Synthesis and antiviral activities of novel 2′,4′- or 3′,4′-doubly branched carbocyclic nucleosides as potential antiviral agents

Article abstract of DOI:10.1002/ardp.200400875

In this study, a series of 2′,4-′ or 3′,4′-doubly branched carbocyclic nucleosides (11, 12, 19, and 20) were synthesized from simple acyclic ketone derivatives as starting materials. The installation of the 4′-quaternary carbon needed was carried out usin

Full text of DOI:10.1002/ardp.200400875

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 457−463
Novel Doubly Branched Carbocyclic Nucleosides 457
Chang Hyun Oha,
Synthesis and Antiviral Activities of Novel
2Ј,4Ј- or 3Ј,4Ј-Doubly Branched Carbocyclic
Nucleosides as Potential Antiviral Agents
Joon Hee Hongb
a Medicinal Chemistry
Research Center,
Korea Institute of Science
and Technology,
Seoul, Republic of Korea
b College of Pharmacy,
Chosun University,
In this study, a series of 2Ј,4-Ј or 3Ј,4Ј-doubly branched carbocyclic nucleosides
(11, 12, 19, and 20) were synthesized from simple acyclic ketone derivatives as
starting materials. The installation of the 4Ј-quaternary carbon needed was car-
ried out using a [3,3]-sigmatropic rearrangement. In addition, the introduction of
a methyl group in the 2Ј- or 3Ј-position was accomplished by either Grignard
reaction or Horner-Wadsworth-Emmons reaction with triethyl-2-phosphonopro-
pionate, respectively. Bis-vinyl was successfully cyclized using a Grubbs’ cata-
lyst II. The natural bases (adenine, cytosine) were coupled efficiently using a
Pd(0) catalyst. Although all the synthesized compounds were assayed against
several viruses, only the cytosine analogue 20 showed moderate antiviral ac-
tivity against the human cytomegalovirus.
Kwangju, Republic of Korea
Keywords: Carbocyclic nucleosides; [3,3]-sigmatropic rearrangement; Horner-
Wadsworth-Emmons reaction, Antiviral agents
Received: February 4, 2004; Accepted March 25, 2004 [FP875]
DOI 10.1002/ardp.200400875
specific target for the reversible hydrolysis of the Ad-
oHcy linkage to adenosine and homocysteine [12, 13].
The inhibition of the enzyme in intact cellular systems
Introduction
Carbocyclic nucleosides [1, 2, 3], where a carbon
atom replaces the oxygen atom of the sugar moiety,
have emerged as a promising class of nucleosides
with interesting antiviral and antitumor activities. Natu-
ral as well as synthetic carbocyclic nucleosides such
as abacavir [4] and entecavir [5] (Figure 1) have
shown interesting antitumor activities and antiviral ac-
tivities against the human cytomegalovirus (HCMV)
[6], herpes virus [7], hepatitis B virus [8], and human
immunodeficiency virus (HIV) [9, 10].
results in the accumulation of AdoHcy; a higher con-
centration of AdoHcy suppresses the enzyme activity
by acting as a product inhibitor of the AdoMet-depen-
dent methylation reaction [14, 15]. Methyltransferases
are essential for the maturation of mRNA. Therefore,
inhibiting the methyl transferases by blocking the
AdoHcy metabolism can disrupt viral mRNA maturation.
AdoHcy inhibitors usually display a broad-spectrum of
antiviral activity. In view of these interesting biological
However, the toxicitiy associated with these nucleo-
sides as well as the emergence of resistant viral strains
has prompted nucleoside chemists to search for ad-
ditional novel and structurally diverse compounds with
minimal overlapping resistance and toxicity profiles.
These compounds have a higher metabolic stability
against the nucleoside phosphorylases [11] because
of the absence of a glycosidic bond. Various carbo-
cyclic nucleosides are also believed to be potent in-
hibitors of the cellular enzyme, S-adenosyl-L-homo-
cysteine (AdoHcy) hydrolase, which is very important
in regulating the S-adenosylmethionine (SAM)-depen-
dent methylation reactions, and has emerged as a
Correspondence: Joon Hee Hong, College of Pharmacy,
Chosun University, Kwangju 501-759, Republic of Korea.
