Syntheses and structure-activity relationships of novel apio and thioapio dideoxydidehydronucleosides as anti-HCMV agents

Article abstract of DOI:10.1021/jm000342f

On the basis of the fact that apio dideoxynucleosides, in which the furanose oxygen and the C2 of the 2,3-dideoxyribose are transposed, exhibited potent anti-HIV activity and 2′,3′-dideoxy-2′,3′-didehydronucleosides also showed potent anti-HIV activity, we synthesized apio dideoxydidehydronucleosides in which the oxygen atom and the double bond of the 2,3-dideoxy-2,3-didehydroribose are exchanged. The thioapio dideoxydidehydronucleosides were also synthesized since sulfur serves as a bioisostere of oxygen. Apio dideoxydidehydronucleosides 13a-f were synthesized starting from 1,3-dihydroxyacetone, utilizing phenylselenenyl chemistry as a key step. The ratio of the anomeric mixture was variable from 1:1 to 5:1 during the condensation of nucleosidic bases with the phenylselenyl acetate 11 in the presence of a Lewis acid. This is in contrast with other glycosyl donors such as 5-O-(tert-butyldiphenylsilyl)-2-phenylselenenyl-2,3-dideoxyribosyl acetate which shows excellent neighboring group effect (α:β = 1:99). Thioapio dideoxydidehydronucleosides 22a,b were synthesized from the lactone 9 via thiolactone 17 as a key intermediate which was synthesized from dicyclohexylcarbodiimide coupling of the mercapto acid produced from the basic hydrolysis of thioacetate 16. The majority of apio analogues synthesized in this study exhibited moderate to potent anti-HCMV activity, among which the 5-fluorouracil derivative 13c was found to be the most potent against HCMV, while thioapio analogues showed no activity against HCMV. However, all synthesized compounds did not exhibit any significant activities against HIV-1, HSV-1, and HSV-2. The fact that apio dideoxydidehydronucleosides were active against HCMV suggests that the apio dideoxydidehydro sugar moiety can serve as a novel template for the development of new antiviral agents.

Full text of DOI:10.1021/jm000342f

806
J . Med. Chem. 2001, 44, 806-813
Syn th eses a n d Str u ctu r e-Activity Rela tion sh ip s of Novel Ap io a n d Th ioa p io
Did eoxyd id eh yd r on u cleosid es a s An ti-HCMV Agen ts
Lak Shin J eong,*,† Hea Ok Kim,‡ Hyung Ryong Moon,† J oon Hee Hong,† Su J eong Yoo,† Won J un Choi,†
Moon Woo Chun,§ and Chong-Kyo Lee|
Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea, College of
Medicine, Yonsei University, Seoul 120-752, Korea, College of Pharmacy, Seoul National University, Seoul 151-742, Korea, and
Korea Research Institute of Chemical Technology, Yusong, Taejon 305-600, Korea
Received August 8, 2000
On the basis of the fact that apio dideoxynucleosides, in which the furanose oxygen and the
C2 of the 2,3-dideoxyribose are transposed, exhibited potent anti-HIV activity and 2′,3′-dideoxy-
2′,3′-didehydronucleosides also showed potent anti-HIV activity, we synthesized apio dideoxy-
didehydronucleosides in which the oxygen atom and the double bond of the 2,3-dideoxy-2,3-
didehydroribose are exchanged. The thioapio dideoxydidehydronucleosides were also synthesized
since sulfur serves as a bioisostere of oxygen. Apio dideoxydidehydronucleosides 13a -f were
synthesized starting from 1,3-dihydroxyacetone, utilizing phenylselenenyl chemistry as a key
step. The ratio of the anomeric mixture was variable from 1:1 to 5:1 during the condensation
of nucleosidic bases with the phenylselenyl acetate 11 in the presence of a Lewis acid. This is
in contrast with other glycosyl donors such as 5-O-(tert-butyldiphenylsilyl)-2-phenylselenenyl-
2,3-dideoxyribosyl acetate which shows excellent neighboring group effect (R:â ) 1:99). Thioapio
dideoxydidehydronucleosides 22a ,b were synthesized from the lactone 9 via thiolactone 17 as
a key intermediate which was synthesized from dicyclohexylcarbodiimide coupling of the
mercapto acid produced from the basic hydrolysis of thioacetate 16. The majority of apio
analogues synthesized in this study exhibited moderate to potent anti-HCMV activity, among
which the 5-fluorouracil derivative 13c was found to be the most potent against HCMV, while
thioapio analogues showed no activity against HCMV. However, all synthesized compounds
did not exhibit any significant activities against HIV-1, HSV-1, and HSV-2. The fact that apio
dideoxydidehydronucleosides were active against HCMV suggests that the apio dideoxydide-
hydro sugar moiety can serve as a novel template for the development of new antiviral agents.
In tr od u ction
A number of 2′,3′-dideoxynucleosides (ddNs) and 2′,3′-
dideoxy-2′,3′-didehydronucleosides (d4Ns) have been
synthesized and evaluated for antiviral activities against
human immunodeficiency virus (HIV)-11 and hepatitis
B virus (HBV).2 These classes of compounds exhibit
their antiviral activities by inhibiting the reverse tran-
scription process of the viral replicative cycle and/or by
being incorporated into the viral DNA chain, resulting
in viral DNA chain termination.3-5 2′,3′-Dideoxycytidine
(1, ddC, Zalcitabine)1 (Figure 1) is a potent anti-HIV
agent being clinically used for the treatment of AIDS
patients. However, this class of compounds show dose-
dependent toxicity like peripheral neuropathy by inhib-
iting DNA polymerase γ.6 2′,3′-Dideoxy-2′,3′-didehy-
F igu r e 1. Rationale to the design of apio and thioapio d4Ns.
drothymidine (2, d4T, Starvudine) is the only nucleoside
approved by the Food and Drug Administration (FDA)
among d4Ns for the treatment of AIDS and AIDS-
related complexes (ARC), but it also suffers from
adverse effects such as peripheral neuropathy and the
appearance of resistant strains.7,8
Iso dideoxynucleosides (iso ddNs, 3)9 and apio dideoxy-
nucleosides (apio ddNs, 4)10 belong to a novel class of
nucleosides in that the oxygen atom of the furanose ring
moves to the C3 or C2 position, respectively. These
compounds not only showed antiviral activities compa-
rable to those of parent ddNs but also have metabolic
advantages such as resistance to adenosine deaminase
and glycosyl bond hydrolysis.
A preliminary account has been published: J eong, L. S.; Lee, Y.
A.; Moon, H. R.; Yoo, S. J .; Kim, S. Y. Total synthesis of (()-iso-d4T as
potential antiviral agents. Tetrahedron Lett. 1998, 39, 7517-7520.
* To whom correspondence should be addressed. Tel: 82-2-3277-
3466. Fax: 82-2-3277-2851. E-mail: lakjeong@mm.ewha.ac.kr.
† Ewha Womans University.
‡ Yonsei University.
Based on the potent antiviral activity of apio ddNs
and d4Ns, it was interesting to transpose the double
§ Seoul National University.
| Korea Research Institute of Chemical Technology.
