Selective inhibition of Human Immunodeficiency Virus type 1 (HIV-1) by a novel family of tricyclic nucleosides

Article abstract of DOI:10.1016/j.antiviral.2011.05.002

Nucleoside 1, with an unusual tricyclic carbohydrate moiety, specifically inhibits HIV-1 replication while being inactive against HIV-2 or other (retro) viruses. In an attempt to increase the inhibitory efficacy against HIV-1, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on this prototype compound. These include substitution of the ethoxy group at the C-4″ position by alkoxy groups of different length, branching, conformational freedom or functionalization. In addition, the 4″-ethoxy group has been removed or substituted by other functional groups. The role of the tert-butyldimethylsilyl (TBDMS) group at the 2' position has also been studied by preparing the corresponding 2'-deprotected derivative or by replacing it by other silyl (tert-hexyldimethylsilyl) or acyl (acetyl) moieties. Finally, the thymine of the prototype compound has been replaced by N-3-methylthymine, uracil or thiophenyl. Some of these compounds were endowed with a 6- to 7-fold higher selectivity than the prototype 1. The tricyclic nucleosides here described represent a novel type of selective anti HIV-1 inhibitors, targeted at the HIV-1-encoded reverse transcriptase.

Full text of DOI:10.1016/j.antiviral.2011.05.002

Antiviral Research 92 (2011) 37–44
Contents lists available at ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Selective inhibition of Human Immunodeficiency Virus type 1 (HIV-1)
by a novel family of tricyclic nucleosides
María-Cruz Bonache a, Alessandra Cordeiro a, Ernesto Quesada a, Els Vanstreels b, Dirk Daelemans b,
María-José Camarasa a, Jan Balzarini b, Ana San-Félix a,
⇑
a Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
b Rega Institute for Medical Research Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Nucleoside 1, with an unusual tricyclic carbohydrate moiety, specifically inhibits HIV-1 replication while
being inactive against HIV-2 or other (retro) viruses. In an attempt to increase the inhibitory efficacy
against HIV-1, and to further explore the structural features required for anti-HIV-1 activity, different
types of modifications have been carried out on this prototype compound. These include substitution
of the ethoxy group at the C-400 position by alkoxy groups of different length, branching, conformational
freedom or functionalization. In addition, the 400-ethoxy group has been removed or substituted by other
functional groups. The role of the tert-butyldimethylsilyl (TBDMS) group at the 20 position has also been
studied by preparing the corresponding 20-deprotected derivative or by replacing it by other silyl (tert-
hexyldimethylsilyl) or acyl (acetyl) moieties. Finally, the thymine of the prototype compound has been
replaced by N-3-methylthymine, uracil or thiophenyl. Some of these compounds were endowed with a
6- to 7-fold higher selectivity than the prototype 1. The tricyclic nucleosides here described represent
a novel type of selective anti HIV-1 inhibitors, targeted at the HIV-1-encoded reverse transcriptase.
Ó 2011 Elsevier B.V. All rights reserved.
Received 21 January 2011
Revised 27 April 2011
Accepted 5 May 2011
Available online 12 May 2011
Keywords:
Antiviral agents
AIDS
HIV-1 reverse transcriptase inhibitors
Nucleosides
Carbohydrates
Glycosylation
1. Introduction
et al., 2001). However, no biological evaluation against HIV of these
compounds have been reported so far.
In the last few years the design and synthesis of nucleosides
with bicyclic carbohydrate moieties have attracted considerable
attention to restrict the conformational flexibility of the nucleoside
into determined conformations which were ideal for nucleic acid
recognition (Mathé and Périgaud, 2008; Kaur et al., 2007; Jepsen
et al., 2004; Kumar et al., 2006; Gagneron et al., 2005). On the other
hand, a number of structurally diverse bicyclic nucleosides have
been synthesized in order to identify conformational preferences
of some receptors and enzymes involved in the metabolism or
polymerization of nucleosides and to study the interaction of such
compounds with their enzymatic targets (Marquez et al., 2006; Co-
min et al., 2003). Some bicyclic nucleosides have shown to moder-
ately inhibit HIV replication (Chuanzheng and Chattopadhyaya,
2009; Díaz-Rodríguez et al., 2009; Tronchet et al., 1994, 1995)
although none of them are specific for HIV-1.
As a part of an ongoing work in our laboratories directed to the
synthesis and biological evaluation of bi- and tricyclic nucleosides,
we found that the tricyclic nucleoside 1 (Bonache et al., 2004)
(Fig. 1) inhibited HIV-1 replication while being inactive against
HIV-2 or other (retro)viruses. The selectivity displayed by this
compound prompted us to perform systematic modifications on
this prototype with the aim of determining the minimal structural
features essential for HIV-1 inhibition. This structure–activity
study constitutes the subject matter of this paper.
First, the ethoxy group at the C-400 position has been replaced by
alkoxy groups of different length, branching, conformational free-
dom or functionalization. In addition, the ethoxy group has been
removed or substituted by other functional groups. Next, the role
of the tert-butyldimethylsilyl (TBDMS) group at the 20 position
has been studied by preparing the corresponding 20-deprotected
derivative or by replacing it by other silyl (tert-hexyldimethylsilyl)
or lipophilic (acetyl) moieties.
Modifications on the nucleobase have been also examined.
Thus, the N-3 methyl analog of 1 has been prepared and the thy-
mine of the prototype compound has been replaced by uracil or
by an aromatic moiety such as thiophenyl.
In order to further restrict the conformation of the bicyclic
nucleosides, the introduction of additional bridges generating tri-
cyclic nucleosides was also undertaken (Neogi et al., 2006; Ravn
⇑
Corresponding author. Tel.: +34 91 5622900; fax: +34 91 5644853.
