4″-benzoylureido-TSAO derivatives as potent and selective non-nucleoside HCMV inhibitors. Structure-activity relationship and mechanism of antiviral action

Article abstract of DOI:10.1021/jm800050t

Analogues of the 4″-benzoyl-ureido-TSAO derivative (1) modified at different positions have been prepared and evaluated against wild-type strains of HCMV and murine cytomegalovirus (MCMV) in cell culture. In addition, the activity of the most active deriv

Full text of DOI:10.1021/jm800050t

J. Med. Chem. 2008, 51, 5823–5832
5823
4′′-Benzoylureido-TSAO Derivatives as Potent and Selective Non-Nucleoside HCMV Inhibitors.
Structure-Activity Relationship and Mechanism of Antiviral Action
Sonia de Castro,† M. Teresa Peromingo,† Lieve Naesens,‡ Graciela Andrei,‡ Robert Snoeck,‡ Jan Balzarini,‡
Sonsoles Vela´zquez,*,† and Mar´ıa-Jose´ Camarasa*,†
Instituto de Qu´ımica Me´dica (C.S.I.C.), Juan de la CierVa 3, E-28006 Madrid, Spain, Rega Institute for Medical Research, K. U. LeuVen,
Minderbroedersstraat 10, B-3000 LeuVen, Belgium
ReceiVed January 18, 2008
Analogues of the 4′′-benzoyl-ureido-TSAO derivative (1) modified at different positions have been prepared
and evaluated against wild-type strains of HCMV and murine cytomegalovirus (MCMV) in cell culture. In
addition, the activity of the most active derivatives against several drug-resistant HCMV mutants has been
determined. A stringent structure-antiviral activity relationship was observed for the 4′′-benzoylureido-
TSAO derivatives for which the concomitant presence of a highly lipophilic substituent at both 2′- and
5′-positions was required to fully preserve the antihuman cytomegalovirus efficacy. Time-of-addition studies
and HCMV immediately early and early gene expression studies revealed a target at the time of viral DNA
synthesis, although direct inhibition of HCMV-encoded DNA polymerase could not be observed in cell-
free assays. Lack of cross-resistance against a broad variety of mutant HCMV strains points to an antiviral
target that is different from those drugs that are currently approved for clinical use.
Scheme 1a
Introduction
Although it rarely causes symptomatic disease in immuno-
competent individuals, human cytomegalovirus (HCMVa) is an
opportunistic pathogen responsible for a variety of severe, often
life-threatening diseases (i.e., pneumonia, retinitis) in immu-
nocompromised or immunosuppressed hosts (i.e., transplant
recipients, AIDS patients).1 HCMV is also a major cause of
congenital malformation in newborn children.2 HCMV is a
ubiquitous member of the herpes virus family. Except for the
antisense RNA formivirsen, antiviral agents approved for the
systemic treatment of HCMV infections are viral DNA poly-
merase inhibitors,3 i.e., a pyrophosphate analogue [foscarnet
(PFA)], the nucleoside analogues ganciclovir (GCV) and
acyclovir (ACV) and their valine ester pro-drugs valganciclovir
and valacyclovir,4-6 and the acyclic nucleotide phosphonate
cidofovir (CDV or HPMPC).6 However, these drugs suffer from
a number of drawbacks and limitations such as dose-limiting
bone marrow (ganciclovir) and kidney (foscarnet and cidofovir)
toxicities,4,6 unfavorable pharmacokinetic properties, as well as
the emergence of single and multiple drug resistance.7-11
Although there are other anti-HCMV drugs in clinical develop-
ment (i.e., marabivir),6 novel and potent anti-HCMV agents,
with a good selectivity, safety profile, and oral bioavailability
are needed. Either inhibitors of the viral DNA polymerase with
a different structure than that of the approved anti-HCMV drugs
or compounds with a different mechanism of action would be
a Reagents and conditions: (i) HCl 1N, methanol, 0 °C; (ii) TBAF, THF,
r.t.; (iii) TBDMSCl, Py, r.t.
good candidates to make progress in reducing the consequences
of HCMV infection in the susceptible populations.We recently
reported12 that some 4′′-substituted-TSAO derivatives (TSAO
compounds are a particular group of potent and highly specific
and selective inhibitors of HIV-1 replication)13 lost the anti-
HIV-1 activity. Surprisingly, several of these compounds gained
inhibitory activity against replication of HCMV in cell culture
showing pronounced antiviral activity at concentrations in the
lower µM range (i.e., 0.29-2.0 µM), that is at a concentration
well below their toxicity threshold. In particular, compound 1
(Scheme 1) showed the highest anti-HCMV activity lacking
appreciable cytotoxicity or antiproliferative activity against HEL
cell cultures.12 The activity of the 4′′-benzoyl-ureido derivative
(1) against HCMV replication belonged to the same order of
magnitude as ganciclovir.12 The highly modified structure of
these TSAO molecules (i.e., containing blocked 5′- and 2′-
positions in the ribose moiety) points to a different mechanism
of inhibition of HCMV than that of currently used anti-HCMV
nucleoside analogues. However, it is currently unclear whether
these compounds are targeted against HCMV DNA polymerase
(acting as non-nucleoside inhibitors) or inhibit another viral (or
cellular) function necessary for virus replication. Further studies
should be pursued with these molecules to address the structural
* To whom correspondence should be addressed. For M.-J.C. and S.V.:
phone,(+34)-912587458;fax,(+34)-915644853;E-mail,mj.camarasa@iqm.csic.es
(M.-J.C.) and iqmsv29@iqm.csic.es (S.V.).
†
Instituto de Qu´ımica Me´dica (C.S.I.C.).
Rega Institute for Medical Research, K. U. Leuven.
‡
a Abbreviations: HCMV, human cytomegalovirus; AIDS, acquired
immunodeficiency syndrome; PFA, foscarnet; GCV, ganciclovir; ACV,
acyclovir; CDV or HPMPC, cidofovir; TSAO, (tert-butyldimethylsilyl-ꢀ-
D-ribofuranosyl)-3′-spiro-4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide; HIV, hu-
man immunodeficiency virus; HEL, human embrionic lung; MCMV, murine
cytomegalovirus; TBDMS, tert-butyldimethylsilyl; TDS, thexyldimethyl-
silyl; TIPS, triisopropylsilyl; DMAP, 4-dimethylaminopyridine; DMF, N,N-
dimethylformamide; SAR, structure-activity relationship; IE, immediately
early; E, early; VZV, varizella zoster virus.
10.1021/jm800050t CCC: $40.75
2008 American Chemical Society
Published on Web 08/16/2008

5824 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
de Castro et al.
Scheme 2a
Scheme 3a
a Reagents and conditions: (i) RNCO, NaH, acetonitrile, r.t.
a Reagents and conditions: (i) PhCONCO, acetonitrile, 100 °C; (ii) NH3,
methanol, 0 °C; (iii) TBDMSCl, DMAP, r.t.
DMAP gave the fully protected compound 18 (78% yield). The
synthesis of the 4′′-benzoylureido-TSAO-m3T derivative 19
bearing a 5′-O-benzyl group (Scheme 2) was next performed.
The required 5′-O-benzyl TSAO intermediate 1117 was obtained
in good yield (87%) by treatment of the 5′-deprotected TSAO-
m3T15 with 1.1 equiv of benzyl bromide at -40 °C in dry DMF
in the presence of 1.1 equiv of K2CO3 as base for 15 min. It
should be noted that under these conditions, alkylation of the
3′′-C position of the sultone moiety was completely avoided,
the reaction time was shortened (15 min vs 12 h), and the yield
of 11 was significantly improved (87% vs 47%) compared with
the previously described classical Williamson procedure in the
presence of NaH as base.17 Unfortunately, treatment of the 5′-
deprotected TSAO-m3T15 with less reactive alkyl halides, such
as isobutyl bromide or neopentyl bromide (in order to prepare
other 5′-carbo analogues of 4′′-benzoylureido-TSAO deriva-
tives), in the presence of K2CO3 or NaH as base, were
unsuccesful and unreacted starting material was recovered.
Finally, acylation of the 5′-benzyl TSAO derivative 11 with
benzoyl isocyanate, under similar conditions described above,
afforded the corresponding 4′′-benzoylureido-TSAO-m3T de-
rivative 19 in 59% yield.
requirements for anti-HCMV activity and to reveal their
mechanism of inhibition of viral replication.
In this report, we describe the synthesis and biological studies
of analogues of the 4′′-benzoyl-ureido-TSAO derivative (1)
modified at different positions. Initial structure-activity rela-
tionships have been characterized for the O5′, O2′ (TBDMS,
TDS, TIPS, Bz, Bn, Ac, H), and 4′′-ureido substituents (Bz,
Ph, Bn, H, Et, CO2Et, CH2CO2, Et) as well as the significance
of the nucleobase (N3-methylthymine). The compounds have
been evaluated against wild-type strains of HCMV and murine
cytomegalovirus (MCMV) in cell culture. In addition, the
activity of the most active derivatives against several drug-
resistant HCMV mutants has been determined. To elucidate the
mode of action of these derivatives, time-of-addition experi-
ments, HCMV immediately early and early gene expression
studies, and HCMV DNA polymerase assays have also been
performed.