Phone: +82 62 230-6378, Fax: +82 62 222-5414, e-mail:
hongjh@chosun.ac.kr
Figure 1. Structures of olefinic carbocyclic nucleo-
sides and target nucleosides
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

458 Hong et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 457−463
Scheme 1. Synthesis of 3Ј,4Ј-doubly branched carbocyclic nucleosides.
and chemical properties of the olefinic carbonucleo-
sides, we have synthesized novel 2Ј,4Ј- or 3Ј,4Ј-
doubly branched olefinic carbonucleosides and as-
sayed them as potential antiviral agents.
relative stereochemistry of compounds 7␣ and 7β was
unambiguously determined based on the NOE corre-
lations between the proximal hydrogen and methylene
group (H-1, vs. H-5). Unlike compound 7β, the NOE
correlation was observed between the H-1 vs. H-5 of
compound 7␣.
Chemistry
In order to couple the cyclopentenol with the bases
(A = adenine, C = cytosine) using a palladium(0) cata-
lyst [23, 24], the cyclopentenol 7β was transformed to
the ethoxycarbonyl derivative 8 using ethyl chloro-
formate in a high yield of 80%. Compound 8 was cou-
pled with an adenine anion generated by NaH/DMSO
with the [tris(dibenzylidene-acetone)-dipalladium(0)-
chloroform] adduct to give the compounds 9 and 10.
The required stereochemistry of the nucleosides 9 and
10 were successfully controlled from the β-configura-
tion of compounds 8 via a Pd(0) catalyzed πϪallyl
complex mechanism. Compounds 9 and 10 were
desilylated by treating the compounds with tetrabutyl-
ammonium fluoride (TBAF) to give the final nucleos-
ides 11 and 12 in high yield.
As shown in Scheme 1, for the synthesis of the 3Ј,4Ј-
doubly branched target nucleosides, α,β-unsaturated
ester derivative 1 was selected as the starting com-
pound, which was readily synthesized from 2-hydroxy-
acetophenone by a method reported previously [16,
17]. Without separation, the ester 1 was reduced using
diisobutylaluminum hydride (DIBALH) and the re-
sulting alcohol was subjected to a [3,3]-sigmatropic re-
arrangement [18, 19] using triethylorthoacetate to give
the g,d-unsaturated esters 3 in a 71% two-step yield.
The addition of DIBALH to a solution of the esters 3
in CH2Cl2 at Ϫ10°C gave the alcohol 4, which was
subjected to oxidation conditions using PCC (pyridin-
ium chlorochromate). The resulting aldehyde 5 was
subjected to a Grignard reaction by CH2 = CHMgBr to
yield the bis-olefin 6 as a stereoisomeric mixture.
The synthesis of the 2Ј,4Ј-doubly branched carbo-
cyclic nucleosides was initiated using aldehyde 13 as
the starting material, which was also readily prepared
from 1,3-dihydroxyacetone with a similar procedure
described for synthesizing compound 5 [16, 17]. As
shown in Scheme 2, introduction of the methyl branch
The bis-olefin 6 was subjected to the well-known ring-
closing metathesis conditions [20, 21, 22] using a
Grubbs’ catalyst II [(Im)Cl2PCy3RuCHPh] to provide
the stereoisomers, 7␣ and 7β, in equal amounts. The
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 457−463
Novel Doubly Branched Carbocyclic Nucleosides 459
Scheme 2. Synthesis of 2Ј,4Ј-doubly branched carbocyclic nucleosides.
in the 2Ј-position could be accomplished by adding
isopropenylmagnesium bromide [CH2 = C(CH3)MgBr]
to aldehyde 13. Using a similar procedure as de-
scribed for compound 8, the allylic coupling substrate
16 could be synthesized via successive ring-closing
metathesis and ethoxycarbonylation reactions. The
desired nucleosides 19 and 20 were successfully syn-
thesized by the coupling of the bases, adenine and
cytosine, using a Pd(0) catalyst and desilylation.