10.1021/jm000342f CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/26/2001

Apio/ Thioapio Nucleosides as Anti-HCMV Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 807
Sch em e 1
which two protons (δ 3.64) of CH2OTBDMS moved more
upfield than those of 5-CH2 (δ 4.20 and 4.39). In
compound (()-9, two protons (δ 3.70) of CH2OTBDPS
also shifted more upfield than those of 5-CH2 (δ 4.26
and 4.42). Treatment of (()-9 with LiHMDS at -78 °C
followed by trapping of the enloate with trimethylsilyl
chloride at room temperature gave the silyl enol ether,
which without isolation was treated with phenylsele-
nenyl bromide at -78 °C to give the desired (()-10a as
a major product (80%) and the undesired (()-10b as a
minor product (10%) after silica gel column chromatog-
raphy.13 The major formation of (()-10a was achieved
because the phenylselenenyl group was added from the
opposite side to the bulky TBDPS group as reported by
Campbell12 and Chu.13 In addition to the explanation
of the stereochemistry of the phenylselenenyl group by
steric effect, a 1H NOE experiment was employed to
confirm the configuration of the phenylselenenyl group
in (()-10a and (()-10b. It was found that a NOE effect
(2.5%) between 3-H and 4-H of the 3,4-trans isomer (()-
10a was smaller than that (6.6%) of the 3,4-cis isomer
(()-10b. To explain the fact that the 3,4-trans compound
Sch em e 2a
3
(()-10a has a smaller J between 3-H and 4-H (5.3 Hz)
than the 3,4-cis compound (()-10b (8.0 Hz), the pre-
ferred conformations of (()-10a and (()-10b were
obtained from MM2 energy minimization. The energy-
minimized structure of 3,4-trans (()-10a appears to
adopt predominantly the N (3-exo/4-endo) conformation
in which the dihedral angle between H3-C3-C4-H4 is
close to 110°, giving a small coupling constant (5.3 Hz)
from the vicinal Karplus correlation. However, 3,4-cis
(()-10b seems to take the S (3-endo/4-exo) conformation
in which the dihedral angle between H3-C3-C4-H4 is
about 25°, giving a large coupling constant (8.0 Hz). This
conformational difference between (()-10a and (()-10b
appears to be due to the gauche effect between the furan
oxygen and the electron-withdrawing selenium.14 The
desired (()-10a was reacted with DIBAL at -78 °C to
give the lactol, which without purification was treated
with acetic anhydride in pyridine to afford the key
intermediate (()-11 which is ready for condensation
with nucleosidic bases.
a
Reagents: (a) H2, Pd/C, 99%; (b) LiHMDS, THF, -78 °C, then
TMSCl, rt; (c) PhSeBr, -78 °C; (d) DIBAL, -78 °C, then A2O,
pyridine, rt, 85%.
bond and furanose ring oxygen of d4Ns for the design
of apio dideoxydidehydronucleosides (5, apio d4Ns). We
also designed thioapio analogues (6) of apio d4Ns since
the sulfur atom serves as a bioisostere of the oxygen.
Here, we report the first syntheses and structure-
activity relationship (SAR) study of novel apio d4Ns 5
and thioapio d4Ns 6 as antiviral agents.
Synthesis of the target apio d4Ns (()-13a -(()-13f
from (()-11 is illustrated in Scheme 3. The glycosyl
donor (()-11 was condensed with silylated uracil, thy-
mine, and 5-halo-substituted uracils in the presence of
TMSOTf as a Lewis acid in anhydrous 1,2-dichloro-
ethane to give the protected nucleosides (()-12a -(()-
12f as inseparable anomeric mixtures, respectively
whose ratios were variable from 1:1 to 5:1, indicating
that neighboring group effect by the phenylselenenyl
group was zero or not as great in this ring system. This
result contrasts with another glycosyl donor, 5-O-(tert-
butyldiphenylsilyl)-2-phenylselenenyl-2,3-dideoxyribo-
syl acetate, which showed excellent neighboring group
effect (R:â ) 1:99) through the participation of the C2-
phenylselenenyl group to the oxonium ion generated at
the anomeric position during the condensation with
silylated thymine.13 It is presumed that the lack of a
neighboring group effect by the phenylselenenyl group
in this ring system might be due to the repulsive effect
between incoming nucleosidic bases and the pseudoaxial
bulky hydroxymethyl substituent as illustrated in Fig-
ure 2, which drove the reaction equlibrium to the
Resu lts a n d Discu ssion
Ch em istr y. Our original synthetic plan was to syn-
thesize a cyclic allylic acetate 8 and then condense with
nucleosidic bases as seen in Scheme 1. However, when
R,â-unsaturated lactone 7,11 which was easily synthe-
sized from 1,3-dihydroxyacetone, was reduced with
DIBAL at -78 °C followed by acetylation with acetic
anhydride in pyridine, it did not give the desired cyclic
allylic acetate 8 but afforded the linear allylic acetate
7a as a major product. With the assumption that no
formation of 8 occurred due to the rigidity of the double
bond, we decided to insert a double bond at the nucleo-
side stage using phenylselenenyl chemistry as shown
in Scheme 2.
R,â-Unsaturated lactone 7 was reduced to the satu-
rated lactone (()-9 by catalytic hydrogenation in quan-
titative yield. The proton signals of (()-9 were assigned
based on the known TBDMS-protected lactone,12 in

808 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
J eong et al.
Attempts to synthesize cytosine and 6-substituted
purine derivatives were made, but we could not obtain
the final products. Although the condensation of the
acetate (()-11 with silylated N4-benzoylcytosine under
TMSOTf followed by syn elimination by hydrogen
peroxide produced the protected cytosine nucleoside in
very good yield, deprotection of the TBDPS group
resulted in the extensive decomposition on TLC. Con-
densation of (()-11 with silylated 6-chloropurine or
adenine also did not afford the desired product as in
the case of cytosine.
Synthesis of bioisosteric nucleosides, (()-22a and (()-
22b, of apio d4Ns in which the oxygen atom was
replaced by a sulfur atom is shown in Scheme 4. The
lactone (()-9 was treated with 1 N NaOH followed by
addition of dimethyl sulfate to yield the hydroxy ester
(()-14 (78%). The primary hydroxyl group was con-
verted to the mesylate (()-15 by reacting with mesyl
chloride in pyridine. To synthesize the desired thiolac-
tone (()-17, we first treated (()-15 with sodium disul-
fide in DMF at 110 °C, but only small amounts of the
desired product (()-15 were produced under this condi-
tions. Therefore, we obtained thiolactone (()-17 by a
three-step procedure in good yield from (()-15: treat-
ment of (()-15 with potassium thioacetate in DMF to
give the thioacetyl ester (()-16, which was reacted with
aqueous NaOH in ethanol, followed by cyclization of the
F igu r e 2. Neighboring group effect by the phenylselenenyl
group is affected by the bulky TBDPS substituent as shown
in the transition state.
Sch em e 3
1
resulting mercapto acid with DCC. When the H NMR
of thiolactone (()-17 was compared to that of lactone
(()-9, their coupling patterns and chemical shifts were
almost identical except for the different chemical shifts
of the 5-H’s. Two protons (δ 3.35 and 3.47) of the 5-H’s
in (()-17 were more shielded than those (δ 4.26 and
4.42) of the 5-H’s in (()-9 because sulfur is less elec-
tronegative than oxygen; thus, in compound (()-17, two
protons of CH2OTBDPS moved more downfield (δ 3.69)
than those of 5-CH2 (δ 3.35 and 3.47) in contrast to
compound (()-9. Introduction of the phenylselenenyl
group at the R position of the thiolactone (()-17 was
achieved using the same procedure shown in Scheme 2
to give the inseparable diastereomeric mixture of (()-
18 (cis/ trans ) 1/10 as determined by 1H NMR).
Reduction of (()-18 with DIBAL in THF gave the lactol
that was treated with acetic anhydride to afford the key
intermediate (()-19. Condensation of (()-19 with sily-
lated uracil and thymine in the presence of TMSOTf
produced (()-20a and (()-20b, respectively. Smooth
conversion of the phenylselenenyl group in (()-20a and
(()-20b to the double bond under the presence of the
sulfur atom was achieved using mCPBA and pyridine
at -30 °C to give (()-21a and (()-21b, respectively.15
In this reaction condition, only the 3,4-trans isomer
underwent syn elimination, while 3,4-cis isomer re-
mained intact as selene oxide due to the absence of a
syn hydrogen. TBDPS group was deprotected under the
standard conditions to give the final thioapio analogues
(()-22a and (()-22b, respectively. As in the case of apio
d4Ns, attempts to synthesize the thioapio cytosine
oxonium ion intermediate (I), not the episelenonium ion
intermediate (II).
The protected nucleosides (()-12a -(()-12f were
treated with hydrogen peroxide with a catalytic amount
of pyridine to give the syn-eliminated products, which
were deprotected with tetra-n-butylammonium fluoride
in THF to afford the final apio d4Ns, (()-13a -(()-13f,
1
1
analogue were not successful. When H NMR of thioapio
respectively. From the H NMR analysis of the synthe-
nucleosides was compared with that of apio nucleosides,
their coupling patterns and chemical shifts were very
similar except for the chemical shifts of the 5-H’s. It is
interesting to note that only one proton of 5-H’s in all
sized nucleosides, two methylene protons of CH2-
OTBDPS or CH2OH in (()-12a -(()-12f or (()-13a -(()-
13f were found to always shift more upfield than the
5-H’s, as in the case of compound (()-9.