E-mail addresses: jan.balzarini@rega.kuleuven.be (J. Balzarini), anarosa@iqm.c-
sic.es (A. San-Félix).
0166-3542/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2011.05.002

38
M.-C. Bonache et al. / Antiviral Research 92 (2011) 37–44
Thymine-N3-CH3
Uracil
were infected with HIV-1(IIIB) or HIV-2 (ROD) at ꢂ100 CCID50
(50% cell culture infective dose) per milliliter of cell suspension.
Then an amount of 100 l of the infected cell suspension was
transferred to microtiter plate wells and mixed with 100 l of
O
N
NH
l
RO
H
l
O
HN
CH3CH2O
SPh
the appropriate dilutions of the test compounds. Giant cell forma-
tion (CEM) and HIV-induced cytopathicity (MT-4) was recorded
microscopically (CEM) and by trypan blue dye exclusion (MT-4)
in the HIV-infected cell cultures after 4 days (CEM) or 5 days
(MT-4). The 50% effective concentration (EC50) of the test com-
pounds was defined as the compound concentration required to in-
hibit virus-induced cytopathicity (CEM) or to reduce cell viability
(MT-4) by 50%. The 50% cytostatic concentration (CC50) was de-
fined as the compound concentration required to inhibit CEM cell
proliferation by 50%.
O
2'
4"
O
OTBDMS
OH
S
O
O
O-tert-hexyl
dimethylsilyl
other functional
groups
1
OAc
Fig. 1. Modifications carried out on the tricyclic nucleoside 1.
2. Materials and methods: chemistry
2.2.2. Reverse transcriptase assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and
purified as described (Auwerx et al., 2002). The RT assay is per-
formed with the EnzCheck reverse transcriptase assay kit (Molecu-
lar Probes, Invitrogen), as described by the manufacturer. The assay
is based on the dsDNA quantitation reagent PicoGreen. This re-
agent shows a pronounced increase in fluorescence signal upon
binding to dsDNA or RNA-DNA heteroduplexes. Single-stranded
nucleic acids generate only minor fluorescence signal enhance-
ment when a sufficiently high dye:basepair ratio is applied (Singer
et al., 1997). This condition is met in the assay.
2.1. General methods
Commercial reagents and solvents were used as received from
the suppliers without further purification unless otherwise stated.
Dichloromethane and acetonitrile were dried prior to use by distil-
lation from CaH2 and stored over Linde type activated 4 Å molecu-
lar sieves as described.
Analytical thin-layer chromatography (TLC) was performed on
aluminum plates pre-coated with silica gel 60 (F254, 0.25 mm).
Products were visualized from the TLC by exposure to ultraviolet
light (254 nm) or by heating on a hot plate (aprox. 200 °C), directly
or after treatment with a 5% solution of phosphomolybdic acid or
vanillin in ethanol. Separations were performed by preparative
flash column chromatography on silica gel 60 (PF254, 230–400
mesh) or by preparative Centrifugal Circular Thin-Layer Chroma-
tography (CCTLC) (Kiesegel 60 PF254 gipshaltig, layer thickness of
1 mm, flow rate 4 mL minꢀ1). Mass spectra were registered in a
quadrupole mass spectrometer 1100 equipped with an electro-
spray source.
A poly(rA) template of approximately 350 bases long, and an
oligo(dT)16 primer, are annealed in
(60 min. at room temperature). Fifty-two ng of the RNA/DNA is
brought into each well of a 96-well plate in a volume of 20 l poly-
merization buffer (60 mM Tris–HCl, 60 mM KCl, 8 mM MgCl2,
13 mM DTT, 100 M dTTP, pH 8.1). 5 l of RT enzyme solution, di-
a molar ratio of 1:1.2
l
l
l
luted to a suitable concentration in enzyme dilution buffer (50 mM
Tris–HCl, 20% glycerol, 2 mM DTT, pH 7.6), is added. The reactions
are incubated at 25 °C for 40 min and then stopped by the addition
of EDTA (15 mM fc). Heteroduplexes are then detected by addition
of PicoGreen. Signals are read using an excitation wavelength of
490 nm and emission detection at 523 nm using a spectrofluorom-
eter (Safire2, Tecan). To test the activity of compounds against RT,
2.1.1. NMR procedures
Monodimensional 1H and 13C NMR spectra were obtained using
standard conditions and were registered in CDCl3 or acetone-d6 as
solvents with 300, 400 or 500 NMR spectrometers. Solvents were
used with higher deuteration degree than 99.5% and were filtered
through a pad of neutral alumina in order to eliminate traces of
water and acid prior to use. Chemical shifts for protons are re-
ported in parts per million (ppm) downfield from tetramethylsil-
ane referred to protonated residual peaks or specific signals due
to deuterated solvents as internal references. Chemical shifts for
proton-decoupled carbons are reported in parts per million
(ppm) referenced to the deuterated solvents as internal standards.
Carbon and proton assignments were based on DEPT, HSQC,
HMBC and NOE-differential experiments.
1 ll of compound in DMSO is added to each well before the addi-
tion of RT enzyme solution. Control wells without compound con-
tain the same amount of DMSO. Results are expressed as relative
fluorescence i.e. the fluorescence signal of the reaction mix with
compound divided by the signal of the same reaction mix without
compound.