Results and Discussion
Chemistry. First, analogues of 1 modified at the 5′ and/or
2′-O positions were pursued (Schemes 1 and 2). Thus, treatment
of 112 with a 0.1N solution of HCl in methanol at 0 °C gave,
selectively, the 5′-O-deprotected ureido derivative 2 in 59% yield
(Scheme 1). Reaction of 112 with tetrabutylammonium fluoride
in THF afforded the fully deprotected derivative 3 (72% yield)
that was silylated (TBDMSCl/pyridine) to give the 5′-O-
TBDMS compound 4. Next, compounds substituted with other
silyl groups at 5′ or 2′ positions (Scheme 2), such as 5′-O- or
2′-thexyldimethylsilyl (TDS) (12 or 13) and 5′-O-triisopropyl-
silyl (TIPS) (14), were prepared in good yields (64-98%) by
reaction of the corresponding 5′-O- or 2′-O-TBDMS-protected
TSAO derivatives 5,14 or 6,15 or 714 with benzoyl isocyanate,
in dry acetonitrile in a pressure tube at 100 °C. The 2′,5′-bis-
O-thexyldimethylsilyl derivative (15) was prepared from the
2′,5′-O-deprotected TSAO derivative 816 as follows. 816 was
reacted with TDSCl/DMAP at 100 °C in dry acetonitrile to give
compound 9 (50% yield), which was treated with benzoyl
isocyanate, under similar conditions described above, to yield
the target compound 15 in 75% yield. 4′′-Benzoylureido-TSAO-
m3T derivatives bearing other groups at 2′- and/or 5′-positions
were also prepared. Thus, reaction of 10 (prepared in 71% yield
from 2′-O-acetyl-5′-O-benzoyl-TSAO-T16 and methyl iodide)
with benzoyl isocyanate gave the 2′-acetyl-5′-benzoyl-4′′-
benzoylureido derivative 16 (94% yield). Treatment of 16 with
NH3 in methanol at 0 °C gave the 5′-benzoyl-2′-O-deprotected
derivative 17 in 90% yield. Reaction of 17 with TBDMSCl/
4′′-Substituted TSAO-m3T derivatives, bearing different
ureido groups at the 4′′-position (21-23), were also prepared
(Scheme 3.) When phenylisocyanate was reacted with 20, under
similar acylation conditions described above (acetonitrile, pres-
sure tube, 100 °C), only the starting material was recovered.
However, when this reaction was carried out with an excess of
conveniently functionalized isocyanates, in the presence of NaH
in dry THF, a mixture of 21 and 22 were isolated in 58 and
21% yield, respectively (Scheme 3). Formation of 22 could be
explained by the attack of a second molecule of phenylisocy-
anate to the initially formed 4′′-phenylureido derivative 21.
Similarly, reaction of 20 with benzylisocyanate gave compound
23 in good yield (75%). Compounds 24-27 were prepared as
previously described12 and are included here for comparative
purposes in the SAR studies.
To determine whether the base part or the sugar part were
necessary for anti-HCMV activity, the abasic analogue 29
(Scheme 4) and the 4-benzoylureido-sultone 33 (Scheme 5) were
prepared. Reaction of the N-acetyl derivative 2818 (Scheme 4)
with benzoyl isocyanate in dry acetonitrile in an Ace pressure
tube at 100 °C gave the desired compound 29 in 90% yield.
Finally, the 4-benzoylureido-sultone 33 was prepared in 67%
yield as shown in Scheme 5. Thus, treatment of the ketone
derivative 3019 with sodium cyanide in a two-phase ethyl ether/
water system in the presence of sodium bicarbonate gave a
racemic mixture of cyanohydrines that were used in the next

4′′-Benzoylureido-TSAO DeriVatiVes as HCMV Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5825
Scheme 4a
5′-positions of the 3′-spiro ribose ring. Removal of the base
part (i.e., 29, 33) of 1 resulted in an inactive compound (data
not shown). Also, removal of the TBDMS moiety at 2′ (4) or
5′ (2) or both 2′- and 5′-position (3) annihilated the antiviral
activity of the parent compound. When one of the silylated
substituents at 2′ or 5′ were replaced by the silicium containing
TDS (12, 13, 15) or TIPS (14) substituents, the anti-HCMV
activity of the test compounds was preserved. As also observed
for the prototype compound 1, the silylated derivatives 12-15
were devoid of significant cytotoxic or cytostatic activity in the
HEL cell cultures. Whereas replacing of the silyl group at the
5′-position of the molecule by a benzoyl moiety (18) preserved
the antiviral activity (but resulted in a higher cytotoxic activity),
the antiviral activity of the compound was annihilated when
the 2′-TBDMS was replaced by an acetyl (16) or a benzyl (19)
or did not contain a substituent at all (free 2′-OH) (17). Thus,
it seems that the presence of silyl groups at the 2′- and
5′-position are concomitantly required or can only be replaced
by a benzoyl at the 5′-position to preserve antiviral activity of
the compounds in the HCMV-infected cell cultures. In this
respect, the structure-activity relationship (SAR) is remarkably
similar to that for the anti-HIV-1 activity of the TSAO
derivatives.13
a Reagents and conditions: (i) PhCONCO, acetonitrile, 100 °C.
Scheme 5a
a Reagents and conditions: (i) NaCN, diethyl ether/water, r.t.; (ii) MsCl,
TEA, dichloromethane, -20 to 0 °C; (iii) Cs2CO3, acetonitrile, r.t.; (iv)
PhCONCO, acetonitrile, 100 °C.
If the benzoylureido group at the 3′-spiro moiety of the
prototype 1 was replaced by other groups, lacking the CdO
entity linked to the urea such as a phenyl (21), benzyl (23),
ethyl (25), ethylcarboxylate (26), ethylcarboxymethyl (27), or
phenylcarboxamide phenyl (22), or when there is no substituent
at all at the ureido group (24), the compounds lost antiviral
activity or showed antiviral activity at concentrations that are
close to the toxicity threshold. Because the mechanism of
antiviral (HIV-1) action of TSAO derivatives is inhibition of
the virus-encoded reverse transcriptase, the DNA polymerase
of HCMV20,21 was investigated as a potential target for the test
compounds. However, when examined for its inhibitory activity
against the viral enzyme in the presence of activated DNA using
either dGTP or dATP as the radiolabeled substrate, no ap-
preciable inhibitory activity was observed at compound (12 and
15) concentrations as high as 100 µM. Under similar experi-
mental conditions, foscarnet (PFA) proved inhibitory at a
concentration of 5-7.5 µM. Therefore, it could be concluded
that most likely the compounds are not directly targeted against
the HCMV UL54-encoded DNA polymerase. However, it
should be kept in mind that DNA synthesis in virus-infected
cells requires interaction of the HCMV UL54-encoded catalytic
subunit with accessory proteins. These proteins were not present
in the reaction mixture of the HCMV DNA polymerase assay,
and thus, it cannot be excluded that the compounds indirectly
inhibit HCMV DNA polymerase activity by interfering with
one of these accessory proteins. Indeed, time-of-addition studies
at which the test compounds had been added to the virus-
infected cell cultures at different time-points post virus infection
revealed that antiviral activity was lost when the compounds
were added at the time point when viral DNA synthesis occurred
(Figure 1.) Thus, a delay of administration of compounds 1,
12, and 15 for up to 56 h postinfection did not affect the antiviral
efficacy of the drugs. At longer delayed time periods, the three
drugs, similarly to (S)-HPMPC (cidofovir) and GCV (ganci-
clovir) that are known to target the viral DNA polymerase,
started to lose their potential to markedly reduce virus-induced
cytopathicity.
Table 1. Anti-HCMV Activity of Test Compounds in HEL Cell
Cultures
a
EC50 (µM)
c
compd
Davis
AD-169
MCCb (µM)
CC50 (µM)
1
2
3
4
2.4
>4
>100
>100
1.4
3.9
4.2
1.5
>100
>100
2.2
>50
0.38
>10
1.1
2.0
2.0
2.8
11
>4
>100
>100
1.1
12.1
6.3
g80
20
>200
16
>50
22
>50
g100
>100
>50
>100
>100
100
>50
1.3
13
4.1
11
10
14
13
>100
>100
>100
>100
g100
g100
g100
g100
>100
>100
g5 (100)
>50
1.4
46
4.0
16
g3.2
10
12
13
14
15
16
17
18
19
21
22
23
24
25
26
27
29
33
1.2
>100
>100
2.2
>50
g0.14
15
1.3
>3.2
1.8
2.3
>3.2
>20
>100
>3.2
>20
>100
16
100
>100
a Effective concentration required to reduce virus-induced cytopathicity
by 50%. b Minimum cytotoxic concentration that causes a microscopically
detectable alteration of cell morphology. c Cytotoxic concentration required
to reduce cell growth by 50%.
step without further purification. Reaction of this cyanohydrine
mixture with mesyl chloride in dry dichloromethane in the
presence of triethylamine afforded the methyl sulfonate deriva-
tive 31. Reaction of 31 with Cs2CO3 gave the ꢀ-amino-γ-sultone
derivative 32 in 75% yield. Finally, treatment of 32 with
benzoylisocyanate, as described for the synthesis of compounds
12-19 (acetonitrile, 100 °C), gave the 4-benzoylureido-sultone
derivative 33 in 67% yield.
Biological Evaluation. The compounds have been evaluated
against two different (AD-169 and Davis) strains of HCMV in
HEL cell cultures (Table 1,) and compared with the parent
compound 1, which contain a TBDMS entity at both 2′- and
Expression of immediately early (IE) and early (E) genes were
determined in compound 12-treated cells and compared to the
reference compounds dextran sulfate (an inhibitor of viral entry)

5826 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
de Castro et al.
revealed that direct interaction of the TSAO compounds with
the E antigen is not required for the eventual antiviral activity.
Thus, besides the viral DNA polymerase, this protein can also
be excluded as a potential target for the test compounds. These
findings point to a novel mechanism of action of the compounds.
Further studies are ongoing to reveal their antiviral molecular
target.
Experimental Section
Chemical Procedures. Microanalyses were obtained a Heraeus
CHN-O-RAPID instrument. Electrospray mass spectra were mea-
sured on a quadropole mass spectrometer equipped with an
electrospray source (Hewlett-Packard, LC/MC HP 1100). 1H NMR
spectra were recorded with, a Varian XL-300 and a Bruker 300
Avance spectrometer operating at 300 MHz with Me4Si as internal
standard. 13C NMR spectra were recorded with a VARIAN XL-
300 and a Bruker 300 Avance spectrometer operating at 75 MHz
with Me4Si as internal standard. Analytical thin-layer chromatog-
raphy (TLC) was performed on silica gel 60 F254 (Merck).
Separations on silica gel were performed by preparative centrifugal
thin-layer chromatography (CCTLC) on a Chromatotron (Kiesegel
60 PF254 gypsiferous (Merck), layer thickness (1 mm), flow rate (5
mL/min). Flash column chromatography was performed with silica
gel 60 (230-400 mesh)(Merck). Dichloromethane and acetonitrile
were dried by refluxing over calcium hydride. Tetrahydrofurane
was dried by refluxing over sodium-benzophenone. Ace pressure
tubes (15 mL) were purchased from Sigma-Aldrich (catalogue no.