Apio/ Thioapio Nucleosides as Anti-HCMV Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 809
Sch em e 4a
a
Reagents: (a) 1 N NaOH, 1 h then (CH3)2SO4, DMSO, 1 h, 89%; (b) MsCl, pyridine, rt; (c) KSAc, DMF, rt, 7 h, 86% from (()-14; (d)
NaOH, H2O, EtOH, 50 °C, 2.5 h, then DCC, DMAP, CH2Cl2, 30 min, 70%; (e) i. LiHMDS, THF, -78 °C, 1 h, then TMSCl, 30 min, ii.
PhSeBr, -78 °C, 30 mim, 99%; (f) i. DIBAL, THF, -78 °C, ii. Ac2O, pyridine, 84%; (g) TMSOTf, ClCH2CH2Cl, rt, 74% for (()-20a , 72%
for (()-20b; (h) mCPBA, CH2Cl2, -30 °C, 71% for (()-21a , 75% for (()-21b; (i) n-Bu4NF, THF, 0 °C, 1 h, 76% for (()-22a , 71% for (()-
22b.
Ta ble 1. Anti-HCMV Activity16,17 of Synthesized Apio d4Ns
and Thioapio d4Ns
showed moderate to potent anti-HCMV activity without
cytotoxicity, among which the 5-fluorouracil derivative
(()-13c was found to be the most potent and other
analogues showed moderate anti-HCMV activity. All
apio d4Ns were more active than forscarnet (PFA) but
less potent than ganciclovir (GCV) which was used for
the control in both AD-169- and Davis-infected cells.
Thioapio analogues (()-22a and (()-22b did not exhibit
any significant antiviral activities. This result indicates
that although the oxygen is similar to the sulfur in
valence electrons, differences in size and electronega-
tivity may play a key role in phosphorylation by kinases
or in binding to the viral polymerases.
In summary, we accomplished the first syntheses of
apio and thioapio d4Ns starting from 1,3-dihydroxy-
acetone using phenylselenenyl chemistry for the inser-
tion of a double bond into the sugar moiety. When the
synthesized final compounds were tested against several
viruses such as HIV-1, HSV-1, HSV-2, and HCMV, they
were only active against HCMV, indicating this virus
allows the rigid sugar moiety unlike other viruses.
These results suggest that the apio dideoxydidehydro
sugar moiety can serve as a novel template for the
development of new antiviral agents.
EC50 (µg/mL)
AD-169 Davis
compd
CC50 (µg/mL)
13a (X ) O, Y ) H)
13b (X ) O, Y ) CH3)
13c (X ) O, Y ) F)
13d (X ) O, Y ) Cl)
13e (X ) O, Y ) Br)
13f (X ) O, Y ) I)
22a (X ) S, Y ) H)
22b (X ) S, Y ) CH3)
foscarnet (PFA)
11.7
33.3
6.9
>33.3
33.3
>100
>100
63.3
>100
>100
>100
>100
>300
>10
16.6
2.81
41.7
33.7
44.1
25.1
25.4
27.2
>100
>100
64.9
1.09
>100
>100
38.7
0.61
ganciclovir (GCV)
of the apio and thioapio nucleosides showed allylic
coupling (J = 2 Hz) probably because of the rigid sugar
moiety.
An tivir a l Activity. All synthesized compounds were
tested against several viruses such as HIV-1 (MT-4
cells), HSV-1 (CCL81 cells), HSV-2 (CCL81 cells), and
HCMV (AD-169 and Davis cells). All compounds exhib-
ited neither antiviral activity nor cytotoxicity when
tested up to 100 µg/mL against HIV-1, HSV-1, and HSV-
2. However, it is interesting to note that apio d4Ns are
only active against HCMV (Table 1), indicating this
virus may allow the conformationally rigid sugar moiety
for phosphorylation as well as for DNA polymerase
unlike other viruses although the hydroxymethyl group
orientation of the apio sugar is greatly different from
that of the normal dideoxyribo sugar. For the evaluation
of anti-HCMV activity, HCMV strains AD-169 (ATCC
VR-538) and Davis (ATCC VR-807) were used and
standard CPE inhibition assay was used.14 HEL 299
(human embryonic lung fibroblast) cells were used for
the cytotoxic assay. As shown in Table 1, most apio d4Ns
Exp er im en ta l Section
Ultraviolet (UV) spectra were recorded on a Beckman DU-
68 spectrophotometer; 1H and 13C NMR spectra were recorded
on a Varian-400 spectrometer, using CDCl3 or DMSO-d6, and
chemical shifts are reported in parts per million (ppm) down-
field from tetramethylsilane as internal standard. FAB mass
spectra were recorded on J EOL HX 110 spectrometer. Elemen-
tal analyses were performed by the General Instrument
Laboratory, Ewha Womans University, Korea. TLC was
performed on Merck precoated 60F254 plates. Column chro-
matography was performed using silica gel 60 (230-400 mesh,
Merck). All the anhydrous solvents were distilled over CaH2
or P2O5 or Na/benzophenone prior to use.
(()-4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-4H,5H-d ih y-
d r ofu r a n -2-on e (9). To a solution of 7 (2.0 g, 5.7 mmol) in
ethyl acetate (10 mL) was added 10% Pd/C (10 mg) and the
mixture was stirred at ambient temperature for 48 h under
H2 atmosphere. The mixture was filtered through a Celite pad

810 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
J eong et al.
and washed with ethyl acetate and methanol successively.
Solvents were evaporated in vacuo and the residue was dried
under high vacuum to give 9 (2.0 g, 99%): Rf ) 0.54 (hexanes:
ethyl acetate ) 4:1); 1H NMR (CDCl3) δ 1.02 (s, 9 H, tert-butyl),
2.35 (dd, 1 H, J ) 6.3 and 17.5 Hz, 3-Ha), 2.61 (dd, 1 H, J )
8.7 and 17.5 Hz, 3-Hb), 2.72-2.81 (m, 1 H, 4-H), 3.70 (d, 2 H,
J ) 5.8 Hz, SiO-CH2), 4.26 (dd, 1 H, J ) 5.4 and 9.1 Hz, 5-Ha),
4.42 (dd, 1 H, J ) 6.6 and 9.1 Hz, 5-Hb), 7.41-7.68 (m, 10 H,
2xPh). Anal. (C21H26O3Si) C, H.
TMSOTf (0.75 mmol) was added and the mixture was allowed
to stir at ambient temperature for 1 h. The reaction mixture
was poured into CH2Cl2, neutralized with saturated NaHCO3
solution and stirred for 10 min. The organic layers were
separated, washed once with saturated NaHCO3 solution and
brine, dried (MgSO4), filtered and evaporated. The residue was
purified by silica gel column chromatography (hexanes:ethyl
acetate ) 2:1) to give the protected nucleosides 12a -f,
respectively.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
selen ylt et r a h yd r ofu r a n -2-yl]-1H -p yr im id in e-2,4-d ion e
(12a ): yield 90% (3:1 anomeric mixture); Rf ) 0.58 (hexanes:
ethyl acetate ) 1:1); UV (MeOH) λmax 260 nm; 1H NMR (CDCl3)
δ 1.06 (s, 2.25 H, tert-butyl), 1.09 (s, 0.75 H, tert-butyl), 2.40-
2.48 (m, 0.75 H, 4-H), 2.50-2.60 (m, 0.25 H, 4-H), 3.56-3.95
(m, 2 H, SiO-CH2), 3.97-4.08 (m, 2 H, 5-H), 4.21-4.39 (m, 1
H, 3-H), 5.54 (d, 0.75 H, J ) 8.0 Hz, H-5), 5.69 (d, 0.25 H, J )
8.0 Hz, H-5), 6.10 (d, 0.75 H, J ) 8.0 Hz, 2-H), 6.21 (d, 0.25 H,
J ) 4.9 Hz, 2-H), 7.07-7.66 (m, 16 H, 3xPh and H-6), 8.21 (br
s, 0.25 H, NH), 8.52 (br s, 0.75 H, NH). Anal. (C31H34N2O4-
SeSi) C, H, N.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
selen yltetr ah ydr ofu r an -2-yl]-5-m eth yl-1H-pyr im idin e-2,4-
d ion e (12b): yield 63% (5:1 anomeric mixture); Rf ) 0.45
(hexanes:ethyl acetate ) 2:1); UV (MeOH) λmax 265 nm; 1H
NMR (CDCl3) δ 1.09 (s, 1.5 H, tert-butyl), 1.14 (s, 7.5 H, tert-
butyl), 1.98 (s, 0.5 H, CH3), 2.08 (s, 2.5 H, CH3), 2.39-2.80
(m, 1 H, 4-H), 3.55-3.85 (m, 2 H, SiO-CH2), 3.98-4.26 (m, 3
H, 3-H and 5-H), 6.13 (d, 0.83 H, J ) 8.5 Hz, 2-H), 6.24 (d,
0.17 H, J ) 5.4 Hz, 2-H), 7.25-7.70 (m, 16 H, 3xPh and H-6),
7.75 (br s, 1 H, NH). Anal. (C32H36N2O4SeSi) C, H, N.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