2.2.3. RNase H assay
The RNase H assay was developed also employing the dsDNA
quantitation reagent PicoGreen (Parniak et al., 2003). A RNA/DNA
heteroduplex is formed by annealing a 40 bases long RNA oligonu-
cleotide (50-CCAGCAGGAAACAGCUAUGACGAUCUGAGCCUGGGAG-
CU-30) and 120 bases long DNA oligonucleotide (50-AGCTC
CCAGGCTCAGATCGTCATAGCTGTTTCCTGCTGGCAGCTCCCAGGCT-
CAGATCGTCATAGCTGTTTCCTGCTGGCAGCTCCCAGGCTCAGATCGT-
CATAGCTGTTTCCTGCTGGC-30) in a 4:1 M ratio.
2.2. Biological methods
2.2.1. Activity assay of test compounds against HIV-1 and HIV-2 in cell
cultures
An amount of 76 ng of the annealed complex is brought into
The antiviral (i.e. HIV-1 and HIV-2) activity of the compounds
was independently investigated in parallel in two different (CEM
and MT-4) T-lymphocyte cell lines. The MT-4 cells are transformed
by HTLV-I and exquisitely sensitive to HIV-1 infection whereas the
CEM cells were not transformed by HTLV-I. The virus input for pro-
ductive infection of MT-4 cell cultures is much lower (>100-fold)
than required for CEM cell cultures. Therefore, it is of interest to re-
veal the antiviral activity of the test compounds in both cell lines. A
total number of 4 ꢁ 105 CEM or 3 ꢁ 105 MT-4 cells per milliliter
each well of a 96-well plate in a volume of 20
(60 mM Tris–HCl, 60 mM KCl, 8 mM MgCl2, 13 mM DTT, pH 8.1)
and 5 l of suitably diluted RT solution is added. Reactions are
stopped after 60 min at 25 °C by the addition of EDTA (15 mM
fc). PicoGreen is then added to measure the amount of heterodu-
plexes and thus the decrease in signal upon RNA hydrolysis in
the presence of enzyme activity and signals are read as described
for the reverse transcriptase assay. The results are expressed
relative to the amount of heteroduplexes measured in a negative
ll RNase H buffer
l

M.-C. Bonache et al. / Antiviral Research 92 (2011) 37–44
39
control sample without RT enzyme, after subtraction of the back-
ground signal obtained with only ssDNA.
2,2-dimethyl-1-propanol afforded the corresponding tricyclic
derivatives: 3 (58%), the prototype 1 (70%), 4 (60%) (Bonache
et al., 2004), 5 (78%), 6 (79%) (Bonache et al., 2004), 7 (72%)
(Bonache et al., 2004) and 8 (83%) (Bonache et al., 2004) in satisfac-
tory yields (58–83%).
To test compounds for activity against RNase H, 1 ll of com-
pound in DMSO is added to each well before the addition of RT en-
zyme solution. Control wells without compound contain the same
amount of DMSO. Positive compounds were tested for autofluores-
cence in a separate test.
Also, compounds 9 (80%) and 10 (76%) with a double or triple
bond, respectively were prepared (Scheme 1). On the other hand,
the functionalization of the alkoxy moiety was studied by prepar-
ing compounds 11 (74%) and 12 (67%), with an hydroxy or phenoxy
moiety at the terminal position of the alkoxy moiety, or com-
pounds 13 (80%) or 14 (67%) with benzyloxy or tetrahydrofuranyl-
methoxy groups at the C-400 position (Scheme 1). These compounds
were prepared following a similar procedure to that described for
the synthesis of 1. Thus, the reaction of 2 with 2-propen-1-ol, 2-
propyn-1-ol, 1,2-ethanediol, 2-phenoxy-1-ethanol, benzyl alcohol
and tetrahydro-2-furanmethanol in refluxing acetonitrile afforded
the corresponding tricyclic nucleosides 9–14 in high yields (67–
80%).
2.2.4. Drug resistance studies
Compound 24 was exposed to HIV-1(IIIB)-infected CEM cell
cultures in a dose-escalating manner (starting at 40
virus-infected cultures were subcultured every 5 days by transfer-
ring 20 l virus-infected cell suspension to fresh 180 l CEM cell
lM). The
l
l
cultures containing new test compound. As soon as a full cyto-
pathic effect was recorded the compound dose was increased for
the next subcultivation (i.e. 100, 200, 400
sage, the cell cultures were split in two parallel subcultivations,
transferring either 20 l culture medium supernatant or 20 l cell
suspension to the next fresh cell cultures. At the 10th subcultiva-
tion, when in both cultures 400 M test compound was reached,
lM). After the 6th pas-
l
l
In addition, the 400-O-tert-butyldimethylsilyl derivative 16 was
prepared in 73% yield by treatment of the 400-hydroxy derivative
15 (Bonache et al., 2009) with tert-butyldimethylsilyl chloride in
pyridine at room temperature (Scheme 1).
As it was previously determined for the prototype 1, these
transformations proceeded with total regio- and stereoselectivity.
The geometry of the new stereocentre created on C-400 in this series
of compounds was unequivocally determined as S on the basis of
NOE difference experiments.
l
genotypic (RT gene) and phenotypic (drug sensitivity) character-
ization of the virus was performed according to previously
reported procedures (Balzarini et al., 1993b).
3. Results and discussion
3.1. Chemical results
Compound 17 in which the ethoxy moiety at the C-400 position
has been removed was also prepared (Scheme 1). The synthesis
of this compound was attempted first by hydrogenation of 2
(Bonache et al., 2004) using 10% palladium on charcoal as catalyst.
However, under these conditions, the starting compound 2 re-
mained unchanged, even upon prolonged reaction times. Finally,
when platinum oxide was used as catalyst, compound 17 was ob-
tained in 30% yield.
We studied first the effect of the substitution of the ethoxy moi-
ety of the prototype compound 1 by flexible alkoxy moieties of dif-
ferent length and branching. These compounds were readily
prepared in one step by reaction of 2 (Bonache et al., 2004) with
the corresponding alcohol in refluxing acetonitrile (Scheme 1).