bited neither excellent antiviral activity nor any cytotox-
icity when tested up to 100 µg/mL. However, it is inter-
esting to note that only the cytosine analogue 20
exhibited moderate antiviral activity against the HCMV
(Table 1), indicating that this virus might allow the con-
formationally rigid sugar moiety for phosphorylation as
well as for DNA polymerase, which is unlike other vi-
ruses. In addition, the adenine analogue 11 showed
weak antiviral activity against the HSV-1. For the
evaluation of anti-HCMV activity, HCMV strains AD-
169 (ATTCC VR-583) and Davis (ATCC VR-807) and
a standard CPE inhibition assay were used. HEL 299
cells (human embryonic lung fibroblast) served for the
cytotoxicity assay.
Antiviral activity studies
All synthesized compounds were tested against sev-
eral viruses such as the HIV (MT-4 cells), HSV-1
(CCL81 cells), HSV-2 (CCL-81 cells), and HCMV (AD-
169, Davis cells). All the compounds synthesized exhi-
In summary, a concise synthetic method for synthesiz-
ing 2Ј,4Ј- or 3Ј,4Ј-doubly branched carbocyclic nucleo-
Table 1. The antiviral activities of the synthesized compounds.
compound
HIV-1
HSV-1
HSV-2
HCMV
cytotoxicity
EC50(µg/mL)
EC50(µg/mL)
EC50(µg/mL)
EC50(µg/mL)
IC50(µg/mL)
11
12
19
20
>100
>100
>100
>100
0.001
ND
>56.2
>100
>100
>100
ND
>100
>100
>100
>100
ND
>100
>100
>100
>21.6
ND
>100
>100
>100
>100
1.15
AZT
Ganciclovir
Ribavirin
1.66
ND
1.66
ND
ND
15.50
>10
300.00
ND
NDϪnot determined.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

460 Hong et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 457−463
( )-3-(t-Butyldimethylsilyloxymethyl)-3-phenyl-4-methyl-pent-
4-enoic acid ethyl ester (3)
sides from simple a-hydroxy ketone derivatives was
developed in this study. The scope of this method-
ology along with its application to other nucleosides
is currently under investigation. When the synthesized
compounds were tested against several viruses such
as the HIV-1, HSV-1, HSV-2, and HCMV, only the cyto-
sine analogue 20 exhibited moderate activity against
HCMV.
A solution of the allylic alcohol 2 (10 g, 34.18 mmol) in triethyl
orthoacetate (150 mL) and 1.0 mL of propionic acid was
heated at 135Ϫ140°C overnight with constant stirring to allow
for the removal of ethanol. An excess of triethyl orthoacetate
was removed by distillation and the residue was purified by
silica gel column chromatography (EtOAc/hexane, 1:40) to
give compound 3 (9.66 g, 78%) as a colorless oil: 1H NMR
(CDCl3, 300 MHz) δ 7.51Ϫ7.24 (m, 5H), 5.09 (s, 1H), 4.94
(s, 1H), 4.29 (d, J = 9.0 Hz, 1H), 4.10 (d, J = 9.0 Hz, 1H),
4.05 (q, J = 6.9 Hz, 1H), 3.22 (d, J = 6.9 Hz, 2H), 2.95 (d, J =
6.9 Hz, 1H), 1.61 (s, 3H), 1.20 (t, J = 6.9 Hz, 3H), 0.87 (s,
9H), 0.03 (s, 6H); 13C NMR (CDCl3, 75 MHz) δ 171.78,
147.14, 143.40, 127.85, 126.48, 126.19, 112.04, 65.84,
59.87, 51.38, 37.91, 25.67, 18.47, 17.84, 14.12, Ϫ5.83; Anal
calc. for C21H34O3Si: C, 69.56; H, 9.45. Found: C, 69.31; H,
9.50.