selen yltetr a h ydr ofu r a n -2-yl]-5-flu or o-1H-p yr im id in e-2,4-
d ion e (12c): yield 62% (1:1 anomeric mixture); Rf ) 0.56
(hexanes:ethyl acetate ) 1:1); UV (MeOH) λmax 268 nm; 1H
NMR (CDCl3) δ 1.06 (s, 4.5 H, tert-butyl), 1.08 (s, 4.5 H, tert-
butyl), 2.35-2.60 (m, 1 H, 4-H), 3.55-3.80 (m, 2 H, SiO-CH2),
3.88-4.12 (m, 2 H, 5-H), 4.23-4.39 (m, 1 H, 3-H), 6.07 (dd,
0.5 H, J ) 1.7 and 6.4 Hz, 2-H), 6.17 (dd, 0.5 H, J ) 1.6 and
3.9 Hz, 2-H), 7.26-7.66 (m, 17 H, 3xPh, NH and H-6). Anal.
(C32H36FN2O4SeSi) C, H, N.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
selen yltetr ah ydr ofu r an -2-yl]-5-ch lor o-1H-pyr im idin e-2,4-
d ion e (12d ): yield 69% (1:1 anomeric mixture); Rf ) 0.58
(hexanes:ethyl acetate ) 1:1); UV (MeOH) λmax 272 nm; 1H
NMR (CDCl3) δ 1.07 (s, 4.5 H, tert-butyl), 1.09 (s, 4.5 H, tert-
butyl), 2.35-2.60 (m, 1 H, 4-H), 3.61-3.80 (m, 2 H, SiO-CH2),
3.86-4.04 (m, 2 H, 5-H), 4.12-4.35 (m, 1 H, 3-H), 6.07 (d, 0.5
H, J ) 8.2 Hz, 2-H), 6.19 (d, 0.5 H, J ) 5.3 Hz, 2-H), 7.18-
7.68 (m, 16 H, 3xPh and H-6), 7.95 (br s, 1 H, NH). Anal.
(C31H33ClN2O4SeSi) C, H, N.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
selen yltetr ah ydr ofu r an -2-yl]-5-br om o-1H-pyr im idin e-2,4-
d ion e (12e): yield 91% (1:1 anomeric mixture); Rf ) 0.60
(hexanes:ethyl acetate ) 1:1); UV (MeOH) λmax 278 nm; 1H
NMR (CDCl3) δ 1.07 (s, 4.5 H, tert-butyl), 1.10 (s, 4.5 H, tert-
butyl), 2.35-2.58 (m, 1 H, 4-H), 3.62-3.79 (m, 2 H, SiO-CH2),
3.90-4.13 (m, 2 H, 5-H), 4.29-4.35 (m, 1 H, 3-H), 6.07 (d, 0.5
H, J ) 8.3 Hz, 2-H), 6.17 (d, 0.5 H, J ) 5.3 Hz, 2-H), 7.18-
7.66 (m, 16 H, 3xPh and H-6), 8.17 (br s, 1 H, NH). Anal.
(C31H33BrN2O4SeSi) C, H, N.
(()-3,4-tr a n s-4-(ter t-Bu t yld ip h en ylsilyloxym et h yl)-3-
p h en ylselen yl-4H,5H-d ih yd r ofu r a n -2-on e (10a ) a n d Its
Cis Isom er (10b). To a solution of 9 (1.72 g, 4.9 mmol) in
anhydrous THF (5 mL) was added LiHMDS (1 M solution in
THF, 5.93 mL, 5.93 mmol) at -78 °C and the mixture was
stirred at the same temperature for 1 h. Chlorotrimethylsilane
(1.43 mL, 5.9 mmol) was added to the reaction mixture and
the mixture was allowed to stir at ambient temperature for
30 min. The mixture was again cooled to -78 °C and a solution
of phenylselenenyl bromide (1.9 g, 7.35 mmol) in THF (2 mL)
was added dropwise to the mixture. When dark brown color
disappeared, reaction was stopped. The mixture was poured
into ether (100 mL) and the ether layer was washed with H2O
(20 mL), dried over anhydrous MgSO4, filtered and evaporated.
The residue was purified by silica gel column chromatography
(hexanes:ethyl acetate) 5:1) to give 10a (2.13 g, 77%) as a
colorless syrup and 10b (0.21 g, 7.7%) as a colorless syrup.
10a : Rf ) 0.67 (hexanes:ethyl acetate ) 5:1); 1H NMR
(CDCl3) δ 1.03 (s, 9 H, tert-butyl), 2.54-2.61 (m, 1 H, 4-H),
3.61 (dd, 1 H, J ) 4.8 and 10.5 Hz, SiO-CHa), 3.70 (dd, 1 H,
J ) 5.3 and 10.5 Hz, SiO-CHb), 3.86 (d, 1 H, J ) 5.3 Hz, 3-H),
4.10 (d, 2 H, J ) 6.2 Hz, 5-H), 7.25-7.64 (m, 15 H, 3xPh).
Anal. (C27H30O3SeSi) C, H.
10b: Rf ) 0.45 (hexanes:ethyl acetate ) 5:1); 1H NMR
(CDCl3) δ 1.03 (s, 9 H, tert-butyl), 2.81-2.90 (m, 1 H, 4-H),
3.86 (dd, 1 H, J ) 4.8 and 10.4 Hz, SiO-CHa), 3.89 (dd, 1 H,
J ) 4.0 and 10.4 Hz, SiO-CHb), 4.01 (d, 1 H, J ) 8.0 Hz, 3-H),
4.06 (dd, 1 H, J ) 6.4 and 9.2 Hz, 5-Ha), 4.32 (dd, 1 H, J ) 7.2
and 9.2 Hz, 5-Hb), 7.21-7.79 (m, 15 H, 3xPh). Anal. (C27H30O3-
SeSi) C, H.
(()-2-O-Acetyl-3,4-tr a n s-4-(ter t-bu tyld ip h en ylsilyloxy-
m eth yl)-3-p h en ylselen yltetr a h yd r ofu r a n (11). To a solu-
tion of 10a (2.05 g, 3.68 mmol) in anhydrous toluene (15 mL)
at -78 °C was added DIBAL (1 M solution in toluene, 5.52
mL, 5.52 mmol) and the mixture was stirred at -78 °C for 2
h. The reaction mixture was quenched with MeOH (2 mL) and
CHCl3 (5 mL) and stirred for 10 min at -78 °C. To the reaction
mixture were added 10% Na,K tartarate solution (20 mL) and
CHCl3 (100 mL). The organic layer was washed with brine (20
mL), dried (MgSO4), filtered and evaporated. The crude lactol,
without further purification, was dried under high vacuum for
1 h and acetylated by treating with acetic anhydride (0.69 mL,
7.36 mmol) and pyridine (10 mL) at room temperature over-
night. After removal of the solvent under reduced pressure,
the residue was dissolved in CH2Cl2 (50 mL), washed with
brine (20 mL), dried over anhydrous MgSO4, filtered and
evaporated. The residue was purified by flash silica gel column
chromatography (hexanes:ethyl acetate ) 8:1) to give an
anomeric mixture (1:1) of 11 (1.7 g, 85%) as a pale yellow
syrup: Rf ) 0.68 (hexanes:ethyl acetate ) 3:1); 1H NMR
(CDCl3) δ 1.00 (s, 4.5 H, tert-butyl), 1.05 (s, 4.5 H, tert-butyl),
1.92 (s, 1.5 H, COCH3), 2.09 (s, 1.5 H, COCH3), 2.35-2.64 (m,
1 H, 4-H), 3.55-3.61 (m, 2 H, SiO-CH2), 3.72-3.77 (m, 1 H,
3-H), 3.86-4.14 (m, 2 H, 5-H), 6.28 (d, 0.5 H, J ) 1.9 Hz, 2-H),
6.43 (d, 0.5 H, J ) 4.4 Hz, 2-H), 7.21-7.63 (m, 15 H, 3xPh).