Thus, reaction of different primary alcohols such as methanol,
ethanol, propanol, butanol, pentanol, isobutyl alcohol and
O
N
O
N
O
N
O
N
NH
NH
NH
NH
O
O
O
O
v
iv
HN
4"
HN
4"
HN
i
HN
O
O
O
O
RO
RO
H
4"
O
OTBDMS
O
OTBDMS
O
OH
O
OTBDMS
S
S
S
S
O
O
O
O
O
O
O
O
prototype
1: R = CH2CH3
18: R = CH2CH3
19: R = CH3
17
2
3: R = CH3
4: R = (CH2)2CH3
5: R = (CH2)3CH3
6: R = (CH2)4CH3
7: R = CH2CH(CH3)2
8: R = CH2C(CH3)3
9: R = CH2CH=CH2
20: R = (CH2)2CH3
21: R = (CH2)3CH3
22: R = (CH2)4CH3
23: R = CH2CH(CH3)2
24: R = CH2C(CH3)3
25: R = CH2CH2OH
26: R = CH2CH2OPh
27: R = CH2Ph
ii
O
N
10: R = CH2C≡ CH
11: R = CH2CH2OH
12: R = CH2CH2OPh
13: R = CH2Ph
NH
28: R = CH2
O
O
HN
4"
14: R = CH2
O
O
RO
O
OTBDMS
S
O
O
15; R = H
16; R = TBDMS
iii
Scheme 1. Synthesis of tricyclic nucleosides 1 and 3–28. Reagents and conditions: (i) ROH, CH3CN, 80 °C (ii) H2O, CH3CN/AcOH (pH 5–6), 80 °C (iii) Si(Me)2tButCl, pyridine, rt
(iv) H2/PtO2, AcOEt (v) NH4F, MeOH, rt.

40
M.-C. Bonache et al. / Antiviral Research 92 (2011) 37–44
A NOE difference experiment carried out on 17, showed that the
trifluoroacetic acid, followed by reaction with acetic anhydride/
pyridine afforded a 1:1.5 mixture of the two anomeric forms (
and b) of the diacetate derivative 33 in 90% yield (Scheme 4). Con-
densation of 33 with silylated uracil under modified Vorbrüggen
(Vorbrüggen and Höfle, 1981) conditions afforded the 30-cyano me-
syl b-nucleoside 34 in 58% yield. The coupling constant value
signal at d 4.54 ppm, corresponding to the H-400 proton, correlates
with the signal of the H-20 proton of the sugar, at d 4.90 ppm, indi-
cating that the H-400 proton is on the upper side of the pyrrolidine
ring (Fig. 2).
a
Next, modifications at the 20 position were investigated
(Schemes 1 and 2). First, the 20 deprotected derivatives 18–28 were
obtained in 65–83% yield by reaction of the corresponding 20-
TBDMS derivatives with ammonium fluoride in methanol
(Scheme 1). In addition, the 20 acetyl derivative 29 was obtained
in 86% yield by acetylation of the 20 deprotected derivative 18 with
acetic anhydride/pyridine. Reaction of 18 with tert-hexyl dimethyl-
silyl chloride in acetonitrile at room temperature afforded the cor-
responding 20-O-silylated nucleoside 30 in 75% yield (Scheme 2).
With respect to the modifications on the nucleobase, the intro-
duction of a methyl group at the N-3 position of thymine was first
carried out (Scheme 3). Thus, compound 1 was transformed into
the corresponding 3-N-methyl nucleoside 31 (75%) by selective
N-3-alkylation using methyl iodide in the presence of potassium
carbonate.
0
0
J1 ,2 = 7 Hz observed for this compound is in good agreement with
the data for other b-cyanomesyl nucleosides previously described
by our group (Camarasa et al., 1992).
On the other hand, reaction of 33 with thiophenol in the
presence of boron trifluoride diethyl etherate afforded the b aryl
thioglycoside 36 in 62% yield together with the
a aryl thioglyco-
side 35 as a very minor compound (1%) (Scheme 4). In these su-
gar derivatives the similar values of the coupling constant J1,2
observed for the
a (J1,2 = 5.9 Hz) and b anomers (J1,2 = 5.8 Hz)
precluded the unambiguously assignment of their anomeric con-
figuration. These, were unequivocally determined by
experiment (Fig. 2).
a NOE
Thus, for the major isomer 36, irradiation of H-1 caused
enhancement of the signals for H-4 indicating that both protons,
Next, compounds 41 and 46 in which the thymine of the proto-
type has been replaced by uracil or thiophenyl moieties, respec-
tively, were prepared using a convergent strategy, in which the
5-O-tosyl-3-cyano mesyl ribofuranose 33 was employed as a com-
mon sugar precursor (Scheme 4).
H-1 and H-4, are at the lower face (a face) of the furanose ring,
and therefore the anomeric configuration of this compound is b.
For the minor isomer 35, irradiation of H-1 causes enhancement
of the signals H-2 and H-5 indicating that all of these protons are
at the upper face (b face) of the furanose ring, and therefore the
In turn, compound 33 was prepared from the previously de-
scribed 5-O-tosyl derivative 32 (Cordeiro et al., 2006). Thus,
hydrolysis of the 1,2-O-isopropylidene moiety of 32, with aqueous
anomeric configuration of this compound is a.
Once formed, nucleoside 34 was transformed into the desired
uracil nucleoside 41 as shown in Scheme 5.