Acknowledgments
We thank Dr. C.-K Lee (KRICT) for antiviral evaluation.
Experimental
( )-3-(t-Butyldimethylsilyloxymethyl)-3-phenyl-4-methyl-pent-
4-enol (4)
All the chemicals were of reagent grade and were used as
purchased. All the moisture-sensitive reactions were per-
formed in an inert atmosphere with either N2 or Ar using dis-
tilled dry solvents. The elemental analyses were performed
using an Elemental Analyzer System (Profile HV-3). The
NMR spectra were recorded on a Bruker 300 MHz Fourier
transform spectrometer (Bruker, Karlsruhe, Germany).
To a solution of compound 3 (8.0 g, 22.06 mmol) in CH2Cl2
(200 mL), DIBALH (46.33 mL, 1.0 M solution in hexane) was
added slowly at Ϫ10°C, and stirred for 30 min at the same
temperature. To the mixture, methanol (40 mL) was then ad-
ded. The mixture was stirred at room temperature for 2 h,
and the resulting solid was filtered through a Celite pad. The
filtrate was concentrated under vacuum, and the residue was
purified by silica gel column chromatography (EtOAc/hexane,
1:20) to give compound 4 (6.93 g, 98%) as a colorless oil:
1H NMR (CDCl3, 300 MHz) δ 7.46Ϫ7.34 (m, 5H), 5.25 (s,
1H), 5.14 (s, 1H), 4.21 (d, J = 9.3 Hz, 1H), 4.02 (d, J = 9.3
Hz, 1H), 3.80 (dd, J = 6.9, 6.6 Hz, 2H), 2.61 (dd, J = 13.5,
6.9 Hz, 1H), 2.38 (dd, J = 13.2, 6.6 Hz, 1H), 1.68 (s, 3H),
1.00 (s, 9H), 0.01 (s, 6H); 13C NMR (CDCl3, 75 MHz) δ
147.59, 144.17, 127.93, 127.06, 126.05, 112.46, 67.53,
59.58, 51.29, 35.42, 25.69, 20.61, 18.06, Ϫ5.90; Anal calc.
for C19H32O2Si: C, 71.19; H, 10.06. Found: C, 70.98; H, 9.89.
(E) and (Z)-4-(t-Butyldimethylsilyloxy)-3-phenyl-2-methyl-but-
2-enoic acid ethyl ester (1)
To a suspension of sodium hydride (60% in mineral oil,
1.11 g, 27.7 mmol) in distilled THF at 0°C, triethyl 2-phos-
phonopropionate (5.94 mL, 27.7 mmol) was added dropwise
and with constant stirring at room temperature for 30 min.
2-(t-Butyldimethylsilyloxy)-acetophonone (6.93 g, 27.7 mmol)
was added to this mixture and stirred for 2 h. The solution
was neutralized with saturated ammonium chloride and ex-
tracted with EtOAc. The organic layer was washed with brine
and dried over anhydrous MgSO4, filtered, and evaporated.
The residue was purified by silica gel column chromatogra-
phy (EtOAc/hexane, 1:20) to give 1 (7.8 g, 85%) as a color-
less oil: 1H NMR (CDCl3, 300 MHz) as mixture δ 7.54Ϫ7.19
(m, 5H), 4.71, 4.53 (s, s, 2H), 4.33 (q, J = 6.9 Hz, 2H), 3.89
(q, J = 6.9 Hz, 2H), 2.14, 0.84 (s, s, 3H), 1.21, 0.97 (dt, J =
6.9, 6.9 Hz, 3H), 0.85, 0.67 (s, s, 9H), 0.04, 0.02 (s, s, 6H);
Anal calc. for C19H30O3Si: C, 68.22; H, 9.04. Found: C, 68.34;
H, 8.90.