Z181064-1EA).
Figure 1. Time-of-addition experiments performed using cytopathic
effect reduction assays in HEL cell cultures and comparing compounds
1, 12, and 15 to the reference drugs ganciclovir (GCV) and cidofvofir
(HPMPC) and to the adsorption inhibitor dextran sulfate (DS), data
are in µM.
and ganciclovir (GCV) and cidofovir (HPMPC), two inhibitors
of the viral DNA polymerase. Similar to HPMPC and GCV,
compound 12 was not able to inhibit IE antigen expression at
48 h postinfection as measured by flow cytometry (Figure 2.)
IE antigen expression during the first cycle of viral replication,
which is known to take approximately 72 h, was strongly
inhibited by dextran sulfate that has been shown to inhibit viral
adsorption. IE antigen expression reflects input virus and is
maintained during the entire viral replicative cycle. No expres-
sion of E antigen at 48 h postinfection was detected, indicating
that viral DNA synthesis have not occurred yet. After one or
two complete cycles of viral replication, IE and E antigen
expression was significantly inhibited by compound 12, GCV,
and HPMPC as well as by dextran sulfate. These results are in
agreement with the time-of-addition experiments and suggest
that compound 12 does not inhibit an early step in the HCMV
replicative cycle but a step around the onset of DNA synthesis.
The 4′′-benzoylureido-TSAO derivatives 12 and 15 showed
also potent activity against murine cytomegalovirus (MCMV),
with EC50 values of 0.66 and 0.54 µM, respectively. In contrast
with the potent inhibitory activity of the lead compounds against
human and murine cytomegalovirus, a markedly lower potency
was observed against the R-herpes virus varicella zoster virus
(VZV) (EC50 values in the order of 11-16 µM for both wild-
type and thymidine-kinase deficient VZV strains). The com-
pounds 12 and 15 were not antivirally active against herpes
simplex virus types 1 and 2, vaccinia virus, or a variety of RNA
viruses.
Compounds 12 and 15 were evaluated against a panel of
HCMV drug-resistant mutants either isolated in vitro (Table 2)
or recovered from immunocompromised patients (Table 3.)
These virus strains bear mutations either in the DNA polymerase
and/or in the UL97 gene, which encodes for a HCMV-specified
protein kinase responsible for the phosphorylation of ganciclovir
and acyclovir in HCMV-infected cells. Interestingly, all mutants
remained fully sensitive to the inhibitory activity of compounds
12 and 15, indicating a mode of action for the 4′′-benzoylureido-
TSAO derivatives different from that of the clinically used
reference compounds.
Thus, although we were not yet able to pinpoint the exact
mechanism of antiviral action of the compounds, they may most
likely act at a target that is operative during the infection cycle
around the onset of viral DNA synthesis. In fact, because the E
antigen is a virus-encoded DNA binding protein, our data also
[1-[(2′-O-tert-Butyldimethylsilyl)-ꢀ-D-ribofuranosyl]-3-N-me-
thylthymine]-3′-spiro-5′′-[4′′-(3-benzoylureido)-1′′,2′′-oxathiole-
2′′,2′′-dioxide] (2). To a solution of 112 (50 mg, 0.06 mmol) in
methanol (2 mL) at 0 °C, 1N HCl in methanol (1.0 mL) was slowly
added. The mixture was stirred at 0 °C for 4 h. The reaction was
treated with a 1N solution of NaOH in methanol until pH ∼ 6 at
0 °C, salts were filtered, and the solvent was evaporated to dryness.
The residue was purified by CCTLC on the Chromatotron (hexane/
1
ethyl acetate, 2:3) to give 2 (22 mg, 59%) as a white foam. H
NMR [300 MHz, (CD3)2CO] δ: 0.21(s, 6H, 2CH3-Si), 0.83 (s,
t
9H, Bu), 1.19 (s, 3H, CH3-5), 3.28 (s, 3H, CH3-3), 4.00 (m, 2H,
H-5′), 4.48 (m, 1H, H-4′), 5.05 (d, 1H, J ) 7.9 Hz, H-2′), 5.77 (bs,
1H, OH), 6.25 (d, 1H, J ) 7.9 Hz, H-1′), 7.58 (s, 1H, H-3′′), 7.60
(m, 2H, Ph), 7.70 (m, 1H, Ph), 8.09 (d, 2H, J ) 7.2, Ph), 8.19 (s,
1H, H-6), 10.37 (bs, 1H, NH), 11.66 (bs, 1H, NH). 13C NMR [75
MHz, (CD3)2CO] δ: -0.16 (CH3-Si), -0.04 (CH3-Si), 13.3 (CH3-
5), 18.3 (C-tBu-Si), 25.6 (tBu-Si), 28.0 (CH3-3), 61.1 (C-5′), 75.5
(C-2′), 85.8 (C-4′), 88.0 (C-3′), 95.5, 108.4 (C-1, C-3′′), 111.3 (C-
5), 129.1, 129.7, 132.8, 134.4 (Ph), 135.1 (C-6), 141.8 (C-4′′), 151.5,
152.3 (C-2, CO), 163.5 (C-4), 168.6 (CO). MS (ESI+) m/z 637 (M
+ H)+, 659 (M + Na)+. Anal. (C27H36N4O10SSi) C, H, N, S.
[1-(ꢀ-D-Ribofuranosyl)-3-N-methylthymine]-3′-spiro-5′′-[4′′-
(3-benzoylureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (3). To a solu-
tion of 112 (50 mg, 0.06 mmol) in THF (2 mL), a 1 M solution of
TBAF in THF was slowly added (0.12 mL, 0.12 mmol). The
mixture was stirred at room temperature for 5 min. The solvent
was evaporated to dryness and the residue was purified by CCTLC
on the Chromatotron (dichloromethane/methanol, 99.5:0.5) to give
3 (31 mg, 72%) as a white foam. 1H NMR [300 MHz, (CD3)2CO]
δ: 1.91 (s, 3H, CH3-5), 3.26 (s, 3H, CH3-3), 3.83 (m, 2H, H-5′),
4.38 (m, 1H, H-4′), 4.99 (m, 1H, H-2′), 5.58 (bs, 1H, OH), 6.28
(d, 1H, J ) 8.4 Hz, H-1′), 6.63 (bs, 1H, OH), 7.55 (s, 1H, H-3′′),
7.66 (m, 3H, Ph), 8.03 (d, 2H, J ) 7.5, Ph), 8.21 (s, 1H, H-6),
10.28 (bs, 1H, NH), 11.42 (bs, 1H, NH). MS (ESI+) m/z 523.0 (M
+ H)+. Anal. (C21H22N4O10S) C, H, N, S.
[1-[(5′-O-tert-Butyldimethylsilyl)-ꢀ-D-ribofuranosyl]-3-N-me-
thylthymine]-3′-spiro-5′′-[4′′-(3-benzoylureido)-1′′,2′′-oxathiole-
2′′,2′′-dioxide] (4). To a solution of 3 (50 mg, 0.10 mmol) in dry
pyridine (3 mL), tert-butyldimethylsilyl chloride (59 mg, 0.38
mmol) was added. The mixture was stirred at room temperature
for 24 h. The solvent was evaporated and ethyl acetate (5 mL) was
added. The organic layer was successively washed with 0.1 N HCl

4′′-Benzoylureido-TSAO DeriVatiVes as HCMV Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5827
Figure 2. Inhibition of immediately early (IE) and early (E) antigen expression on days 2, 5, and 7 postinfection as measured by flow cytometry.
The percentage of cells expressing IE and E antigens in treated and untreated infected cells is expressed after withdrawal of the unspecific fluorescence
of uninfected cells.
Table 2. Inhibitory Activity of Compounds 12 and 15 against Drug-Resistant HCMV Mutants Selected in HEL Cell Cultures
EC50 (µg/mL)a
HCMV strain
(S)-HPMPC
(S)-HPMPA
GCV
ACV
PFA
PMEA
PMEDAP
compd 12
compd 15
wild-type (AD-169)
0.12
0.59
2.2
3.1
0.10
0.069
0.28
2.0
3.3
6.2
0.11
0.081
0.77
7.6
4.9
9.1
1.5
22
4.8
12
7.9
173
130
18
17
14
35
135
37
48
5.8
18
9.1
128
137
15
1.2
0.90
1.0
3.1
0.78
1.1
2.4
3.3
2.6
5.6
2.7
2.7
GCVr (DNA pol mutant)
(S)-HPMPCr (DNA pol mutant)
(S)-HPMPAr (DNA pol mutant)
PFAr (DNA pol mutant)
ACVr (DNA pol mutant)
1.6
5.2
2.8
44
0.54
38
a Effective concentration-50, required to reduce virus-induced cytopathicity by 50%.
Table 3. Activity of Compounds 12 and 15 against Drug-Resistant HCMV Clinical Isolates
EC50 (µg/mL)a
HCMV strain
(S)-HPMPC
(S)-HPMPA
GCV
ACV
PFA
PMEA
PMEDAP
compd 12
compd 15
wild-type (Davis strain)
wild-type (AD-169 strain)
CMV-6 (UL97 mutant)
CMV-521(UL97 + DNA pol mutant)
CMV-530 (UL97 + DNA pol mutant)
0.20
0.12
0.49
0.79
0.62
0.52
0.28
0.91
2.3
0.83
0.77
9.4
6.0
6.2
11
22
91
7.7
22
14
18
34
29
31
30
48
90
20
42
10
15
26
6.7
12
1.1
1.2
0.86
0.67
1.3
4.3
2.4
5.5
2.9
5.7
1.2
a Effective concentration-50, required to reduce virus-induced cytopathicity by 50%.