O
O
N
O
N
5
CH3
NH
NH
N
N
O
O
O
5
i
HN
TsO
NC
H
TsO
NC
HN
CH3CH2O
HN
CH3CH2O
SPh
H
O
O
O
1
O
O
H
2'
1
4
4''
R
H
2
H
SPh
OAc
H
H3C
O
OTBDMS
H3C
O
OTBDMS
O OTBDMS
O
S
S
S
O
OAc
S
S
O
O
O
O
O
O
O
O
O
O
36
1
31
17
35
Scheme 3. Synthesis of 31. Reagents and conditions: (i) MeI, K2CO3, dry acetone,
80 °C.
Fig. 2. Observed NOE’s for compounds 17, 35 and 36.
O
O
NH
NH
N
O
N
O
HN
CH3CH2O
HN
CH3CH2O
i
O
O
O
OH
O OCOCH3
S
S
O
O
O
18
O
ii
29
O
N
NH
O
(TDS = ter t-hexyldimethylsilyl)
HN
O
CH3CH2O
O
OTDS
S
O
O
30
Scheme 2. Synthesis of 29 and 30. Reagents and conditions: (i) (CH3CO)2O, pyridine, rt (ii) Si(Me)2tHexCl, DMAP, dry acetonitrile, rt.

M.-C. Bonache et al. / Antiviral Research 92 (2011) 37–44
41
O
O
N
NH
NH
N
O
O
TsO
NC
HN
CH3CH2O
O
O
i
4''
H3C
OAc
O
O OTBDMS
S
S
O
O
O
O
TsO
NC
TsO
NC
34
41
O
O
OAc
OAc
O
H3C
H3C
O
O
O
S
S
O
O
O
O
33
32
ii
SPh
HN
CH3CH2O
TsO
NC
TsO
NC
SPh
O
O
O
+
SPh
OAc
4''
H3C
H3C
OAc
36
Scheme 4. Synthesis of carbohydrates 33–36. Reagents and conditions: (i) Uracil, TMSTriflate, dry acetonitrile (ii) PhSH, BF3.OEt2, dry dichloromethane.
O
OTBDMS
O
O
S
S
S
O
O
O
O
O
O
35
46
B
B
B
TsO
NC
TsO
NC
TsO
NC
O
i
O
ii
O
H3C
H3C
H3C
OAc
OH
OTBDMS
O
O
O
S
S
S
O
O
O
O
O
O
34: B = Uracil-1-yl
36: B = SPh
38: B = Uracil-1-yl
43: B = SPh
37: B = Uracil-1-yl
42: B = SPh
iii
B
iv
v
B
B
TsO
O
HN
HN
CH3CH2O
O
O
H2N
O OTBDMS
O OTBDMS
S
O
O
O
OTBDMS
S
S
O
O
O
O
39: B = Uracil-1-yl
44: B = SPh
41: B = Uracil-1-yl
46: B = SPh
40: B = Uracil-1-yl
45: B = SPh
Scheme 5. Synthesis of 37–46. Reagents and conditions: (i) NH3, MeOH, 0 °C, 10 min (ii) TBDMSCl, DMAP, 80 °C, 1 h (iii) DBU, dry acetonitrile (iv) K2CO3, dry acetonitrile, 80 °C
(v) CH3CH2OH, 80 °C.
Thus, 20 deprotection of nucleoside 34 with saturated metha-
nolic ammonia gave the deprotected nucleoside 37 (90%). Reac-
tion of 37 with tert-butyldimethylsilyl chloride at 80 °C for 1 h
afforded the 20-O-silylated nucleoside 38 (73%). It should be
mentioned that the substitution of the 50-tosyl group by chloride
was observed upon prolonged reaction times as a secondary
reaction. Replacement of the tosyl moiety at 50 position by chlo-
ride was determined in the 1H NMR spectrum by the disappear-
ance of the signals corresponding to the tosyl group and the
upfield shift (ꢂd 0.4 ppm) of the signals corresponding to the
H-50 protons with respect to the same signals in the tosyl deriv-
ative (d 4.40 and 4.50 ppm). However, the 50-chloro derivative
obtained behaves exactly as the corresponding 50-O-tosyl deriva-
tive 38 and for this reason when the substitution takes place the
resulting 50-O-tosyl and 50-Cl- mixture was used directly without
separation. Nucleoside 38 was treated with DBU to give the spiro
derivative 39 (74%). Reaction of 39 with potassium carbonate at
80 °C for 5 h afforded the cyclic enamine sulfonate 40 in 73%
yield that was treated with ethanol to afford 41 in 70% yield.
A similar synthetic sequence was followed with the thiophenyl

42
M.-C. Bonache et al. / Antiviral Research 92 (2011) 37–44
Table 1
O
O
N
Inhibitory effects of test compounds on HIV-1 and HIV-2 replication in MT-4 and CEM
cell culture.
NH
NH
a
b
N
O
O
CAM EC50
(
l
M)
CC50
CEM
(lM)
HN
HN
i
O
O
MT-4
HIV-1
CEM
MT-4
R
R
HIV-2 HIV-1
HIV-2
4"
4"
O
OH
O
OTBDMS
3
4
5
6
7
8.4 3.9
5.1 0.4
>10
P5
5.2 0.6
>50
>10
>10
>5
>10
>10
>10
>25
>25
>5
5.5 0.7
1.4 0.8
>10
>5
8.0 2.8
>10
15.0 5.0
>25
11 4.0
>5
P10
>50
>250
>2
30.0 7.1
>250
35.0 21.2 >250
>250
P125
160 79.4 >250
>250
22.5 3.5
>250
>250
P100
20.0 0.0
>250
>10
3.0 1.4
>50
>10
32.5 3.5
>50
>125
30.0 0.0
>250
>50
>10
>10
>5
>10
>10
>50
>25
>25
>5
>10
>50
>250
>2
>125
>250
111 14.8
28.5 2.0
34.8 13.7 18.0 0.64
63 24
21.6 1.6
S
S
O
O
O
O
10.1 0.1
21.1 3.6
21.5 1.6
57.8 9.3
66.2 4.9
73 10
10.4 0.7
20.4 1.1
72.0 1.7
>250
7.04 1.8
>125
P250
>250
>250
>125
>250
>250
>250
>250
P250
>250
P250
9.0 1.4
16 2.1
12 0.42
24 0.57
49 2.1
59 11
4.8 1.4
6.4 0.98
29 1.8
>250
9.1
P125
>250
>250
>250
>125
P250
>250
237 18
>250
P250
>250
P250
>250
50: R = SCH2CH3
51: R = CN
47: R = SCH2CH3
48: R = CN
8
9
>10
>10
>25
52: R = CONH2
49: R = CONH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
41
46
47
48
49
50
51
52
1
Scheme 6. Synthesis of 50–52. Reagents and conditions: (i) NH4F, MeOH, rt.
