 (m, 1H), 2.94 (m, 1H), 2.15 (dd, J ϭ 12.4, 6.0 Hz, 1H), 1.82
(dd, J ϭ 12.4, 5.4 Hz, 1H), 1.31 (s, 3H); 13C NMR (DMSO-d6, 75
MHz) d 164.82, 151.33, 134.27, 104.2, 64.67, 62.44, 59.23, 33.76,
29.56, 12.13; Anal. calc. for C11H16N2O4S: C, 48.52; H, 5.92; N,
10.29. Found: C, 48.39; H, 6.10; N, 10.57.
Evaluation of anti-HIV-1 activity
The MT-4 cells in the stationary phase were infected with the virus
at a 2-4 CCID50 (Cell Culture Infective Dose 50) multiplicity of
infection (MOI) per well in 96-well plates. After 2 h incubation at
37°C for the viral adsorption, the medium was aspirated off to re-
move the non-adsorbed viral particles, and 100 µL MEM contain-
ing 2% FBS and each compound was added to each well and
further incubated for 6 days. The antiviral activity was measured
microscopically or fluorometrically. For the microscopic obser-
vations, the cells were fixed with 70% ethanol and stained with a
2.5% Giemsa solution for 2 h, rinsed with distilled water and air-
dried. The antiviral activity is expressed as the IC50, which is the
concentration required to inhibit the virus-induced CPE (cytopathic
effect) by 50%. The IC50 values were estimated from the semi logar-
ithmic graphic plots of the percentage of CPE as a function of the
concentration of the test compound. For the fluorometric assay, the
cells were washed twice with 100 µL of phosphate-buffered saline
(PBS). 100 µL of a 5 µg/mL fluorescein diacetate solution (FDA,
Sigma, St. Louis, MO, USA) was added to each well and the plates
were incubated for 30 min at 37°C. The FDA solution was removed
by aspiration and the wells were washed with 100 µL PBS. The
fluorescence intensity (as absolute fluorescent units, AFU) in each
well was measured using a fluorescent microplate reader that was
equipped with a 485 nm excitation filter and a 538 nm emission fil-
ter.
(
)-1-[3,3Ј-C-Bis(tert-butyldimethylsilyloxymethyl)tetra-
hydrothiophen-2-yl] cytosine 17
Compound 13 (102.6 mg, 0.174 mmol) was treated with saturated
methanolic ammonia (5 mL) overnight at rt. The solvent was evapo-
rated under reduced pressure and the residue was purified by silica
gel column chromatography (EtOAc/hexane/MeOH, 3:1:0.5) to give
compound 17 (67 mg, 80%); 1H NMR (CDCl3, 300 MHz) d 7.43
(d, J ϭ 7.6 Hz, 1H), 5.99 (s, 1H), 5.69 (d, J ϭ 7.6 Hz, 1H), 3.80 (d,
J ϭ 9.0 Hz, 1H), 3.70 (d, J ϭ 9.0 Hz, 1H), 3.22 (d, J ϭ 10.0 Hz,
1H), 3.00 (d, J ϭ 10.2 Hz, 1H), 2.17 (m, 2H), 0.89 (s, 18H), 0.02 (s,
12H); 13C NMR (CDCl3, 75 MHz) d 165.57, 156.62, 143.28, 99.36,
64.72, 62.56, 58.40, 33.56, 29.54, 25.81, 18.56, -5.60; Anal. calc. for
C
22H43N3O3SSi2: C, 54.39; H, 8.92; N, 8.65. Found: C, 54.10; H,
9.11; N, 8.79.