(2 × 5 mL) and brine (2 × 5 mL), dried (Na2SO4), filtered, and
evaporated to dryness. Purification of the residue by CCTLC on
the Chromatotron (dichloromethane/methanol 30:1) afforded 29 mg
(47%) of 4 as a white amorphous solid. 1H NMR [300 MHz,
(CD3)2SO] δ: 0.19 (s, 6H, 2 CH3), 0.82 (s, 9 H, t-Bu), 1.90 (s, 3H,
CH3-5), 3.27 (s, 3H, CH3-3), 4.18 (dd, 1H, J ) 5.5, 10.4 Hz, H-5′a),
4.29 (dd, 1H, J ) 6.2, 10.4 Hz, H-5′b), 4.40 (dd, 1H, J ) 5.5, 6.2
Hz, H-4′), 4.92 (d, 1H, J ) 6.9 Hz, H-2′), 6.11 (d, 1H, J ) 6.9 Hz,
H-1′), 7.29 (s, 1H, H-3′′), 7.56 (m, 2H, Ph), 7.69 (m, 1H, Ph), 7.75
(s, 1H, H-6), 8.11 (d, 2H, J ) 7.5 Hz, Ph), 10.64 (bs, 1H, NH),

5828 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
de Castro et al.
11.45 (bs, 1H, NH). MS (ESI+) m/z 537.6 (M + H)+, 659.5 (M +
Na)+. Anal. (C27H36N4O10SSi) C, H, N, S.
(ESI+) m/z 522.1 (M + H)+, 544.0 (M + Na)+. Anal.
(C22H23N3O10S) C, H, N, S.
[1-[5′-O-(tert-Butyldimethylsilyl)-2′-O-(thexyldimethylsilyl)-
ꢀ-D-ribofuranosyl]-3-N-methylthymine]-3′-spiro-5′′-[4′′-amino-
1′′,2′′-oxathiole-2′′,2′′-dioxide] (6). A solution of 2′-O-deprotected
TSAO-m3T15 (100 mg, 0.2 mmol), thexyldimethylsilyl chloride (83
µL, 0.4 mmol), and DMAP (51 mg, 0.4 mmol) in dry acetonitrile
(4 mL) was stirred at room temperature for 24 h, and the solvent
was evaporated at dryness. The residue was redissolved in ethyl
acetate and was successively washed with 1N HCl (2 × 10 mL),
water (2 × 10 mL), and brine (2 × 10 mL). The organic phase
was dried (Na2SO4), filtered, and evaporated. The residue was
purified by CCTLC on the Chromatotron (hexane/ethyl acetate, 2:1)
[1-(5′-O-Benzyl-2′-O-(tert-butyldimethylsilyl)-ꢀ-D-ribofurano-
syl)-3-N-methylthymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) (11).17 To a solution of 5′-O-deprotected TSAO-
m3T15 (100 mg, 0.2 mmol) in dry DMF (6 mL), K2CO3 (30 mg,
0.22 mmol) was added and the resulting mixture was stirred for 15
min at -40 °C. Then, benzyl bromide (27 µL, 0.22 mmol) was
added,l and the reaction was stirred at -40 °C for 15 min. The
solvent was evaporated to dryness, and ethyl acetate (5 mL) was
added. The organic layer was washed with brine (2 × 5 mL), dried
(Na2SO4), filtered, and evaporated to dryness. Purification of the
residue by CCTLC on the Chromatotron (dichloromethane/methanol
1
to give 40 mg (30%) of 6 as a white foam. H NMR [300 MHz,
1
20:1) afforded 100 mg (87%) of 1117 as a white foam. H NMR
(CD3)2CO] δ: -0.02 (s, 3H, CH3-Si), 0.14 (s, 3H, CH3-Si), 0.20
(s, 3H, CH3-Si), 0.22 (s, 3H, CH3-Si), 0.74 (s, 3H, CH3), 0.78
(s, 3H, CH3), 0.81 (d, 3H, J ) 6.9 Hz, CH3-CH-Si), 0.82 (d, 3H,
[300 MHz, (CD3)2CO] δ: -0.10 (s, 3H, CH3-Si), 0.04 (s, 3H,
CH3-Si), 0.83 (s, 9 H, t-Bu), 1.60 (s, 3H, CH3-5), 3.25 (s, 3H,
CH3-3), 3.90 (dd, 1H, J ) 2.9, 11.8 Hz, H-5′a), 4.00 (dd, 1H, J )
1.9 Hz, H-5′b), 4.46 (m, 1H, H-4′), 4.72 (d, 1H, H-2′), 4.86 (s, 2H,
CH2Ph), 5.80 (s, 1H, H-3′′), 6.25 (d, 1H, J ) 8.2 Hz, H-1′), 6.48
(bs, 2H, NH2), 7.40 (m, 5H, Ph), 7.72 (s, 1H, H-6).
t
J ) 6.9 Hz, CH3-CH-Si), 0.97 (s, 9H, Bu-Si), 1.58 (m, 1H, CH3-
CH-Si), 1.94 (s, 3H, CH3-5), 3.27 (s, 3H, CH3-3), 4.08 (m, 2H,
H-5′a and H-5′b), 4.33 (t, 1H, J ) 3.6 Hz, H-4′), 4.69 (d, 1H, J )
7.8 Hz, H-2′), 5.77 (s, 1H, H-3′′), 6.08 (d, 1H, J ) 7.8 Hz, H-1′),
6.49 (bs, 2H, NH2), 7.51 (s, 1H, H-6). 13C NMR [75 MHz,
(CD3)2CO] δ: -4.4 (CH3-Si), -3.8 (CH3-Si), -2.1 (CH3-Si),
-0.8 (CH3-Si), 14.1 (CH3-5), 15.5 (C-Si), 19.7 (CH3), 20.0 (CH3),
20.9 (CH3), 21.5 (CH3), 26.6 (C-Si), 27.4 (tBu-Si), 29.1 (CHTDS),
35.6 (CH3-3), 64.1 (C-5′), 76.2 (C-2′), 86.1 (C-4′), 89.3 (C-3′), 93.2
(C-1′), 93.3 (C-3′′), 112.3 (C-5), 135.5 (C-6), 153.1 (C-2), 153.3
(C-4′′), 164.4 (C-4). MS (ESI+) m/z 633.1 (M + H)+, 655.1 (M +
Na)+. Anal. (C27H49N3O8SSi2) C, H, N, S.
General Procedure for the Synthesis of Substituted 4′′-
Ureido TSAO-m3T Derivatives (12-19). A solution of the
corresponding TSAO compound (0.16 mmol) in dry acetonitrile
(8 mL) was reacted with benzoyl isocyanate (80 µL, 0.64 mmol)
in an Ace pressure tube at 100 °C overnight. After evaporation,
the residue was purified by CCTLC on the Chromatotron. The
chromatography eluent, yield, and analytical and spectroscopic data
of the isolated products are indicated below for each reaction.
[1-[2′,5′-Bis-O-(thexyldimethylsilyl)-ꢀ-D-ribofuranosyl]-3-N-
methylthymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-
dioxide) (9). A solution of 2′,5′-O-deprotected TSAO-m3T (8)16
(150 mg, 0.4 mmol), thexyldimethylsilyl chloride (393 µL, 2 mmol),
and DMAP (195 mg, 1.6 mmol) in dry acetonitrile (5 mL) was
stirred at 100 °C for 24 h. Then, the solvent was evaporated at
dryness. The residue was redissolved in ethyl acetate and washed
with 1N HCl (2 × 10 mL), water (2 × 10 mL), and brine (2 × 10
mL). The organic phase was dried (Na2SO4), filtered, and evapo-
rated. The residue was purified by CCTLC on the Chromatotron
(hexane/ethyl acetate, 2:1) to yield 131 mg (50%) of 9 as a white
foam. 1H NMR [300 MHz, (CD3)2CO] δ: -0.02 (s, 3H, CH3-Si),
0.14 (s, 3H, CH3-Si), 0.21 (s, 3H, CH3-Si), 0.22 (s, 3H, CH3-Si),
0.75 (s, 3H, CH3), 0.79 (s, 3H, CH3), 0.81 (d, 3H, J ) 6.9 Hz,
CH3-CH-Si), 0.83 (d, 3H, J ) 6.9 Hz, CH3-CH-Si), 0.91 (m, 12H,
TDS), 1.57 (sept, 1H, J ) 6.9 Hz, CH3-CH-Si), 1.68 (sept, 1H, J
) 6.9 Hz, CH3-CH-Si), 1.94 (s, 3H, CH3-5), 3.27 (s, 3H, CH3-3),
4.02 (dd, 1H, J ) 5.1 and 12.3 Hz, H-5′a), 4.11 (dd, 1H, J ) 3.6
and 12.3 Hz, H-5′b), 4.32 (m, 1H, H-4′), 4.77 (d, 1H, J ) 7.8 Hz,
H-2′), 5.74 (s, 1H, H-3′′), 6.00 (d, 1H, J ) 7.8 Hz, H-1′), 6.49 (bs,
2H, NH2), 7.53 (s, 1H, H-6). 13C NMR [75 MHz, (CD3)2CO] δ:
-2.2 (CH3-Si), -1.9 (CH3-Si), -0.9 (CH3-Si), 14.1 (CH3-5),
19.6 (CH3-Si), 19.7 (CH3-Si), 19.9 (CH3-Si), 20.0 (CH3-Si),
20.9 (CH3-Si), 21.5 (CH3-Si), 21.6 (CH3-Si), 21.8 (CH3-Si),
26.5 (C-Si), 26.9 (C-Si), 29.1 (CH), 35.6 (CH-TDS), 35.7 (CH3-
3), 63.8 (C-5′), 75.8 (C-2′), 86.2 (C-4′), 89.9 (C-3′), 92.6, 92.8 (C-
1′, C-3′′), 112.2 (C-5), 136.0 (C-6), 153.1 (C-2), 153.6 (C-4′′), 164.3
(C-4). MS (ESI+) m/z 660.3 (M + H)+. Anal. (C29H53N3O8SSi2)
C, H, N, S.