Anal. (C29H34O4 SeSi) C, H.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
selen yltetr a h yd r ofu r a n -2-yl]-5-iod o-1H-p yr im id in e-2,4-
d ion e (12f): yield 68% (3:1 anomeric mixture); Rf ) 0.63
(hexanes:ethyl acetate ) 1:1); UV (MeOH) λmax 283 nm; 1H
NMR (CDCl3) δ 1.07 (s, 2.25 H, tert-butyl), 1.12 (s, 6.75 H,
tert-butyl), 2.39-2.46 (m, 1 H, 4-H), 3.61-3.79 (m, 2 H, SiO-
CH2), 3.89-4.17 (m, 2 H, 5-H), 4.31-4.38 (m, 1 H, 3-H), 6.05
(d, 0.75 H, J ) 8.3 Hz, 2-H), 6.16 (d, 0.25 H, J ) 5.3 Hz, 2-H),
7.18-7.66 (m, 16 H, 3xPh and H-6), 7.85 (br s, 1 H, NH). Anal.
(C31H33IN2O4SeSi) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 12a -f: Con -
d en sa tion . A suspension of nucleosidic base (0.75 mmol) and
ammonium sulfate (catalytic amount) in hexadimethyldisila-
zane (HMDS; 6 mL) was heated at 140-150 °C until a clear
solution was obtained. The reaction mixture was cooled to room
temperature and HMDS was removed under reduced pressure
with exclusion of moisture. To this residue was added a
solution of 11 (0.5 mmol) in anhydrous 1,2-dichloroethane
under nitrogen and the reaction mixture was cooled to 5 °C.
Gen er a l P r oced u r e for th e Syn th esis of 13a -f: Syn
Elim in a tion a n d Dep r otection . To a solution of protected
nucleosides 12a -f (0.15 mmol) in CH2Cl2 (3 mL) were added

Apio/ Thioapio Nucleosides as Anti-HCMV Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 811
30% aqueous hydrogen peroxide (1.9 mmol) and 1 drop of
pyridine and the mixture was stirred at ambient temperature
for 1 h, respectively. The mixture was poured into saturated
NaHCO3 solution (10 mL) and CH2Cl2 (30 mL) and the organic
layer was separated, washed with brine (20 mL), dried
(MgSO4), filtered and evaporated. The residue was purified
by silica gel column chromatography (hexanes:ethyl acetate
) 2:1) to give the syn-eliminated nucleosides, respectively. A
solution of the syn-eliminated nucleosides (0.17 mmol) in THF
(5 mL) was treated with tetra-n-butylammonium fluoride (1
M solution in THF, 0.23 mL, 0.23 mmol) at 0 °C and the
mixture was stirred at the same temperature for 30 min. The
mixture was evaporated and the residue was purified by silica
gel column chromatography (chloroform:methanol ) 10:1) to
give the final nucleosides 13a -f, respectively.
(()-1-(4-H yd r oxym e t h yl-2,5-d ih yd r ofu r a n -2-yl)-1H -
p yr im id in e-2,4-d ion e (13a ): yield 84%; Rf ) 0.38 (chloroform:
methanol ) 10:1); mp 326 °C (methanol/ether); UV (H2O) λmax
259 nm (ꢀ 7850); 1H NMR (DMSO-d6) δ 4.17 (br s, 2 H,
HOCH2), 4.52 (d, 1 H, J ) 13.2 Hz, 5-Ha), 4.75 (ddd, 1 H, J )
2.0, 5.2 and 13.2 Hz, 5-Hb), 5.12 (br s, 1 H, OH, D2O
exchangeable), 5.61 (d, 1 H, J ) 8.0 Hz, H-5), 5.64 (br d, 1 H,
J ) 1.6 Hz, 3-H), 6.80 (m, 1 H, 2-H), 7.35 (d, 1 H, J ) 8.0 Hz,
H-6), 11.1 (br s, 1 H, NH, D2O exchangeable); 13C NMR
(DMSO-d6) δ 56.6, 74.8, 90.1, 101.9, 116.9, 140.6, 149.8, 150.5,
163.1. Anal. (C9H10N2O4) C, H, N.
D2O exchangeable), 5.63 (d, 1 H, J ) 1.5 Hz, 3-H), 6.76 (m, 1
H, 2-H), 7.77 (s, 1 H, H-6), 11.7 (br s, 1 H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 56.6, 56.7, 70.1, 74.9,
90.7, 116.6, 144.6, 150.1, 160.4. Anal. (C9H9IN2O4) C, H, N.
(()-3-(ter t-Bu t yld ip h en ylsilyloxym et h yl)-4-h yd r oxy-
m eth ylbu tyr ic Acid Meth yl Ester (14). To a solution of 9
(11.9 g, 33.4 mmol) in H2O (36 mL) and ethanol (140 mL) was
added NaOH (1.4 g, 35 mmol) and the reaction mixture was
stirred at ambient temperature for 1 h and concentrated in
vacuo. Without further purification, the resulting residue was
dissolved in DMSO (40 mL) and toluene (10 mL). To this
mixture was added dimethyl sulfate (3.8 mL, 40.2 mmol)
slowly and stirred at ambient temperature for another 1 h.
The mixture was evaporated and the residue was poured into
ethyl acetate (200 mL) and water (50 mL). The organic layer
was separated, washed with brine (50 mL) and dried (MgSO4),
filtered, and evaporated. The residue was purified by silica
gel column chromatography (hexanes:ethyl acetate ) 4:1) to
give 14 (11.5 g, 89%) as a colorless oil: 1H NMR (CDCl3) δ
1.05 (s, 9 H, tert-butyl), 2.42 (dd, 1 H, J ) 6.8 and 17.6 Hz,
2-Ha), 2.44 (br s, 1 H, OH), 2.56 (dd, 1 H, J ) 8.8 and 17.6 Hz,
2-Hb), 2.73 (m, 1 H, 3-H), 3.65 (s, 3 H, OCH3), 3.67-3.78 (m,
2 H, SiO-CH2), 4.23 (dd, 1 H, J ) 5.6 and 9.6 Hz, HO-CHa),
4.39 (dd, 1 H, J ) 7.2 and 9.6 Hz, HO-CHb), 7.38-7.66 (m,
10 H, 2xPh). Anal. (C22H30O4Si) C, H.
(()-4-Ace t ylsu lfa n yl-3-(t er t -b u t yld ip h e n ylsilyloxy-
m eth yl)bu tyr ic Acid Meth yl Ester (16). To a solution of
14 (7.7 g, 19.9 mmol) in anhydrous pyridine (30 mL) was added
mesyl chloride (2.32 mL, 29.9 mmol) at 0 °C and the reaction
mixture was stirred at ambient temperature for 3 h. The
solvent was removed under reduced pressure and the residue
was extracted with ethyl acetate. The residue was purified by
silica gel column chromatography (hexanes:ethyl acetate ) 7:1)
to give the mesylate 15 as a colorless oil, which without further
purification, was treated with potassium thioacetate (2.3 g,
20.5 mmol) in anhydrous DMF (50 mL). The resulting mixture
was stirred at ambient temperature for 7 h and the reaction
mixture was partitioned between ether (200 mL x 3) and water
(100 mL). The organic layer was washed with brine (50 mL)
and dried (MgSO4), filtered, and evaporated. The residue was
purified by silica gel column chromatography (hexanes:ethyl
acetate ) 7.5:1) to give 16 (7.6 g, 86% in two steps) as a
colorless oil: 1H NMR (CDCl3) δ 1.05 (s, 9 H, tert-butyl), 2.31
(s, 3 H, SCOCH3), 2.30-2.42 (m, 2 H, 2-Ha and 3-H), 2.53 (dd,
1 H, J ) 7.4 and 15.6 Hz, 2-Hb), 3.02 (dd, 1 H, J ) 6.1 and
13.6 Hz, 4-Ha), 3.11 (dd, 1 H, J ) 6.5 and 13.6, 4-Hb), 3.59 (d,
2 H, J ) 5.7 Hz, SiO-CH2), 3.64 (s, 3 H, OCH3), 7.25-7.65
(m, 10 H, 2xAr). Anal. (C24H32O4SSi) C, H.