9.5 0.2
>5
derivative 36 to afford the deprotected derivative 42 (65%) that
was successively transformed into 43 (65%), 44 (77%), 45 (73%)
and 46 (79%) as described above (Scheme 5).
>10
>10
>250
>2
>10
>10
>250
>2
Finally, for comparative purposes the 20-deprotected tricyclic
nucleosides 50–52 were prepared in 60–78% yield respectively
by reaction of the corresponding 20-TBDMS derivatives 47 (Bo-
nache et al., 2004), 48 (Bonache et al., 2004) and 49 (Bonache
et al., 2004) with ammonium fluoride in methanol (Scheme 6).
30.4 23.5 >125
–
–
98.9 8.7
>250
>250
>250
>125
>250
>250
>250
>250
>250
>250
>250
>125
>125
143 0.0
>250
24.2 1.9
>250
>250
>250
>250
>250
>250
>250
>250
>10
P50
>50
>50
>50
>50
3.2. Biological results
>250
>250
The tricyclic nucleoside analogs 3–31, 41 and 46 were tested for
their in vitro inhibitory effects on HIV-1 and HIV-2 replication. In
addition, for comparative purposes we tested the tricyclic
nucleosides 47, 48 and 49 with ethylthio, cyano and carboxamide
moieties at the C-400 position that were previously synthesized in
our laboratory (Bonache et al., 2004) and their corresponding
20-deprotected derivatives 50–52.
Table 1 summarizes the results of the biological evaluation of
the test compounds. The inhibitory activity of the compounds
was expressed as EC50 values or compound concentrations re-
quired to inhibit virus-induced giant cell formation (CEM) or cyto-
pathicity (cell destruction) (MT-4) by 50%. We decided to evaluate
the compounds against both virus infection models because both
assays used a different read-out, but should correspond to each
other in terms of antiviral activity of test compounds. The antiviral
data on the prototype compound (1) is also reported as reference.
Whereas several compounds inhibited HIV-1 replication in the
lower micromolar concentration range, none of the compounds
proved active against HIV-2 at subtoxic concentrations (Table 1).
Therefore, the active compounds should be considered as specific
inhibitors of HIV-1 replication. Their anti-HIV activity in CEM cells
were very similar to those observed in HIV-1 infected MT-4 cell
cultures (Table 1).
A structure–activity relationship (SAR) could be recognized for
the 20-TBDMS protected test compounds with regard of their cellu-
lar cytostatic profile. Small alkyl entities were clearly preferable
over more bulky, either linear or branched, groups [order of lesser
toxicity to highest toxicity: R = methyl (3) < ethyl (1) ꢂ propyl
(4) ꢂ butyl (5) < isopropyl (7), isobutyl (8) < pentyl (6)]. The unsat-
urated propenyl (9) and propynyl (10) derivatives showed a 2-fold
lesser cytostatic activity than the propyl derivative (4). Hydroxy-
ethyl (11) resulted in far less cytostatic activity than phenyloxyeth-
yl (12), and the benzyl (13) derivative was 2-fold less cytostatic
than the phenyloxyethyl (12) derivative. The highly bulky OTBDMS
23.9 1.13 >250
>250
–
>250
–
P50
>50
–
>250
27.0 3.6
32.0 2.0
111.0 1.4
131.0 12.7
80.6 8.6
103.0 7.9
P125
–
3.6 2.3
49 2.3
119 16
–
42 14
101 8.1
108 21
208 73
P250
P250
24.8 5.94
>50
–
31.1 16.5 >50
>50
>125
>50
>125
>250
>250
>250
>10
>125
>250
>250
>250
>50
111 3.5
>250
P250
P250
>250
25.0 1.2
>250
>250
7.7 4.0
13.8 5.4
Data are the mean S.D. of at least 2–3 independent experiments.
a
50% effective concentration, or the compound concentration required to inhibit
HIV-induced cytopathicity by 50%.
b
50% cytostatic concentration, or the compound concentration required to
inhibit cell proliferation (CEM) or to reduce cell viability (MT-4) by 50%.
be easily made, since several compounds could not be evaluated
at concentrations higher than 5 or 10 lM.
Nucleosides 3 and 4, with one more or one less carbon atom in
the alkoxy chain than the prototype (1) showed an antiviral activ-
ity that was superior to the antiviral potential of 1. Moreover, 3
was 4-to 5-fold less cytostatic than 1. Consequently, the selectivity
index (ratio CC50 (CEM)/EC50 (CEM)) increased from 3.3, for the
prototype compound 1, to 20–22 for compounds 3 and 4 due to
both a higher antiviral potency and lower cytostatic effect. How-
ever, compounds with longer alkoxy chains (5 or 6) were devoid
of antiviral activity at sub-cytostatic concentrations. Thus, the pro-
pyloxy derivative 4 was the most inhibitory to HIV-1 replication in
CEM cell cultures of this series.