[1-(2′-O-Acetyl-5′-O-benzoyl-ꢀ-D-ribofuranosyl)-3-N-meth-
ylthymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-diox-
ide) (10). To a solution of 2′-O-acetyl-5′-O-benzoyl TSAO-T16 (250
mg, 0.48 mmol) in acetone (10 mL), K2CO3 (33 mg, 0.24 mmol)
and methyl iodide (120 µL, 1.9 mmol) were added. The reaction
mixture was refluxed at 50 °C overnight. After removal of the
solvent, the residue was purified by column chromatography with
dicholoromethane/methanol (30:1) to give 10 (228 mg, 71%) as a
white foam. 1H NMR [300 MHz, (CD3)2CO] δ: 1.84 (s, 3H, CH3-
5), 2.03 (s, 3H, CH3CO), 3.24 (s, 3H, CH3-3), 4.70 (m, 3H, H-5′
and H-4′), 5.83 (s, 1H, H-3′′), 5.93 (d, 1H, J ) 7.2 Hz, H-2′), 6.15
(d, 1H, J ) 7.2 Hz, H-1′), 6.72 (bs, 2H, NH2), 7.51 (m, 2H, H-6
and Ph), 7.62-7.68 (m, 2H, Ph), 8.04 (d, 2H, J ) 7.5 Hz, Ph). MS
[1-[2′-O-(tert-Butyldimethylsilyl)-5′-O-(thexyldimethylsilyl)]-
ꢀ-D-ribofuranosyl]-3-N-methylthymine]-3′-spiro-5′′-[4′′-(3-ben-
zoylureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (12). According to the
general procedure, 514 (100 mg, 0.16 mmol) was reacted with
benzoyl isocyanate. Chromatography of the residue with hexane/
ethyl acetate (3:1) afforded 121 mg (98%) of 12 as a white foam.
1H NMR [300 MHz, (CD3)2CO] δ: -0.15 (s, 3H, CH3-Si), -0.02
(s, 3H, CH3-Si), 0.12 (s, 3H, CH3-Si), 0.13 (s, 3H, CH3-Si),
t
0.80 (s, 15H, CH3-TDS and Bu), 0.81 (d, 6H, J ) 7.5 Hz, CH3-
CH-Si), 1.56 (sept, 1H, J ) 7.2 Hz, CH-TDS), 1.92 (d, 3H, J )
1.2 Hz, CH3-5), 3.28 (s, 3H, CH3-3), 4.03 (dd, 1H, J ) 5.1 and
12.0 Hz, H-5′a), 4.20 (dd, 1H, J ) 7.8 and 12.0 Hz, H-5′b), 4.39
(dd, 1H, J ) 5.1 and 7.8 Hz, H-4′), 5.04 (d, 1H, J ) 7.8 Hz, H-2′),
5.98 (d, 1H, J ) 7.8 Hz, H-1′), 7.41 (s, 1H, H-3′′), 7.64 (m, 3H,
H-6 and Ph), 7.77 (m, 1H, Ph), 8.15 (d, 2H, J ) 7.5 Hz, Ph), 10.62
(bs, 1H, NH), 11.53 (bs, 1H, NH). 13C NMR [75 MHz, (CD3)2CO]
δ: -5.5 (CH3-Si), -5.2 (CH3-Si), -3.6 (CH3-Si), 12.8 (CH3-
5), 18.0, 18.5 (CH3-TDS), 20.1, 20.2 (CH3-TDS), 25.4 (tBu-Si),
25.5 (C-tBu), 27.7 (CH-TDS), 34.4 (CH3-3), 61.5 (C-5′), 73.8 (C-
2′), 84.9 (C-4′), 89.5, 91.6 (C-1′, C-3′), 106.9, 111.1 (C-5, C-3′′),
128.9, 129.6, 131.9, 134.6 (Ph), 135.4 (C-6), 142.0 (C-4′′), 150.9,
151.6 (CO, C-2), 163.1 (C-4), 170.4 (CO). MS (ESI+) m/z 779.2
(M + H)+. Anal. (C35H54N4O10SSi2) C, H, N, S.
[1-[5′-O-(tert-Butyldimethylsilyl]-2′-O-(thexyldimethylsilyl)-
ꢀ-D-ribofuranosyl]-3-N-methylthymine]-3′-spiro-5′′-[4′′-(3-ben-
zoylureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (13). The general pro-
cedure was followed with 615 (100 mg, 0.16 mmol). Chromatography
of the residue with hexane/ethyl acetate (3:1) yielded 13 (80 mg,
1
64%) as a white foam. H NMR [300 MHz, (CD3)2CO] δ: -0.07
(s, 3H, CH3-Si), 0.04 (s, 3H, CH3-Si), 0.12 (s, 3H, CH3-Si),
0.13 (s, 3H, CH3-Si), 0.77 (d, 3H, J ) 7.6 Hz, CH3-CH-Si), 0.82
(d, 3H, J ) 7.0 Hz, CH3-CH-Si), 0.85 (s, 15H, 2CH3-TDS and
tBu), 1.56 (sept, 1H, J ) 7.0 Hz, CH-TDS), 1.96 (d, 3H, J ) 1.1
Hz, CH3-5), 3.29 (s, 3H, CH3-3), 4.05 (dd, 1H, J ) 4.5 and 12.3
Hz, H-5′a), 4.20 (dd, 1H, J ) 6.4 and 12.3 Hz, H-5′b), 4.41 (dd,
1H, J ) 4.5 and 6.4 Hz, H-4′), 5.00 (d, 1H, J ) 7.8 Hz, H-2′),
6.08 (d, 1H, J ) 7.8 Hz, H-1′), 7.44 (s, 1H, H-3′′), 7.64 (m, 3H,
H-6 and Ph), 7.76 (m, 1H, Ph), 8.16 (d, 2H, J ) 7.5 Hz, Ph), 10.65
(bs, 1H, NH), 11.47 (bs, 1H, NH). MS (ESI+) m/z 779.3 (M +
H)+. Anal. (C35H54N4O10SSi2) C, H, N, S.

4′′-Benzoylureido-TSAO DeriVatiVes as HCMV Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5829
[1-[2′-O-(tert-Butyldimethylsilyl)-5′-O-(triisopropylsilyl)-ꢀ-D-
ribofuranosyl]-3-N-methylthymine]-3′-spiro-5′′-[4′′-(3-benzoy-
lureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (14). Following the general
procedure, 714 (100 mg, 0.15 mmol) was reacted with benzoyl
isocyanate (75 µL, 0.60 mmol). Chromatography of the residue
with hexane/ethyl acetate 3:1 yielded 82 mg (69%) of 14 as a white
foam. 1H NMR [300 MHz, (CD3)2CO] δ: -0.15 (s, 3H, CH3-Si),
152.1, 152.9 (C-2, CO), 164.2 (CO), 166.8 (C-4), 171.4 (CO). MS
(ESI+) m/z 627.1 (M + H)+. Anal. (C28H26N4O11S) C, H, N, S.
[1-[5′-O-Benzoyl-2′-O-(tert-butyldimethylsilyl)-ꢀ-D-ribofura-
nosyl]-3-N-methylthymine]-3′-spiro-5′′-[4′′-(3-benzoylureido)-
1′′,2′′-oxathiole-2′′,2′′-dioxide] (18). A solution of 17 (100 mg,
0.16 mmol), tert-butyldimethylsilyl chloride (48 mg, 0.32 mmol),
and DMAP (39 mg, 0.32 mmol) in dry acetonitrile (5 mL) was
stirred at room temperature overnight. The solvent was evaporated
to dryness, and ethyl acetate (5 mL) was added. The organic layer
was successively washed with 0.1 N HCl (2 × 5 mL) and brine (2
× 5 mL), dried (Na2SO4), filtered, and evaporated to dryness.
Purification of the residue by CCTLC on the Chromatotron (hexane/
t
-0.01 (s, 3H, CH3-Si), 0.80 (s, 9H, Bu), 1.00 (m, 21H, 3iPr),
1.91 (s, 3H, CH3-5), 4.12 (dd, 1H, J ) 5.8 and 11.9 Hz, H-5′a),
4.28 (dd, 1H, J ) 7.3 and 11.9 Hz, H-5′b), 4.46 (dd, 1H, J ) 5.8
and 7.3 Hz, H-4′), 5.07 (d, 1H, J ) 7.7 Hz, H-2′), 5.96 (d, 1H, J
) 7.7 Hz, H-1′), 7.41 (s, 1H, H-3′′), 7.65 (m, 3H, H-6 and Ph),
7.74 (m, 1H, Ph), 8.15 (d, 2H, J ) 7.5 Hz, Ph), 10.62 (bs, 1H,
NH), 11.61 (bs, 1H, NH). 13C NMR [75 MHz, (CD3)2CO] δ: -5.2
(CH3-Si), -4.8 (CH3-Si), 12.5 (CH3-5), 13.1 (3CH-iPr), 18.2
(6CH3-iPr), 18.3 (C-tBu), 25.7 (tBu), 27.9 (CH3-3), 62.3 (C-5′), 74.2
(C-2′), 85.4 (C-4′), 90.2, 91.4 (C-1′, C-3′), 107.0, 111.3 (C-5, C-3′′),
129.2, 129.9, 132.3, 135.0 (Ph), 136.0 (C-6), 142.4 (C-4′′), 151.2,
151.9 (CO, C-2), 163.4 (C-4), 170.9 (CO). MS (ESI+) m/z 793.2
(M + H)+, 815.1 (M + Na)+. Anal. (C36H56N4O10SSi2) C, H, N,
S.