( )-3-(t-Butyldimethylsilyloxymethyl)-3-phenyl-4-methyl-pent-
4-enal (5)
To a solution of compound 4 (7.0 g, 21.83 mmol) in CH2Cl2
˚
(150 mL), 4A molecular sieves (12.7 g) and PCC (11.76 g,
54.55 mmol) were added slowly at 0°C, and stirred for 4 h at
room temperature. To the mixture, excess diethyl ether (600
mL) was then added. The mixture was stirred vigorously for 3
h at the same temperature, and the resulting solid was filtered
through a short silica gel column. The filtrate was concen-
trated under vacuum and the residue was purified by silica
gel column chromatography (EtOAc/hexane, 1:50) to give
compound 5 (5.70 g, 82%) as a colorless oil: 1H NMR
(CDCl3, 300 MHz) δ 9.64 (m, 1H), 7.35Ϫ7.21 (m, 5H), 5.15
(d, J = 2.4 Hz, 1H), 5.09 (s, 1H), 4.05 (d, J = 9.6 Hz, 1H),
3.90 (d, J = 9.6 Hz, 1H), 2.99 (dd, J = 13.2, 3.0 Hz, 1H), 2.84
(dd, J = 13.2, 3.2 Hz, 1H), 1.55 (s, 3H), 0.84 (s, 9H), 0.01 (s,
6H); 13C NMR (CDCl3, 75 MHz) δ 203.05, 146.09, 142.60,
127.88, 126.77, 113.53, 68.58, 51.45, 47.38, 25.76, 20.39,
18.17, Ϫ5.84; Anal calc. for C19H30O2Si: C, 71.64; H, 9.49.
Found: C, 71.44; H, 9.40.
(E) and (Z)-4-(t-Butyldimethylsilyloxymethyl)-4-phenyl-2-
methyl-but-2-en-1-ol (2)
To a solution of compound 1 (10 g, 29.89 mmol) in CH2Cl2
(350 mL), DIBALH (62.7 mL, 1.0 M solution in hexane) was
added slowly at Ϫ20°C, and stirred for 1 h at the same tem-
perature. To the resulting mixture, methanol (60 mL) was ad-
ded. The mixture was stirred at room temperature for 3 h,
and the resulting solid was filtered through a Celite pad. The
filtrate was concentrated under vacuum and the residue was
purified by silica gel column chromatography (EtOAc/hexane,
1:7) to give the allylic alcohol 2 (7.95 g, 91%) as a colorless
oil: as a isomeric mixture for 1H NMR (CDCl3, 300 MHz) δ
7.44-7.02 (m, 5H), 4.51 (d, J = 5.4, 2H), 4.36 (d, J = 4.2 Hz,
1H), 4.02 (s, 1H), 0.89 (s, 6H), 0.82 (s, 3H), 0.04 (s, 4H), 0.02
(s, 2H); Anal calc. for C17H28O2Si: C, 69.81; H, 9.65. Found:
C, 69.61; H, 9.54.
(rel)-(3R and 3S,5S)-5-(t-Butyldimethylsilyloxymethyl)-5-
phenyl-6-methyl-hepta-1,6-dien-3-ol (6)
To a solution of compound 5 (6.0 g, 18.83 mmol) in dry THF
(200 mL) vinylmagnesium bromide (22.59 mL, 1.0 M solution
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 457−463
Novel Doubly Branched Carbocyclic Nucleosides 461
in THF) was added slowly at -78°C. After 3 h, a saturated
NH4Cl solution (22 mL) was added, and the reaction mixture
was warmed slowly to room temperature. The mixture was
extracted with EtOAc (2 ϫ 250 mL). The combined organic
layer was dried over MgSO4, filtered, and evaporated. The
residue was purified by silica gel column chromatography
(EtOAc/hexane, 1:30) to give compound 6 (4.63 g, 71%) as
a colorless oil: as a diastereomeric mixture for 1H NMR
(CDCl3, 300 MHz) δ 7.45Ϫ7.32 (m, 5H), 6.17Ϫ5.97 (m, 1H),
5.43Ϫ5.11 (m, 4H), 4.36Ϫ4.18 (m, 2H), 2.56Ϫ2.42 (m, 1H),
2.36Ϫ2.27 (m, 1H), 1.69, 1.62 (s, s, 3H), 0.97 (s, 9H), 0.03,
0.01 (s, 6H); 13C NMR (CDCl3, 75 MHz) δ 149.34, 146.75,
144.26, 143.95, 141.92, 128.13, 127.15, 126.20, 113.48,
112.34, 70.01, 69.50, 69.03, 66.64, 52.10, 51.42, 41.01,
40.69, 25.72, 20.94, 20.81, 18.11, Ϫ5.70, Ϫ5.92; Anal calc.
for C21H34O2Si: C, 72.78; H, 9.89. Found: C, 72.58; H, 9.74.