The isobutyloxy derivative 7 showed an antiviral activity com-
parable to that of the unbranched derivative 4 in MT-4 cell cul-
tures. It was less potent than 4 in CEM cell cultures.
Replacement of the ethoxy moiety in the prototype by OH or
OTBDMS resulted in compounds (i.e. 15 and 16) that lack anti-
HIV-1 activity. The presence of other functional groups (CN and
CONH2) also gave inactive compounds (48 and 49). In contrast,
nucleoside 17, lacking the ethoxy moiety, or 47 with an ethyl chain
(16) resulted in a most cytostatic compound (IC50: 7
the OH-substituted compound (15) was not cytostatic at all (IC50
>250 M).
lM), whereas
:
l
Given the wide varying range of the cytostatic effect of the indi-
vidual derivatives, an exact comparison of the antiviral potential of
the individual compounds to delineate an antiviral SAR could not

M.-C. Bonache et al. / Antiviral Research 92 (2011) 37–44
43
containing a sulfur atom at C-400 were endowed with anti-HIV-1
activity, although being approximately 2-fold less active than the
prototype compound.
TSAO derivatives both compounds markedly lost inhibitory poten-
tial against the mutant E138K RT and Y181C RT HIV-1 strains
(EC50 > 20
lM). The EC50’s of 3 and 4 for the mutant K103N RT
Substitution of the TBDMS group at the 20 position of the ribo-
furanose by other lipophilic moieties, such as acetyl (29) or tert-
hexyl (30) gave inactive compounds. The 20-deprotected derivative
(18) was also inactive.
HIV-1 strains were 13 and >20
lM, respectively. Thus, the new
compound derivatives act somewhat similar to TSAO derivatives
in terms of (mutant) antiviral spectrum.
Since compound 24 was devoid of any cytostatic effect at
The 3-N-methylthymine derivative 31 was 2- to 4-fold more ac-
tive than its unsubstituted counterpart (1). Substitution of the thy-
mine moiety by uracil or thiophenyl resulted in inactive
compounds (41 and 46), respectively.
250 lM, this compound was chosen to select for drug-resistant
HIV-1 strains. Virus-exposed cell cultures were cultured in the
presence of escalating concentrations of the test compounds. Dur-
ing the first six subcultivations, a cell suspension was transferred
to fresh culture medium containing the compounds. At the sixth
subcultivation, both cell suspension or supernatant was, in parallel,
transferred to fresh culture medium for another 4 subcultivations.
After the 10th subcultivation, the virus in both cell cultures was
isolated and further phenotypically and genotypically analysed.
The two virus strains isolated under dose-escalating cultivation
conditions were found to be insensitive to the inhibitory activity
The 20-deprotected derivatives (18–28 and 50–52) were, as a
rule, not cytostatic at concentrations as high as 250
Also, they showed marginal (i.e. 19, 22, 50), if any, anti-HIV activity
(EC50: P50 M), except for compounds 24 and 28 that proved
moderately inhibitory to HIV-1 replication in MT-4 and CEM cell
cultures (EC50: 20–24 M).
lM (Table 1).
l
l
Our biological results indicates that shorter substituents in the
400 position improve potency when the TBDMS group is present,
while in its absence, branched and bigger alkoxy groups are
needed. Taking in consideration that the bulky 20-OTBDMS and
the OR moiety on the C-400 carbon are respectively at the lower
and the upper side of the furanose ring (Bonache et al., 2004) this
biological result indicates that these two groups might define the
length of the cavity that accommodates the tricyclic nucleosides.
The distance between the ‘‘roof’’ and ‘‘floor’’ of the cavity might
not be larger enough to accommodate simultaneously two bulky
groups at the top and bottom faces of the furanose ring.
The most active compounds 3 and 4 were evaluated against re-
combinant HIV-1 reverse transcriptase using polyA-oligodT as the
template/primer. The activity measurements were based on a
fluorescence (PicoGreen) directed assay. Compounds 3 and 4
dose-dependently inhibited the enzyme reaction. The IC50 (50%
of compound 24 at 250 lM. Interestingly, amino acid mutations
were observed in the reverse transcriptase of these virus strains:
i.e. K101K/E, K102K/E, V108V/I and E138E/K) for the virus isolate
for which cell suspension was transferred during the last four sub-
cultivations (HIV-1/24res#1
)
and K101K/E + V108 V/I + E138E/
K + Y181Y/C for the virus isolate for which supernatant was trans-
ferred during the last four subcultivations (HIV-1/24res#2). The
emergence of mutations lining the NNRTI pocket of RT occurred
at a similar speed as observed for first-generation NNRTIs such
as nevirapine and TSAO-m3T (Balzarini et al., 1993b). In contrast
to TSAO derivatives, that consistently select for a E138K mutation
in the HIV-1 RT, compound 24 seems to select for additional muta-
tions in the 98–108 amino acid stretch of HIV-1 RT (Table 2). It
should be noticed that mutations at amino acid position K102 of
the HIV-1 RT occur rather rarely (Ceccherini-Silberstein et al.,
2007) whereas mutations at amino acid positions 101, 108, 138
and 181 are more common. Our findings (lack of anti-HIV-2 activ-
ity and the appearance of NNRTI-characteristic RT mutations)
strongly suggest that the compounds behave as NNRTIs and likely
bind in the NNRTI-binding pocket (resulting in mutations at amino
acid positions 101, 102, 108, 138 and 181) to escape drug pres-
sure). The amino acid mutations at positions 102 and 181 seem
to have appeared most likely after the amino acid mutations at
positions 101, 108 and 138. It is interesting to observe the appear-
ance of the E138 K mutation in the mutant virus strains. An E138 K
RT mutation is very characteristic for the TSAO derivatives that
contain, beside the oxathioledioxide spiro moiety, also bulky tert-
butyldimethylsilyl groups at C-20 and C-50. The latter groups are
not present in the tricyclic nucleoside 24, pointing to other parts
of the molecule (i.e. the oxathioledioxide ring or the pyrrolidine
inhibitory concentration) was around 100
as a control) also inhibited 50% of the enzyme reaction at
ꢂ0.7 M (Fig. 3). It should be noticed that when using a poly
C-oligo dG template, the RT reaction was also inhibited by nevira-
pine (data not shown), but not by 3 and 4 at 100 M, pointing to a
lM. Nevirapine (used
l
l
certain degree of template preference of the test compounds to in-
hibit the RT reaction. The compounds have also been tested for
their inhibitory activity against RNase H-associated RT but were
found inactive at 100 lM.