(()-1-(4-Hydr oxym eth yl-2,5-dih ydr ofu r an -2-yl)-5-m eth -
yl-1H-p yr im id in e-2,4-d ion e (13b): yield 44%; Rf ) 0.50
(chloroform:methanol ) 10:1); mp 320 °C (methanol/ether); UV
(H2O) λmax 264 nm (ꢀ 10760); 1H NMR (DMSO-d6) δ 1.77 (s, 3
H, CH3), 4.18 (br s, 2 H, HOCH2), 4.51 (d, 1 H, J ) 13.2 Hz,
5-Ha), 4.78 (ddd, 1 H, J ) 2.0, 4.0 and 13.2 Hz, 5-Hb), 5.12 (t,
1 H, J ) 5.6 Hz, OH, D2O exchangeable), 5.61 (br d, 1 H, J )
1.6 Hz, 3-H), 6.82 (m, 1 H, 2-H), 7.16 (s, 1 H, H-6), 11.3 (br s,
1 H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 12.0, 56.6,
74.7, 89.0, 109.7, 117.0, 135.9, 149.5, 150.5, 163.8. Anal.
(C10H12N2O4) C, H, N.
(()-5-F lu or o-1-(4-h yd r oxym eth yl-2,5-d ih yd r ofu r a n -2-
yl)-1H-p yr im id in e-2,4-d ion e (13c): yield 44%; Rf ) 0.45
(chloroform:methanol ) 10:1); mp 260 °C (methanol/ether); UV
1
(H2O) λmax 268 nm (ꢀ 7980); H NMR (DMSO-d6) δ 4.17 (br s,
2 H, OH-CH2), 4.50 (d, 1 H, J ) 13.2 Hz, 5-Ha), 4.80 (ddd, 1
H, J ) 2.4, 5.2 and 13.2 Hz, 5-Hb), 5.10 (t, 1 H, J ) 5.6 Hz,
OH, D2O exchangeable), 5.61 (d, 1 H, J ) 1.6 Hz, 3-H), 6.79
(m, 1 H, 2-H), 7.65 (d, 1 H, J ) 6.8 Hz, H-6), 11.8 (br s, 1 H,
NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 56.6, 56.7,
74.9, 90.6, 116.5, 124.7, 141.3, 150.2, 159.8. Anal. (C9H9FN2O4)
C, H, N.
(()-5-Ch lor o-1-(4-h yd r oxym eth yl-2,5-d ih yd r ofu r a n -2-
yl)-1H-p yr im id in e-2,4-d ion e (13d ): yield 90%; Rf ) 0.44
(chloroform:methanol ) 15:1); mp 333 °C dec (methanol/ether);
UV (H2O) λmax 277 nm (ꢀ 7750); 1H NMR (DMSO-d6) δ 4.17
(br s, 2 H, HOCH2), 4.51 (d, 1 H, J ) 13.2 Hz, 5-Ha), 4.81 (ddd,
1 H, J ) 2.1, 5.1 and 13.2 Hz, 5-Hb), 5.14 (t, 1 H, J ) 5.6 Hz,
OH, D2O exchangeable), 5.63 (d, 1 H, J ) 1.5 Hz, 3-H), 6.78
(m, 1 H, 2-H), 7.66 (s, 1 H, H-6), 11.9 (br s, 1 H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 56.6, 56.7, 75.0, 90.8,
107.6, 116.5, 137.7, 150.2, 159.0. Anal. (C9H9ClN2O4) C, H, N.
(()-4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-4H,5H-d ih y-
d r oth iop h en -2-on e (17). To a solution of 16 (4.4 g, 10.0
mmol) in H2O (100 mL) and EtOH (300 mL) was added NaOH
(1.9 g, 50 mmol) and the reaction mixture was stirred at 50
°C for 2.5 h. After complete removal of the volatile materials,
the residue was acidified to pH ) 3 by adding HCl solution,
which then was poured into methylene chloride (300 mL) and
water (100 mL). The organic layer was washed with brine (50
mL) and dried (MgSO4), filtered, and evaporated. The residue
was purified by silica gel column chromatography (hexanes:
ethyl acetate ) 6:1) to give the crude mercapto acid derivative
as an oil, which without further purification, was treated with
dicyclohexylcarbodiimide (2.1 g, 10.1 mmol) and dimethylami-
nopyridine (6.1 g, 50.1 mmol) in anhydrous methylene chloride
(250 mL). The reaction mixture was stirred at ambient
temperature for 30 min. The resulting solid was filtered and
the filtrate was evaporated. The residue was purified by silica
gel column chromatography (hexanes:ethyl acetate ) 16:1) to
give 17 (2.6 g, 70% in two steps) as a white solid: MS m/z 371
(M + H+); 1H NMR (CDCl3) δ 1.06 (s, 9 H, tert-butyl), 2.45
(dd, 1 H, J ) 9.2 and 16.8 Hz, 3-Ha), 2.58 (dd, 1 H, J ) 6.8
and 16.8 Hz, 3-Hb), 2.68-2.83 (m, 1 H, 4-H), 3.35 (dd, 1 H, J
) 7.7 and 11.2 Hz, 5-Ha), 3.47 (dd, 1 H, J ) 6.6 and 11.2 Hz,
5-Hb), 3.69 (d, 2 H, J ) 5.9 Hz, SiO-CH2), 7.36-7.64 (m, 10
H, 2xPh). Anal. (C21H26O2SSi) C, H.
(()-5-Br om o-1-(4-h yd r oxym eth yl-2,5-d ih yd r ofu r a n -2-
yl)-1H-p yr im id in e-2,4-d ion e (13e): yield 90%; Rf ) 0.45
(chloroform:methanol ) 15:1); mp 292 °C (methanol/ether); UV
1
(H2O) λmax 278 nm (ꢀ 9530); H NMR (DMSO-d6) δ 4.18 (br s,
2 H, OHCH2), 4.51 (d, 1 H, J ) 13.2 Hz, 5-Ha), 4.81 (ddd, 1 H,
J ) 2.1, 5.1 and 13.2 Hz, 5-Hb), 5.15 (t, 1 H, J ) 5.6 Hz, OH,
D2O exchangeable), 5.63 (d, 1 H, J ) 1.6 Hz, 3-H), 6.78 (m, 1
H, 2-H), 7.70 (s, 1 H, H-6), 11.9 (br s, 1 H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 56.6, 56.7, 75.0, 90.8,
96.2, 116.5, 140.0, 150.4, 159.1. Anal. (C9H9BrN2O4) C, H, N.
(()-1-(4-Hyd r oxym eth yl-2,5-d ih yd r ofu r a n -2-yl)-5-iod o-
1H-p yr im id in e-2,4-d ion e (13f): yield 45%; Rf ) 0.46 (chlo-
roform:methanol ) 15:1); mp 241 °C (methanol/ether); UV
(H2O) λmax 283 nm (ꢀ 6810); 1H NMR (DMSO-d6) δ 4.18 (br d,
2 H, HOCH2), 4.52 (d, 1 H, J ) 13.2 Hz, 5-Ha), 4.81 (ddd, 1 H,
J ) 2.2, 5.1 and 13.2 Hz, 5-Hb), 5.16 (t, 1 H, J ) 5.6 Hz, OH,
(()-4-(2-ter t-Bu t yld ip h en ylsilyloxym et h yl)-3-p h en yl-

812 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
J eong et al.