[1-[2′,5′-Bis-O-(thexyldimethylsilyl)-ꢀ-D-ribofuranosyl]-3-N-
methylthymine]-3′-spiro-5′′-[4′′-(3-benzoylureido)-1′′,2′′-oxathi-
ole-2′′,2′′-dioxide] (15). According to the general procedure, 9 (100
mg, 0.16 mmol) was reacted with benzoyl isocyanate to give after
chromatography (hexane/ethyl acetate 3:1) 15 (92 mg, 75%) as a
white foam. 1H NMR [300 MHz, (CD3)2CO] δ: -0.09 (s, 3H,
CH3-Si), 0.03 (s, 3H, CH3-Si), 0.13 (s, 3H, CH3-Si), 0.14 (s,
3H, CH3-Si), 0.80 (m, 24H, 8CH3), 1.56 (m, 2H, 2CH-Si), 1.94
(s, 3H, CH3-5), 3.29 (s, 3H, CH3-3), 4.02 (dd, 1H, J ) 5.1 and
12.1 Hz, H-5′a), 4.21 (dd, 1H, J ) 7.5 and 12.1 Hz, H-5′b), 4.38
(dd, 1H, J ) 5.1 and 7.5 Hz, H-4′), 5.07 (d, 1H, J ) 7.7 Hz, H-2′),
5.97 (d, 1H, J ) 7.7 Hz, H-1′), 7.41 (s, 1H, H-3′′), 7.64 (m, 3H,
H-6 and Ph), 7.76 (m, 1H, Ph), 8.16 (d, 2H, J ) 7.5 Hz, Ph), 10.69
(bs, 1H, NH), 11.55 (bs, 1H, NH). 13C NMR [75 MHz, (CD3)2CO]
δ: -2.2 (2CH3-Si), -2.0 (CH3-Si), -1.4 (CH3-Si), 14.1 (CH3-
5), 19.6 (CH3-TDS), 19.7 (CH3-TDS), 19.8 (CH3-TDS), 19.9 (CH3-
TDS), 21.1 (CH3-TDS), 21.4 (CH3-TDS), 21.5 (CH3-TDS), 21.6
(CH3-TDS), 26.5 (C-TDS), 26.9 (C-TDS), 29.0 (2CH-TDS), 35.6
(CH3-3), 62.8 (C-5′), 74.9 (C-2′), 86.2 (C-4′), 90.9, 92.9 (C-1′, C-3′),
108.3, 112.4 (C-5, C-3′′), 130.2, 130.9, 133.3, 135.9 (Ph), 136.9
(C-6), 143.4 (C-4′′), 152.2, 152.9 (CO, C-2), 164.4 (C-4), 171.7
(CO). MS (ESI+) m/z 807.3 (M + H)+. Anal. (C37H58N4O10SSi2)
C, H, N, S.
1
ethyl acetate, 3:1) gave 92 mg (78%) of 18 as a white foam. H
NMR [300 MHz, (CD3)2CO] δ: -0.10 (s, 3H, CH3-Si), 0.04 (s,
3H, CH3-Si), 0.83 (s, 9 H, t-Bu), 1.86 (s, 3H, CH3-5), 3.28 (s,
3H, CH3-3), 4.82 (m, 3H, H-5′ and H-4′), 5.04 (d, 1H, J ) 7.7 Hz,
H-2′), 6.13 (d, 1H, J ) 7.7 Hz, H-1′), 7.46 (m, 3H, H-3′′ and Ph),
7.56 (m, 3H, H-6 and Ph), 7.74 (m, 2H, Ph), 8.03 (m, 4H, Ph),
10.62 (bs, 1H, NH), 11.70 (bs, 1H, NH). 13C NMR [75 MHz,
(CD3)2SO] δ: -4.2 (CH3-Si), -3.7 (CH3-Si), 14.1 (CH3-5), 19.4
(C-tBu), 26.7 (tBu), 29.1 (CH3-3), 63.3 (C-5′), 75.8 (C-2′), 83.1
(C-4′), 90.7, 91.4 (C-3′, C-1′), 107.8, 112.6 (C-3′′, C-5), 130.1,
130.2, 130.4, 130.7, 131.3, 131.5, 133.2, 134.4, 135.3, 135.9 (Ph),
136.3 (C-6), 152.3, 152.9 (C-2, CO), 164.4 (CO), 167.0 (C-4), 171.8
(CO). MS (ESI+) m/z 741.2 (M + H)+. Anal. (C34H40N4O11SSi)
C, H, N, S.
[1-[5′-O-Benzyl-2′-O-(tert-butyldimethylsilyl)-ꢀ-D-ribofurano-
syl]-3-N-methylthymine]-3′-spiro-5′′-[4′′-(3-benzoylureido)-1′′,2′′-
oxathiole-2′′,2′′-dioxide] (19). Following the general procedure,
1117 (100 mg, 0.15 mmol) was reacted with benzoyl isocyanate
(75 µL, 0.60 mmol). Purification of the residue by CCTLC on the
Chromatotron (hexane/ethyl acetate 3:1) yielded 82 mg (69%) of
19 as a white foam. 1H NMR [300 MHz, (CD3)2CO] δ: -0.10 (s,
3H, CH3-Si), 0.04 (s, 3H, CH3-Si), 0.80 (s, 9 H, t-Bu), 1.93 (s,
3H, CH3-5), 3.29 (s, 3H, CH3-3), 3.90 (m, 2H, 2H-5′), 4.55 (m,
1H, H-4′), 4.96 (m, 3H, H-2′, CH2Ph), 6.33 (d, 1H, J ) 7.9 Hz,
H-1′), 7.27-8.17 (m, 12H, H-3′′, H-6 and 2Ph), 9.85 (bs, 1H, NH),
11.55 (bs, 1H, NH). MS (ESI+) m/z 727.1 (M + H)+. Anal.
(C34H42N4O10SSi) C, H, N, S.
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl)-ꢀ-D-ribofuranosyl]-3-
N-methylthymine]-3′-spiro-5′′-[4′′-(3-phenylureido)-1′′,2′′-oxati-
ole-2′′,2′′-dioxide] and [1-[2′,5′-Bis-O-tert-buthyldimethylsilyl)-
ꢀ-D-ribofuranosyl]-3-N-methylthymine]-3′-spiro-5′′-[4′′-(3-
phenylcarboxamide-3-phenylureido)-1′′,2′′-oxatiole-2′′,2′′-
dioxide] (21 and 22). To a solution of TSAO-m3T (20)16 (70 mg,
0.12 mmol) in dry THF (10 mL), previously degassed under an
argon atmosphere, sodium hydride 60% dispersion in mineral oil
(10 mg, 0.23 mmol) was added. The mixture was stirred at room
temperature for 1 h. Then, phenylisocyanate (0.03 mL, 0.23 mmol)
was added and the resulting mixture was stirred at room temperature
for 2.5 h. Volatiles were removed, and the residue was dissolved
in ethyl acetate (10 mL) and washed, successively, with water (1
× 10 mL) and brine (2 × 10 mL). The organic phase was dried
(Na2SO4), filtered, and evaporated to dryness. The residue was
purified by CCTLC on the Chromatotron (hexane/ethyl acetate, 4:1).
The fastest moving band afforded 21 (49 mg, 58%) as a white foam.
1H NMR [300 MHz, (CD3)2CO] δ: 0.07 (s, 3H, CH3-Si), 0.09 (s,
3H, CH3-Si), 0.10 (s, 3H, CH3-Si), 0.13 (s, 3H, CH3-Si), 0.80
[1-(2′-O-Acetyl-5′-O-benzoyl-ꢀ-D-ribofuranosyl)-3-N-meth-
ylthymine]-3′-spiro-5′′′-[4′′-(3-benzoylureido)-1′′,2′′-oxathiole-
2′′,2′′-dioxide] (16). According to the general procedure, compound
10 (100 mg, 0.19 mmol) was reacted with benzoyl isocyanate (95
µL, 0.76 mmol). Chromatography of the residue with hexane/ethyl
1
acetate (3:1) gave 16 (119 mg, 94%) as a white foam. H NMR
[300 MHz, (CD3)2CO] δ: 1.82 (s, 3H, CH3-5), 2.06 (s, 3H,
CH3-CO), 3.22 (s, 3H, CH3-3), 4.80 (m, 2H, H-5′), 4.90 (m, 1H,
H-4′), 5.83 (d, 1H, J ) 7.9 Hz, H-2′), 6.43 (d, 1H, J ) 7.7 Hz,
H-1′), 7.44-7.94 (m, 8H, H-3′′ and Ph), 8.05 (m, 4H, H-6 and
Ph), 11.34 (bs, 1H, NH), 11.72 (bs, 1H, NH). MS (ESI-) m/z 667.0
(M - H)-. Anal. (C30H28N4O12S) C, H, N, S.
[1-(5′-O-Benzoyl-ꢀ-D-ribofuranosyl)-3-N-methylthymine]-3′-
spiro-5′′-[4′′-(3-benzoylureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide]
(17). A solution of 16 (50 mg, 0.08 mmol) in methanol saturated
with ammonia (2 mL) was stirred at 0 °C for 1 h. The reaction
mixture was evaporated to dryness and the residue was purified by
CCTLC on the Chromatotron (dichloromethane/methanol, 20:1) to
give 17 (36 mg, 90%) as a white foam. 1H NMR [300 MHz,
(CD3)2CO] δ: 1.83 (s, 3H, CH3-5), 3.26 (s, 3H, CH3-3), 4.62-4.83
(m, 3H, H-5′ and H-4′), 5.03 (d, 1H, J ) 7.9 Hz, H-2′), 6.18 (d,
1H, J ) 7.9 Hz, H-1′), 7.43-7.74 (m, 8H, H-3′′, H-6 and Ph),
8.00 (m, 4H, Ph), 8.30 (bs, 1H, NH), 9.55 (bs, 1H, NH). 13C NMR
[75 MHz, (CD3)2SO] δ: 13.8 (CH3-5), 28.8 (CH3-3), 63.1 (C-5′),
74.9 (C-2′), 82.8 (C-4′), 91.1, 91.3 (C-3′, C-1′), 106.9, 111.9 (C-
3′′, C-5), 129.5, 129.9, 130.1, 130.2, 130.4, 130.5, 130.9, 131.2,
132.8, 133.0, 134.9, 135.5 (Ph), 135.8 (C-6, Ph), 143.8 (C-4′′),
t
t
(s, 9H, Bu-Si), 0.85 (s, 9H, Bu-Si), 1.94 (s, 3H, CH3-5), 3.40 (s,
3H, CH3-3), 3.95 (m, 1H, H-5′), 4.13 (m, 2H, H-4′ and H-5′), 5.18
(d, 1H, J ) 6.2 H, H-2′), 5.50 (d, 1H, J ) 6.4, H-1′), 7.10 (m, 1H,
Ph), 7.21 (s, 1H, H-3′′), 7.33 (m, 2H, Ph), 7.55 (m, 2H, Ph), 7.74
(d, 1H, J ) 1.2, H-6), 8.30 (bs, 1H, NH), 9.22 (bs, 1H, NH). 13C
NMR [75 MHz, (CD3)2CO] δ: -4.2 (CH3-Si), -4.1 (CH3-Si),
-3.9 (CH3-Si), -3.7 (CH3-Si), 12.9 (CH3-5), 18.5 (C-tBu), 18.8
(C-tBu), 25.8 (tBu), 26.1 (tBu), 28.1 (CH3-3), 61.4 (C-5′), 73.5 (C-
2′), 84.2 (C-4′), 88.7 (C-3′), 97.7 (C-1′), 100.8 (C-3′′), 111.8 (C-
5), 119.7, 124.3, 129.8, 139.2, 139.6 (C-6), 145.3 (C-4′′), 152.5,
151.4 (C-2, CO), 163.2 (C-4). MS (ESI+) m/z 723.2 (M + H)+,
745.3 (M + Na)+. Anal. (C32H50N4O9SSi2) C, H, N, S.