(
)-9-[3,3Ј-C-Bis(tert-butyldimethylsilyloxymethyl)tetra-
hydrothiophen-2-yl] adenine 18
Compound 14 (306.9 mg, 0.58 mmol) was treated with saturated
methanolic ammonia (15 mL) overnight at 95-100 C in a steel
bomb. After removing the solvent, the residue was purified by silica
gel column chromatography (EtOAc/hexane, 4:1) to give compound
18 (227.7mg, 77%); 1H NMR (CDCl3, 300 MHz) d 8.31 (s, 1H),
7.97 (s, 1H), 6.00 (s, 1H), 3.61 (d, J ϭ 8.6 Hz, 1H), 3.33 (d, J ϭ 8.6
Hz, 1H), 3.17 (d, J ϭ 10.2 Hz, 1H), 2.99 (d, J ϭ 10.2 Hz, 1H), 2.12
(m, 2H), 0.89 (s, 18H), 0.03 (s, 12H); 13C NMR (CDCl3, 75 MHz)
d 155.70, 151.97, 144.63, 141.23, 119.30, 63.45, 62.37, 58.73, 33.82,
29.61, 25.65, 1828, -5.56; Anal. calc. for C23H43N5O2SSi2: C, 54.18;
H, 8.50; N, 13.74. Found: C, 54.01; H, 8.60; N, 13.88.
References
[1] V. Nair, T. S. Jahnke, Antimcrob. Agents Chemother. 1995, 39,
1017Ϫ1029.
[2] D. M. Huryn, M. Okabe, Chem. Rev. 1992, 92, 1745Ϫ1768.
[3] Y. Terao, M. Akamatsu, K. Achiwa, Chem. Pharm. Bull. 1991,
39, 823Ϫ825.
[4] T. B. Sells, V. Nair, Tetrahedron Lett. 1993, 34, 3527Ϫ3530.
[5] C. O-Yang, H. Y. Wu, E. B. Fraser-Smith, K. A. M. Walker,
Tetrahedron Lett. 1992, 33, 37Ϫ40.
[6] H. Maag, R. M. Rydzewski, M. J. McRoberts, D. Crowford-
Ruth, J. P. Verheyden, E. J. Prisbe, J. Med. Chem. 1992, 35,
1440Ϫ1451.
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1992, 36, 1589Ϫ1595.
(
)-1-[3,3Ј-C-Bis(hydroxymethyl)tetrahydrothiophen-2-yl]
cytosine 19
Compound 19 was prepared from 17 using a similar method de-
scribed for synthesizing compound 15: yield 60%; mp. 167-170°C;
UV (H2O) lmax 271.5 nm; IR (KBr) cm-1 3330, 1655, 1620; 1H NMR
(DMSO-d6, 300 MHz) d 7.44 (d, J ϭ 7.2 Hz, 1H), 7.20 (br d, 2H),
5.98 (d, J ϭ 8.0 Hz, 1H), 5.70 (d, J ϭ 7.2 Hz, 1H), 4.99 (t, J ϭ 5.2
Hz, 1H), 4.69 (t, J ϭ 5.4 Hz, 1H), 3.72 (d, J ϭ 8.1 Hz, 1H), 3.47
(d, J ϭ 8.2 Hz, 1H), 3.23 (d, J ϭ 10.2 Hz, 1H), 3.02 (d, J ϭ 11.1,
1H), 2.21 (d, J ϭ 8.4 Hz, 1H), 1.96 (d, J ϭ 8.4 Hz, 1H); 13C NMR
(DMSO-d6, 75 MHz) d 165.60, 155.54, 142.88, 99.63, 64.21, 62.84,
58.48, 34.03, 29.90; Anal. calc. for C10H15N3O3S: C, 46.68; H, 5.88;
N, 16.33. Found: C, 46.50; H, 6.11; N, 16.25.
[10] O. H. Ko, J. H. Hong, Tetrahedron Lett. 2002, 43, 6399Ϫ6402.
[11] Y. Yoshimura, K. Kitano, K. Yamada, H. Satoh, M. Watanabe,
S. Miura, S. Sakata, T. Sasaki, A. Matsuda, J. Org. Chem. 1997,
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