(rel)-(1ЈR,4ЈR)-9-[4-(t-Butyldimethylsilyloxymethyl)-4-phenyl-
3-methyl-cyclopent-2-en-1-yl] adenine (9)
To pure NaH (46.8 mg, 1.96 mmol) in anhydrous DMSO (9.0
mL), adenine (268 mg, 1.96 mmol) was added. The reaction
mixture was stirred for 30 min at 50-55°C and cooled to room
temperature. Simultaneously, P(O-i-Pr)3 (0.96 mL, 2.2 mmol)
was added to a solution of Pd2(dba)3·CHCl3 (46 mg, 25
mmol) in anhydrous THF (8.0 mL), which was stirred for 40
min. To the adenine solution in DMSO, the catalyst solution
of THF and compound 8 (687 mg, 1.76 mmol) dissolved in
anhydrous THF (5 mL) were added slowly. The reaction mix-
ture was stirred overnight at a refluxing temperature and
quenched with water (5 mL). The reaction solvent was re-
moved under reduced pressure. The residue was purified by
silica gel column chromatography (MeOH/CH2Cl2, 1:10) to
give compound 9 (322 mg, 42%) as a white solid: mp
192Ϫ195°C; UV (MeOH) lmax 260.5 nm; 1H NMR (CDCl3,
300 MHz) d 8.33 (s, 1H), 8.03 (s, 1H), 7.36Ϫ7.26 (m, 5H),
5.81 (s, 1H), 5.56 (dd, J = 7.2, 6.0 Hz, 1H), 3.70 (s, 2H), 2.61
(dd, J = 13.8, 7.8 Hz, 1H), 1.92 (dd, J = 13.8, 6.4 Hz, 1H),
1.47 (s, 3H), 0.87 (s, 9H), 0.01 (s, 6H); 13C NMR (CDCl3,
75 MHz) δ 155.46, 152.91, 145.26, 139.31, 138.47, 135.34,
128.34, 126.62, 70.24, 61.38, 57.45, 42.94, 25.98, 18.52,
13.71, Ϫ5.37; Anal calc. for C24H33N5OSi: C, 66.17; H, 7.64;
N, 16.08. Found: C, 65.88; H, 7.51; N, 16.21.
(rel)-(1R,4R)-4-(t-Butyldimethylsilyloxymethyl)-3-methyl-4-
phenyl-cyclopent-2-enol (7β); and (rel)-(1S,4R)-4-(t-Butyldi-
methylsilyloxymethyl)-3-methyl-4-phenyl-cyclopent-2-enol
(7␣)
To a solution of compound 6 (4.0 g, 11.54 mmol) in dry
CH2Cl2 (15 mL), Grubbs’ catalyst II (102 mg 0.12 mmol) was
added. The reaction mixture was stirred overnight, and con-
centrated. The residue was purified by silica gel column chro-
matography (EtOAc/hexane, 1:10) to give the cyclopentenols
7β (1.6 g, 44%) and 7␣ (1.58 g, 43%) as colorless oil. 7β:1H
NMR (CDCl3, 300 MHz) d 7.16-6.95 (m, 5H), 5.67 (s, 1H),
4.47 (t, J = 9.3 Hz, 1H), 3.95 (d, J = 9.6 Hz, 1H), 3.84 (d, J =
9.6 Hz, 1H), 2.27 (dd, J = 14.4, 6.9 Hz, 1H), 1.96 (d, J = 14.1