5830 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
de Castro et al.
The slowest moving band afforded 26 mg (21%) of 22 as a white
foam. 1H NMR [300 MHz, (CD3)2CO] δ: -0.08 (s, 3H, CH3-Si),
0.07 (s, 3H, CH3-Si), 0.14 (s, 3H, CH3-Si), 0.15 (s, 3H, CH3-Si),
0.83 (s, 9H, tBu-Si), 0.92 (s, 9H, tBu-Si), 1.75 (s, 3H, CH3-5), 3.28
(s, 3H, CH3-3), 4.15 (m, 2H, H-5′), 4.42 (m, 1H, H-4′), 4.85 (d,
1H, J ) 8.2, H-2′), 6.10 (d, 1H, J ) 8.2, H-1′), 7.16-7.66 (m,
12H, H-6, H-3′′ and Ph), 8.15 (bs, 1H, NH), 12.30 (bs, 1H, NH).
13C NMR [75 MHz, (CD3)2CO] δ: -3.9 (CH3-Si), -3.7 (CH3-Si),
-3.5 (CH3-Si), -3.3 (CH3-Si), 13.7 (CH3-5), 18.7 (C-tBu), 18.9
(C-tBu), 26.4 (tBu), 27.0 (tBu), 28.7 (CH3-3), 62.8 (C-5′), 75.6 (C-
2′), 86.0 (C-4′), 89.1 (C-1′), 92.7 (C-3′), 106.8 (C-3′′), 112.3 (C-
5), 124.8, 127.1, 130.2, 130.4, 130.5, 131.4, 131.4, 131.9 (Ph), 135.2
(C-6), 137.0 (Ph), 138.3 (Ph), 143.4 (C-4′′), 152.6, 153.1 (C-2, CO),
157.2 (CO), 164.0 (C-4). MS (ESI+) m/z 843.5 (M + H)+, 864.0
(M + Na)+. Anal. (C39H55N5O10SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(tert-buthyldimethylsilyl)-ꢀ-D-ribofuranosyl]-
3-N-methylthymine]-3′-spiro-5′′-[4′′-(3-benzylureido)-1′′,2′′-ox-
atiole-2′′,2′′-dioxide] (23). To a solution of 2016 (70 mg, 0.12
mmol) in dry THF (10 mL), previously degassed under an argon
atmosphere, sodium hydride 60% dispersion in mineral oil (10 mg,
0.23 mmol) was added. The mixture was stirred at room temperature
for 1 h. Then, benzylisocyanate (0.03 mL, 0.23 mmol) was added
and the resulting mixture was stirred at room temperature for 3 h.
Volatiles were removed, and the residue was dissolved in ethyl
acetate (10 mL) and successively washed with water (1 × 10 mL)
and brine (2 × 10 mL). The organic phase was dried (Na2SO4),
filtered, and evaporated to dryness. The final residue was purified
by CCTLC on the Chromatotron (hexane/ethyl acetate, 9:1) to give
64 mg (75%) of 23 as a white foam. 1H NMR [300 MHz,
(CD3)2CO] δ: -0.07 (s, 3H, CH3-Si), 0.07 (s, 3H, CH3-Si), 0.08
(s, 3H, CH3-Si), 0.09 (s, 3H, CH3-Si), 0.83 (s, 9H, tBu), 0.88 (s,
9H, tBu), 1.90 (s, 3H, CH3-5), 3.24 (s, 3H, CH3-3), 3.87-4.24 (m,
3H, H-5′ and H-4′), 4.45 (dd, J ) 5.8 and 6.2, CH2Ph), 5.06 (d,
1H, J ) 6.8, H-2′), 5.58 (d, 1H, J ) 6.8, H-1′), 6.77 (t, 1H, J )
5.8, NHCH2Ph), 7.16 (s, 1H, H-3′′), 7.30 (m, 5H, Ph), 7.70 (s, 1H,
H-6), 9.08 (bs, 1H, NH). 13C NMR [75 MHz, (CD3)2CO] δ: -4.79
(CH3-Si), -4.76 (CH3-Si), -4.70 (CH3-Si), -4.15 (CH3-Si),
13.4, (CH3-5), 18.9 (C-tBu), 18.6 (C-tBu), 26.7 (tBu-Si), 26.2 (tBu-
Si), 28.5 (CH3-3), 44.8 (CH2Ph), 62.2 (C-5′), 74.1 (C-2′), 85.1 (C-
4′), 90.0 (C-3′), 95.8 (C-1′), 101.0 (C-3′′), 112.1 (C-5), 128.4, 128.6,
129.7, 138.9 (Ph), 140.5 (C-6), 145.7 (C-4′′), 152.8, 154.5 (C-2,
CO), 163.7 (C-4). MS (ESI+) m/z 737.1 (M + H)+, 759.2 (M +
Na)+. Anal. (C33H52N4O9SSi2) C, H, N, S.
[N-Acetyl-[2,5-bis-O-(tert-butyldimethylsilyl)-ꢀ-D-ribofurano-
syl]amine]-3-spiro-5′-[4′-3-(benzoylureido)-1′,2′-oxathiole-2′,2′-
dioxide] (29). Following the general procedure described for
compounds 12-19, compound 2818 (50 mg, 0.1 mmol) was reacted
with benzoyl isocyanate (50 µL, 0.4 mmol) in dry acetonitrile (5
mL) in an Ace pressure tube at 100 °C overnight. Chromatography
of the residue on the Chromatotron (hexane/ethyl acetate 5:1) gave
29 (60 mg, 90%) as a white foam. 1H NMR [300 MHz, (CD3)2CO]
δ: 0.03 (s, 3H, CH3-Si), 0.06 (s, 3H, CH3-Si), 0.08 (s, 3H,
CH3-Si), 0.09 (s, 3H, CH3-Si), 0.82 (s, 9H, tBu-Si), 0.87 (s, 9H,
tBu-Si), 1.98 (s, 3H, CH3-CO), 3.94 (dd, 1H, J ) 5.2 and 11.9
Hz, H-5a), 4.01 (dd, 1H, J ) 6.7 and 11.9 Hz, H-5b), 4.17 (dd,
1H, J ) 5.2 and 6.7 Hz, H-4), 4.51 (d, 1H, J ) 6.5 Hz, H-2), 5.61
(dd, 1H, J ) 6.5 and 9.9 Hz, H-1), 7.32 (s, 1H, H-3′), 7.62 (m,
3H, H-6 and Ph), 7.75 (m, 1H, Ph), 8.11 (d, 1H, J ) 7.2 Hz, Ph),
9.84 (bs, 1H, NH), 10.54 (bs, 1H, NH), 11.29 (bs, 1H, NH). 13C
NMR [75 MHz, (CD3)2CO] δ: -5.8 (CH3-Si), -5.7 (CH3-Si),
-5.2 (CH3-Si), -5.0 (CH3-Si), 18.2 (C-tBu), 18.4 (C-tBu), 25.6
(tBu), 25.8 (tBu), 61.4 (C-5), 76.4 (C-2), 82.6 (C-3′), 84.6 (C-4),
90.9 (C-1), 105.9 (C-3), 128.6, 128.8, 129.5, 134.4 (Ph), 153.3 (C-
4′), 160.9 (CO), 170.4 (CO). MS (ESI+) m/z 670 (M + H)+, 692
(M + Na)+. Anal. (C29H47N3O9SSi2) C, H, N, S.
24 h. The organic layer was separated, and the aqueous phase was
further extracted with diethyl ether (2 × 20 mL). The combined
ethereal extracts were dried (Na2SO4) and evaporated to dryness.
The residue was dissolved in dry dichloromethane (3 mL) and
treated with Et3N (1.6 mL, 11.6 mmol) and then methanesulfonyl
chloride (0.38 mL, 5 mmol) was slowly added. The mixture was
stirred at -20 °C for 1 h and at 0 °C for an additional hour. Volatiles
were removed, and the residue was dissolved in ethyl acetate (10
mL) and washed successively with water (1 × 10 mL) and brine
(2 × 10 mL). The organic phase was dried (Na2SO4), filtered, and
evaporated to dryness. The final residue was purified by flash
column chromatography (hexane:ethyl acetate, 60:1). The faster
moving fractions afforded 31 (298 mg, 31%) as an amorphous solid.
1H NMR [300 MHz, (CD3)2CO] δ: 0.14 (s, 6H, 2CH3-Si), 0.19
(s, 6H, 2CH3-Si), 0.95 (s, 9H, Bu-Si), 0.97 (s, 9H, Bu-Si), 2.43
(m, 2H, H-3), 3.34 (s, 3H, CH3-S), 3.97 (m, 2H, H-4), 4.19 (s,
2H, H-1). MS (ESI+) m/z 438.9 (M + 1)+. Anal. (C18H39NO5SSi2)
C, H, N, S.