Compounds 3 and 4 have also been evaluated for their inhibi-
tory activity against mutant K103N RT, E138K RT and Y181C RT
HIV-1 replication in CEM cell cultures. Similarly as observed for
Table 2
Inhibitory activity of 24, NNRTIs and the NRTI tenofovir against mutant HIV-1 strains
in CEM cell cultures.
a
Compound
EC50
(lM)
HIV-1(IIIB) (WT)
32
0.01
0.013 0.004
0.001 0.0007
4.2 1.8
HIV-1/24res#1b
HIV-1/24res#2c
24
8
>250
>250
>20
0.055 0.007
0.012 0.003
11 1.4
TSAO-m3T
Nevirapine
UC-781
Tenofovir
0.45 0.07
0.034 0.03
0.008 0.003
16 5.7
Data are the mean S.D. of at least 2–3 independent experiments.
a
50% effective concentration or compound concentration required to inhibit
virus-induced giant cell formation in CEM cell cultures by 50%.
b
Following amino acid mutations were observed: K101K/E + K102K/E + V108 V/
I + E138E/K.
c
Following amino acid mutations were observed: K101K/E + V108V/I + E138E/
K + Y181Y/C.
Fig. 3. Anti-HIV-1 RT activity of compounds 3, 4 and nevirapine.

44
M.-C. Bonache et al. / Antiviral Research 92 (2011) 37–44
Camarasa, M.J., Pérez-Pérez, M.J., San-Félix, A., Balzarini, J., De Clercq, E., 1992. 3’-
Spironucleosides. A new class of specific Human Immunodeficiency Virus type 1
inhibitors: Synthesis and antiviral activity of [20, 50-Bis-O-(tert-
butyldimethylsilyl)-b-D-xylo- and ribofuranosel-30-spiro-500-(400-amino-100, 200-
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4. Conclusions
In summary, we report on novel derivatives of the prototype tri-
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sition, the nucleobase and the substituent at the 20 position.
Several members of this class of compounds show specific anti-
HIV-1 activity comparable or superior to those of the prototype tri-
cyclic nucleoside. Our studies demonstrate that both, the presence
of thymine and a tert-butyldimethylsilyl group at the 20-position of
the sugar are important structural components since deletion of
either of them is detrimental to the anti-HIV-1 activity. Modifica-
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keep anti-HIV-1 activity. Thus, the ethoxy moiety can be replaced
by methoxy, propyloxy, hydrogen and ethylthio moieties. The
methoxy (3) and propyloxy (4) derivatives were endowed with a
6- to 7-fold higher selectivity than the prototype 1. Introduction
of a methyl group at the position N-3 of the thymine enhanced
the antiviral activity by 2- to 4-fold. Also several 20-deprotected
analogs (24 and 28) were endowed with selective anti-HIV-1 activ-
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selective anti HIV-1 inhibitors, targeting HIV-1 reverse transcrip-
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Acknowledgements
Parniak, M.A., Min, K.L., Budihas, S.R., Le Grice, S.F.J., Beutler, J.A., 2003.
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We thank Susana Ruiz, Ann Absillis and Leen Ingels for excellent
technical assistance. The Spanish MEC/MCINN (Project SAF 2009-
13914-C02-01), the Spanish CSIC (Project PIF 08-019-2) and the
Concerted Research Actions of the K.U. Leuven (GOA 10/014) are
also acknowledged for financial support. The Spanish CSIC (JAE-
Doc Program) and European Science Foundation (ESF) are also
acknowledged for a JAE-Doc contract to M.-C. Bonache.
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Further reading
Balzarini, J., Karlsson, A., Vandamme, A.-M., Pérez-Pérez, M.-J., Zhang, H., Vrang, L.,
Öberg, B., Bäckbro, K., Unge, T., San-Félix, A., Velázquez, S., Camarasa, M.-J., De
Clercq, E., 1993a. Human immunodeficiency virus type
1 (HIV-1) strains
selected for resistance against the HIV-1-specific [20, 50-bis-O-(tert-
butyldimethylsilyl)-30-spiro-500-(400-amino-100, 200-oxathiole-200, 200-dioxide)]-b-
D-pentofuranosyl (TSAO) nucleoside analogues retain sensitivity to HIV-1-
specific nonnucleoside inhibitors. Proc. Natl. Acad. Sci. USA 90, 6952–6956.
Bonache, M.C., Cordeiro, A., Carrero, P., Quesada, E., Camarasa, M.J., Jimeno, M.L.,
San-Félix, A., 2009. One-pot synthesis of polycyclic nucleosides with inusual
molecular skeletons. J. Org. Chem 74, 9071–9081.