selen yl-4H,5H-d ih yd r oth iop h en -2-on e (18). To a solution
of 17 (0.7 g, 1.89 mmol) in anhydrous THF (10 mL) was added
LiHMDS (1 M solution in THF, 2.27 mL, 2.27 mmol) dropwise
at -78 °C and the mixture was stirred for 1 h at the same
temperature. To this mixture was added TMSCl (0.36 mL, 2.8
mmol) slowly and stirred for another 10 min at -78 °C. The
reaction mixture was elevated to ambient temperature and
stirred for 30 min. The reaction mixture was again cooled to
-78 °C and a solution of PhSeBr (670 mg, 2.8 mmol) in THF
(5 mL) was added quickly. The reaction mixture was stirred
for 30 min and quenched by adding a few drops of ethyl
acetate. The reaction mixture was evaporated under reduced
pressure and the residue was diluted with ethyl acetate (150
mL) and water (50 mL). The organic layer was washed with
brine and dried (MgSO4), filtered, and evaporated. The residue
was purified by silica gel column chromatography (hexanes:
ethyl acetate ) 15:1) to give an inseparable mixture of 18 (989
(3 mL) at -25 °C. The reaction mixture was stirred at the same
temperature for 1 h, evaporated, and diluted with ethyl acetate
(40 mL). The organic layer was washed with brine (10 mL)
and dried (MgSO4), filtered, and evaporated. The residue was
purified by silica gel column chromatography (hexanes:ethyl
acetate ) 2.5:1) to give 21a (129 mg, 71%): UV (MeOH) λmax
267 nm; 1H NMR (CDCl3) δ 0.99 (s, 9 H, tert-butyl), 3.65 (d, 1
H, J ) 15.2 Hz, 5-Ha), 3.93 (ddd, 1 H, J ) 1.6, 5.2 and 15.2
Hz, 5-Hb), 4.11 (s, 2 H, SiO-CH2), 5.65 (d, 1 H, J ) 8.0 Hz,
H-5), 5.68 (d, 1 H, J ) 1.6 Hz, 3-H), 7.02 (d, 1 H, J ) 1.6 Hz,
2-H), 7.37-7.69 (m, 11 H, 2xPh and H-6).
(()-1-(4-Hyd r oxym eth yl-2H,5H-d ih yd r oth iop h en -2-yl)-
1H-p yr im id in e-2,4-d ion e (22a ). To a solution of 21a (100
mg, 0.22 mmol) in THF (5 mL) was added tetra-n-butylam-
monium fluoride (1 M solution in THF, 0.3 mL, 0.3 mmol) and
the mixture was stirred at 0 °C for 1 h and evaporated to a
dryness. The residues were purified by silica gel column
chromatography (chloroform:methanol ) 10:1) to give 22a (37
mg, 76%) as a white solid: MS m/z 249 (M + Na+); mp 163 °C
(methanol/ether); UV (H2O) λmax 266 nm (ꢀ 7830); 1H NMR
(DMSO-d6) δ 3.66 (d, 1 H, J ) 15.2 Hz, 5-Ha), 3.94 (ddd, 1 H,
J ) 2.0, 5.2 and 15.2 Hz, 5-Hb), 4.12 (br s, 1 H, HOCH2), 5.19
(t, 1 H, J ) 5.2 Hz, OH, D2O exchangeable), 5.66 (d, 1 H, J )
8.0 Hz, H-5), 5.69 (d, 1 H, J ) 1.7 Hz, 3-H), 6.74 (m, 1 H, 2-H),
7.44 (d, 1 H, J ) 8.0 Hz, H-6); 13C NMR (DMSO-d6) δ 37.88,
60.22, 68.45, 95.64, 122.29, 142.43, 151.53, 155.85, 166.09.
Anal. (C9H10N2O3S) C, H, N.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
selenyltetrahydrothiophen-2-yl]-5-methyl-1H-pyrimidine-2,4-
d ion e (20b). The acetate 19 (0.96 g, 1.68 mmol) was converted
to 20b (0.77 g, 72% with small amounts of cis isomer) as an
anomeric mixture (4:1 ratio determined by 1H NMR) according
to the procedure used for the preparation of 20a : 1H NMR
(CDCl3) δ 1.06 (s, 1.8 H, tert-butyl), 1.12 (s, 7.2 H, tert-butyl),
1.77 (s, 0.6 H, 5-CH3), 1.81 (s, 2.4 H, 5-CH3), 2.14-2.22 (m,
0.8 H, H-4), 2.72-2.78 (m, 0.2 H, H-4), 2.92 (dd, 0.8 H, J )
8.0 and 10.9 Hz, 5-Ha), 3.07 (dd, 0.2 H, J ) 4.0 and 10.9 Hz,
5-Ha), 3.27 (dd, 0.2 H, J ) 6.4 and 10.9 Hz, 5-Hb), 3.37 (t, 0.8
H, J ) 10.9 Hz, 5-Hb), 3.53 (dd, 0.8 H, J ) 10.1 and 12.0 Hz,
3-H), 3.73 (dd, 0.2 H, J ) 6.1 and 7.7 Hz, 3-H), 3.85-3.89 (m,
1 H, SiO-CHa), 4.11-4.18 (m, 1 H, SiO-CHb), 6.33 (d, 0.2 H,
J ) 7.7 Hz, 2-H), 6.42 (d, 0.8 H, J ) 10.1 Hz, 2-H), 7.05-7.72
(m, 16 H, 3xPh and H-6), 7.95 (br s, 0.2 H, NH, D2O
exchangeable), 7.95 (br s, 0.8 H, NH, D2O exchangeable). Anal.
(C32H36N2O3SSeSi) C, H, N.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-2H,5H-d i-
h yd r ot h iop h en -2-yl]-5-m et h yl-1H-p yr im id in e-2,4-d ion e
(21b). Compound 20b (485 mg, 0.76 mmol) was converted to
21b (272 mg, 75%) according to the procedure used for the
preparation of 21a : 1H NMR (CDCl3) δ 0.98 (s, 9 H, tert-butyl),
1.78 (s, 3 H, CH3), 3.65 (d, 1 H, J ) 15.2 Hz, 5-Ha), 3.98 (ddd,
1 H, J ) 2.0, 5.6 and 15.2 Hz, 5-Hb), 4.08 (d, 1 H, J ) 15.2 Hz,
SiO-CHa), 4.12 (d, 1 H, J ) 15.2 Hz, SiO-CHb), 5.65 (d, 1 H,
J ) 1.2 Hz, 3-H), 7.04 (d, 1 H, J ) 1.2 Hz, 2-H), 7.37-7.69 (m,
11 H, 2xPh and H-6), 8.62 (br s, 1 H, NH).
1
mg, 99%, 3,4-trans/3,4-cis ) 10/1 determined by H NMR) as
a colorless oil. Analytical samples were obtained by preparative
TLC to give pure (()-3,4-tr a n s-18 and (()-3,4-cis-18.
(()-3,4-tr a n s-18: 1H NMR (CDCl3) δ 1.03 (s, 9 H, tert-butyl),
2.47-2.59 (m, 1 H, 4-H), 3.32 (br d, 2 H, J ) 6.4 Hz, 5-H),
3.85 (d, 1 H, J ) 7.0 Hz, 3-H), 3.89 (dd, 1 H, J ) 4.9 and 10.4
Hz, SiO-CHa), 4.07 (dd, 1 H, J ) 6.0 and 10.4 Hz, SiO-CHb),
7.25-7.63 (m, 15 H, 3xPh). Anal. (C27H30O2SSeSi) C, H.
1
(()-3,4-cis-18: H NMR (CDCl3) δ 0.98 (s, 9 H, tert-butyl),
2.40-2.43 (m, 1 H, 4-H), 3.05 (dd, 1 H, J ) 2.4 and 10.4 Hz,
5-Ha), 3.25 (dd, 1 H, J ) 4.4 and 10.4 Hz, 5-Hb), 3.86 (m, 3 H,
3-H and SiO-CH2), 7.19-7.72 (m, 15 H, 3xPh). Anal. (C27H30O2-
SSeSi) C, H.