The slowest moving fractions afforded 246 mg (35%) of
unreacted starting material (30).
t
t
4-Amino-5-(tert-butyldimethylsilyloxyethyl)-5-(tert-butyldim-
ethylsilyloxymethyl)-5H-1,2-oxathiole-2,2-dioxide (32). To a
suspension of 31 (270 mg, 0.6 mmol) in dry acetonitrile (6 mL),
cesium carbonate (300 mg, 0.9 mmol) was added and the mixture
was stirred at room temperature for 3 h. The solvent was removed
and the residue, thus obtained, was dissolved in ethyl acetate (20
mL) and washed successively with water (10 mL) and brine (2 ×
10 mL). The organic phase was dried (Na2SO4), filtered, and
evaporated to dryness. The residue was purified by CCTLC on the
Chromatotron (hexane/ethyl acetate, 6:1) to give 32 (200 mg, 75%)
1
as a white foam. H NMR [300 MHz, (CD3)2CO] δ: 0.09 (s, 3H,
CH3-Si), 0.10 (s, 3H, CH3-Si), 0.13 (s, 3H, CH3-Si), 0.14 (s,
t
t
3H, CH3-Si), 0.92 (s, 9H, Bu-Si), 0.94 (s, 9H, Bu-Si), 2.24 (m,
2H, CH2-CH2-O), 3.79-3.94 (m, 2H, CH2-CH2-O), 3.99 (s, 2H,
CH2-O), 5.46 (s, 1H, H-3), 6.10 (bs, 2H, NH2). 13C NMR [75
MHz, (CD3)2CO] δ:-7.0 (2CH3-Si), -6.9 (2CH3-Si), 24.5 (tBu-
Si), 24.6 (tBu-Si), 35.5 CH2-CH2-O), 57.1 CH2-CH2-O), 65.7
(CH2-O), 87.8 (C-3), 89.4 (C-5), 155.3 (C-4). MS (ESI+) m/z 438.0
(M + 1)+, 460.0 (M + Na)+, 897.2 (2 M + Na)+. Anal.
(C18H39NO5SSi) C, H, N, S.
4-Benzoylureido-5-(tert-butyldimethylsilyloxyethyl)-5-(tert-bu-
tyldimethylsilyloxymethyl)-5H-1,2-oxathiole-2,2-dioxide (33). Ac-
cording to the general procedure described for the synthesis of
compounds 12-19, compound 32 (100 mg, 0.22 mmol) was reacted
with benzoyl isocyanate (112 µL, 0.88 mmol) in dry acetonitrile
(8 mL) in an Ace pressure tube at 100 °C overnight. Chromatog-
raphy of the residue on the Chromatotron (hexane/ethyl acetate 3:1)
yielded 86 mg (67%) of 33 as a white foam. 1H NMR [300 MHz,
CDCl3] δ: -0.01 (s, 3H, CH3-Si), 0.01 (s, 3H, CH3-Si), 0.10 (s,
3H, CH3-Si), 0.13 (s, 3H, CH3-Si), 0.81 (s, 9H, tBu-Si), 0.89 (s,
t
9H, Bu-Si), 2.17 (td, 1H, J ) 4.4 and 15.2 Hz, CH2a-CH2-O),
2.40 (ddd, 1H, J ) 5.2, 8.4 and 15.2 Hz, CH2b-CH2-O), 3.84 (m,
2H, CH2-CH2-O), 3.91 and 3.95 (AB system, J ) 10.4 Hz,
CH2-O), 7.00 (s, 1H, H-3), 7.57 (t, 2H, J ) 8.0 Hz, Ph), 7.71 (t,
1H, J ) 7.6 Hz, Ph), 7.96 (d, 2H, J ) 7.6 Hz, Ph), 10.09 (bs, 1H,
NH), 11.30 (bs, 1H, NH). 13C NMR [75 MHz, CDCl3] δ: -5.7
(CH3-Si),-5.6 (CH3-Si), -5.5 (CH3-Si), -5.4 (CH3-Si), 18.2
(C-tBu), 18.5 (C-tBu), 25.7 (tBu-Si), 25.8 (tBu-Si), 36.4
(CH2-CH2-O), 57.2 (CH2-CH2-O), 68.1 (CH2O), 89.8 (C-3),
104.2 (C-5), 127.9, 129.1, 131.1, 134.4 (Ph), 146.4 (C-4), 151.9
(CO), 168.4 (CO). MS (ESI+) m/z 586.3 (M + 1)+, 605.0 (M +
Na)+. Anal. (C26H44N2O7SSi2) C, H, N, S.
Biological Methods. Anticytomegalovirus Assay. For the
HCMV assays, confluent HEL fibroblasts were grown in 96-
well microtiter plates and infected with the human cytomega-
lovirus strains Davis and AD-169 at 100 PFU per well. After a
2 h incubation period, residual virus was removed and the
infected cells were further incubated with medium containing
different concentrations of the test compounds (in duplicate).
After incubation for 7 days at 37 °C, virus-induced cytopatho-
genicity was monitored microscopically after ethanol fixation
2-(Methanesulfonyloxy)-4-(tert-butyldimethylsilyloxy)-2-(tert-
butyldimethylsilyloxymethyl)-2-butyronitrile (31). To a solution
of 1,4-bis[(tert-butyldimethylsilyl)oxy]-2-butanone (30)19 (700 mg,
2.2 mmol) in a (2:1) mixture of diethyl ether/water (12 mL),
NaHCO3 (550 mg, 6.6 mmol) and NaCN (160 mg, 3.3 mmol) were
added. The mixture was vigorously stirred at room temperature for

4′′-Benzoylureido-TSAO DeriVatiVes as HCMV Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5831
and staining with Giemsa. Antiviral activity was expressed as
the EC50 or compound concentration required reducing virus-
induced cytopathogenicity by 50%.
HCMV Antigen Expression Assay. HEL cells were grown in
24-well microtiter plates and infected with HCMV (Davis strain).
At the time of infection, different dilutions of the compounds
were added. Following different times of incubation at 37 °C,
HCMV-infected and uninfected HEL cells were fixed in acetone
at 4 °C. After resuspension of cells in PBS containing 5% gelatin,
the cells were incubated at 37 °C in the presence of the desired
monoclonal antibody (mAb E13 (Biosoft), directed against IE
(immediately early) proteins 1 and 2, and mAb CCH2 (Dako),
which reacts with the delayed early (E) DNA-binding protein
p52). After a washing step, the cells were incubated for 30 min
with a fluorescein-labeled rabbit antimouse IgG and finally fixed
in a solution of 0.5% formaldehyde in PBS. The cells were then
analyzed with a fluorescence-activated cell sorter, as described
earlier.22,23
Compounds 12 and 15 were also evaluated against a panel of
drug-resistant HCMV mutants isolated in vitro after serial
passage of the reference strain AD-169 under increasing
concentrations of anti-HCMV agents and also against several
HCMV drug-resistant isolates recovered from immunocompro-
mised patients. Different reference drugs (i.e., acyclovir (ACV),
ganciclovir (GCV), foscarnet (PFA), (S)-3-hydroxy-2-phospho-
nomethoxypropyl (HPMP) derivatives of adenine (HPMPA) and
cytosine ((S)-HPMPC, cidofovir)), and 2-phosphonomethoxy-
ethyl (PME) derivatives of adenine (PMEA) and 2,6-diaminopu-
rine (PMEDAP) were included in the antiviral assays.
Murine CMV (MCMV) assays were performed with the Smith
strain on confluent C127I cells following the same methodology
as described for HCMV.
Acknowledgment. The authors would like to thank Susana
Ruiz, Lies Van den Heurck, Steven Carmans, Anita Camps,
Lizette van Berckelaer, and Elke Simons for excellent
technical assistance. The Spanish MEC (project SAF2006-
12713-C02), the European Commission (project HPAW-CT-
2002-90001 (Rene´ Descartes Prize-2001)), and the Gecon-
certeerde Onderzoeksacties (GOA 05/15) of the K.U. Leuven
are acknowledged for financial support.
Activity against Other Viruses. Varicella zoster virus (VZV)
drug susceptibility tests were performed on confluent HEL cells
in 96-well microtiter plates by the plaque reduction assay.
Monolayers were infected with 20 PFU of cell-associated virus
per well. For each assay, virus controls (infected-untreated cells)
were included. After a 2 h incubation period, the virus inoculum
was removed and the media replaced by the different dilutions
(in duplicate) of the tested molecules. Serial dilutions of test
compounds were incubated with the infected monolayers for 5
days. After a 5-day incubation period, the cells were fixed and
stained with Giemsa, and the level of virus-induced cytopathic
effect was determined by counting the number of plaques for
each dilution. Activity was expressed as EC50 (effective com-
pound concentration required to reduce virus plaque formation
by 50%) compared to the untreated control.
Cytotoxicity Assays. Cytotoxicity measurements were based
on the inhibition of HEL cell growth. HEL cells were seeded at
a rate of 5 × 103 cells/well into 96-well microtiter plates and
allowed to proliferate for 24 h. Then, medium containing
different concentrations of the test compounds was added. After
3 days of incubation at 37 °C, the cell number was determined
with a Coulter counter. The 50% cytostatic concentration was
calculated as the CC50, the compound concentration required to
reduce cell growth by 50% relative to the number of cells in
the untreated controls. CC50 values were estimated from graphic
plots of the number of cells (percentage of control) as a function
of the concentration of the test compounds. Cytotoxicity was
expressed as minimum cytotoxic concentration (MCC) or the
compound concentration that causes a microscopically detectable
alteration of cell morphology.
Time-of-Drug Addition Studies. Confluent HEL cell cultures
were infected with approximately 100 PFU of HCMV (Davis
strain) per well. A variety of concentrations of compounds were
added to the infected cell cultures at the time of infection (0 h)
or at several time points post infection (one set of microtiter
plates per time point). Seven days after the initial infection of
the cells, the different microtiter plates were fixed with ethanol
and stained with Giemsa. The antiviral effects of the compounds
were microscopically scored, and the EC50 values were calculated.
Enzyme Assay for HCMV DNA Polymerase. The procedure
to determine the direct inhibitory activity of the compounds on
the HCMV UL54-encoded DNA polymerase has been published
elsewhere.20 An UL54-containing expression plasmid (a kind
gift from Dr. T. Cihlar, Gilead Sciences, Foster City, CA) was
used to prepare the catalytic subunit of the DNA polymerase by
in vitro transcription/translation. The enzyme mixture contained
activated calf thymus DNA as the primer/template, 1 µM of [3H]-
dGTP and 100 µM of dATP, dTTP, and dCTP, or 1 µM of
[3H]dATP and 100 µM of dGTP, dTTP, and dCTP and serial
dilutions of the test compounds.21 After 60 min at 37 °C, nucleic
acids were precipitated with trichloroacetic acid and collected
on filters in which incorporated radioactivity was quantified by
scintillation counting.
Supporting Information Available: Elemental analysis data
of of compounds 2-4, 9-19, 21-23, 29 and 31-33. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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