(()-2-O-Acetoxy-4-(ter t-bu tyld ip h en ylsilyloxym eth yl)-
3-p h en ylselen yltetr a h yd r oth iop h en e (19). An inseparable
mixture of 18 (990 mg, 1.83 mmol) was converted to compound
19 (899 mg, 84% with small amounts of cis isomer) as an
anomeric mixture (3:2 determined by 1H NMR) according to
the similar procedure used for the preparation of 11. (()-3,4-
1
tr a n s-19: H NMR (CDCl3) δ 1.01 (s, 5.4 H, tert-butyl), 1.05
(s, 3.6 H, tert-butyl), 1.92 (s, 1.2 H, CH3), 2.09 (s, 1.8 H, CH3),
2.44-2.57 (m, 0.4 H, H-4), 2.58-2.98 (m, 0.6 H, H-4), 2.98-
3.07 (m, 2 H, H-5), 3.61 (dd, 0.6 H, J ) 4.2 and 12.5 Hz, 3-H),
3.58-3.94 (m, 2.4 H, SiO-CH2 and 3-H), 6.14 (d, 0.4 H, J )
4.8 Hz, 2-H), 6.22 (d, 0.6 H, J ) 4.2 Hz, 2-H), 7.20-7.62 (m,
15 H, 3xPh). Anal. (C29H34O3SSeSi) C, H.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-3-p h en yl-
se le n ylt e t r a h yd r ot h iop h e n -2-yl]-1H -p yr im id in e -2,4-
d ion e (20a ). The suspension of uracil (118 mg, 1.05 mmol),
HMDS (10 mL), and ammonium sulfate (catalytic amount) was
refluxed under nitrogen atmosphere for 4 h and excess HMDS
was removed under high vacuum. To the residue were added
dry dichloroethane (5 mL), a solution of 19 (300 mg, 0.52 mmol)
in dry 1,2-dichloroethane (15 mL), and TMSOTf (0.2 mL, 0.96
mmol) at ambient temperature and the resulting reaction
mixture was stirred at ambient temperature for 1 h. Saturated
NaHCO3 (2 mL) solution was added to the reaction mixture
and the mixture was stirred for another 30 min and diluted
with methylene chloride (30 mL). The organic layer was
washed with brine (10 mL) and dried (MgSO4), filtered, and
evaporated. The residue was purified by silica gel column
chromatography (hexanes:ethyl acetate ) 2:1) to give 20a (243
mg, 74% with small amounts of cis isomer) as an anomeric
(()-1-(4-Hyd r oxym eth yl-2H,5H-d ih yd r oth iop h en -2-yl)-
5-m eth yl-1H-p yr im id in e-2,4-d ion e (22b). Compound 21b
was converted to 22b (35.6 mg, 71%) as a white solid according
to the procedure used for the preparation of 22a : MS m/z 263
(M + Na+); mp 185 °C (methanol/ether); UV (H2O) λmax 270
1
1
mixture (4:1 ratio determined by H NMR): UV (MeOH) λmax
nm (ꢀ 11550); H NMR (DMSO-d6) δ 1.77 (s, 3 H, CH3), 3.65
266 nm; 1H NMR (CDCl3) δ 0.98 (s, 1.8 H, tert-butyl), 1.10 (s,
7.2 H, tert-butyl), 2.11-2.24 (m, 0.8 H, 4-H), 2.48-2.53 (m,
0.2 H, 4-H), 2.97 (dd, 0.8 H, J ) 6.8 and 10.4 Hz, 5-Ha), 3.23
(dd, 0.2 H, J ) 4.3 and 10.4 Hz, 5-Ha), 3.35 (dd, 0.2 H, J ) 4.5
and 10.4 Hz, 5-Hb), 3.41 (t, 0.8 H, J ) 10.4 Hz, 5-Hb), 3.55 (m,
1 H, 3-H), 3.90-4.06 (m, 2 H, SiO-CH2), 6.36 (d, 0.2 H, J )
3.6 Hz, 2-H), 6.55 (d, 0.8 H, J ) 9.6 Hz, 2-H), 7.17-7.71 (m,
16 H, 3xPh and H-6), 8.82 (br s, 1 H, NH). Anal. (C31H34N2O3-
SSeSi) C, H, N.
(()-1-[4-(ter t-Bu tyld ip h en ylsilyloxym eth yl)-2H,5H-d i-
h yd r oth iop h en -2-yl]-1H-p yr im id in e-2,4-d ion e (21a ). To a
solution of 20a (200 mg, 0.32 mmol) in anhydrous CH2Cl2 (10
mL) was slowly added m-CPBA (72 mg, 0.4 mmol) in CH2Cl2
(d, 1 H, J ) 15.6 Hz, 5-Ha), 3.99 (ddd, 1 H, J ) 2.4, 5.6 and
15.6 Hz, 5-Hb), 4.12 (br s, 2 H, HOCH2), 5.17 (t, 1 H, J ) 5.7
Hz, OH, D2O exchangeable), 5.65 (br d, 1 H, J ) 1.6 Hz, 3-H),
6.75 (m, 1 H, 2-H), 7.24 (s, 1 H, H-6), 11.4 (br s, 1 H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 12.89, 38.21, 60.22,
67.79, 110.95, 121.64, 137.09, 151.13, 152.06, 164.36. Anal.
(C10H12N2O3S) C, H, N.
An ti-HCMV Assa y. Vir u ses a n d cells: Human cytome-
galovirus (HCMV) strains AD-169 (ATCC VR-538) and Davis
(ATCC VR-807) and HEL 299 (human embryonic lung fibro-
blast) cells (ATCC CCL137) were purchased from American
Type Culture Collection (ATCC). The cells were grown in
minimal essential medium (MEM; Gibco) containing nones-

Apio/ Thioapio Nucleosides as Anti-HCMV Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 813
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sential amino acids, 40 µg/mL gentamycin (Gibco) and 10%
heat-inactivated FBS (MEM/10% FBS; Gibco).
Com p ou n d s: All compounds used were dissolved with
100% dimethyl sulfoxide as 20 mg/mL of stock solution except
phosphonoformic acid (PFA, foscarnet; Sigma) and ganciclovir
(GCV; Synthex) with distilled H2O.
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Eva lu a tion of a n ti-HCMV a ctivity: Standard CPE inhi-
bition assay was used.16 HEL cells in stationary phase were
infected with the virus at a multiplicity of infection of 2-4
CCID50 (50% cell culture inhibitory dose) per well of 96-well
plates. After 2 h adsorption at 37 °C, the liquid was aspirated
off to remove the unadsorbed viruses and 100 µL of MEM/2%
FBS containing a compound was applied to each well in
duplicate for each concentration and further incubated for 6
days. Antiviral activity was measured microscopically or
fluorometrically. For microscopic observation the cells were
fixed with 70% ethanol, stained with 2.5% Giemsa solution
for 2 h, rinsed with distilled water and air-dried. Antiviral
activity was expressed as the EC50, or the concentration
required to inhibit virus-induced CPE by 50%. EC50 values
were estimated from semilogarithmic graphic plots of the
percentage of CPE as a function of the concentration of the
test compound. For fluorometric assay,17 the cells were washed
twice with 100 µL of phosphate-buffered saline (PBS). To each
well 100 µL of 5 µg/mL fluorescein diacetate (FDA; Sigma) was
added and the plates were incubated for 30 min at 37 °C. The
FDA solution was removed by aspiration and each well was
washed with 100 µL of PBS. The fluorescence intensity (as
absolute fluorescence units, AFU) in each well was measured
with a fluorescent microplate reader (fluoroskan ascent, Lab-
system) equipped with a 485-nm excitation filter and a 538-
nm emission filter. The percentage reduction was caculated
as follows: 100 - [AFU(dt) - AFU(cc)/AFU(vc) - AFU(cc) ×
100] ) Y (cc, cell control; vc, virus control; dt, drug-treated).
Cytotoxcicity a ssa y: The effect of the test compounds on
host cell growth and on viability was assayed microscopically
and fluorometrically by using propidium iodide (PI; Sigma).
To measure cytostatic effects, HEL cells were seeded at 3000
cells/well in 96-well plates in 100 µL of MEM/10% FBS. The
cells were allowed to attach to the plates by incubation at 37
°C for 1 day, different dilutions of the test compounds were
added and the cells were further incubated for 3 days at 37
°C. The cells were permeabilized by freezing at -20 °C for 2
h. They were thawed at 37 °C for 30 min, 100 µL of 40 ug/mL
PI diluted with medium was added to each well following
incubation at room temperature for 60 min in the dark to allow
dye penetration. AUF was read with a fluorescence microplate
reader using a 544-nm excitation filter and a 620-nm emission
filter. The concentration of compound responsible for 50%
inhibition of cell growth was calculated and expressed as CS50
(50% cytostatic effect).
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Cytocidal assay was performed as a control experiment for
antiviral assay. It was carried out simultaneously with the
antiviral assay described previously using mock instead of
virus for infection, and cell viability was measured using PI
instead of FDA. The concentration of compound responsible
for 50% reduction of cell viability was calculated and expressed
as CC50 (50% cytocidal effect).
Ack n ow led gm en t. This work was supported by a
grant from the Ministry of Science and Technology
(MOST) through 2000 National R&D Program for
Women’s Universities in Korea.
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J M000342F