Hz, 1H), 1.34 (s, 3H), 0.71 (s, 9H), 0.02 (s, 6H); 13C NMR
(CDCl3, 75 MHz) d 145.73, 145.03, 126.97, 126.30, 126.14,
126.03, 74.47, 64.98, 59.02, 50.29, 25.73, 18.03, 12.99,
Ϫ5.68; Anal calc. for C19H30O2Si: C, 71.64; H, 9.49. Found:
C, 71.56; H, 9.52. 7␣: 1H NMR (CDCl3, 300 MHz) δ
7.26Ϫ7.03 (m, 5H), 5.65 (s, 1H), 4.73 (br s, 1H), 3.91 (d, J =
9.6 Hz, 1H), 3.80 (d, J = 9.6 Hz, 1H), 2.65 (dd, J = 13.8, 7.2
Hz, 1H), 1.77 (dd, J = 18.8, 3.6 Hz, 1H), 1.37 (s, 3H), 0.79
(s, 9H), 0.03 (s, 6H); 13C NMR (CDCl3, 75 MHz) δ 147.64,
145.54, 128.34, 127.20, 126.77, 126.07, 66.12, 59.17, 48.66,
25.76, 18.15, 13.63, Ϫ5.52; Anal calc. for C19H30O2Si: C,
71.64; H, 9.49. Found: C, 71.79; H, 9.30.
(rel)-(1ЈR,4ЈR)-1-[4-(t-Butyldimethylsilyloxymethyl)-4-phenyl-
3-methyl-cyclopent-2-en-1-yl] cytosine (10)
Compound 10 was prepared from compound 8 using the
method described for synthesizing compound 9: Yield 39%;
mp 170Ϫ173°C; UV (MeOH) λmax 272.5 nm; 1H NMR
(CDCl3, 300 MHz) d 7.81 (d, J = 7.2 Hz, 1H), 7.30-7.26 (m,
5H), 6.00 (s, 1H), 5.91 (d, J = 7.2 Hz, 1H), 5.78 (dd, J = 6.8,
5.6 Hz, 1H), 3.62 (dd, J = 13.2, 7.8 Hz, 2H), 2.78 (dd, J =
13.6, 7.6 Hz, 1H), 2.32 (dd, J = 13.6, 5.6 Hz, 1H), 1.70 (s,
3H), 0.90 (s, 9H), 0.04 (s, 6H); 13C NMR (CDCl3, 75 MHz)
δ 165.41, 154.63, 157.48, 146.50, 139.81, 133.89, 128.71,
127.23, 124.44, 98.34, 83.12, 71.67, 58.72, 42.50, 25.71,
18.45, 14.33, Ϫ5.45; Anal calc. for C23H33N3O2Si: C, 67.11;
H, 8.08; N, 10.21. Found: C, 67.32; H, 8.11; N, 10.39.
(rel)-(1ЈR,4ЈR)-9-[4-(Hydroxymethyl)-4-phenyl-3-methyl-
cyclopent-2-en-1-yl] adenine (11)
(rel)-(1R,4R)-1-Ethoxycarbonyloxy-4-(t-butyldimethylsilyloxy-
To a solution of compound 9 (130 mg, 0.298 mmol) in THF
(3 mL), TBAF (0.45 mL, 1.0 M solution in THF) at 0°C was
added. The mixture was stirred at room temperature for 4 h,
and concentrated. The residue was purified by silica gel col-
umn chromatography (MeOH/CH2Cl2, 1:5) to give compound
11 (75 mg, 79%) as a white solid: mp 190Ϫ193°C; UV
(MeOH) λmax 260.0 nm; 1H NMR (DMSO-d6, 300 MHz) δ 8.35
(s, 1H), 8.11 (s, 1H), 7.30Ϫ7.22 (m, 5H), 7.16 (br s, 2H, D2O
exchangeable), 5.83 (s, 1H), 5.59 (br s, 1H), 4.74 (t, J = 6.6
Hz, 1H, D2O exchangeable), 3.82 (d, J = 5.6 Hz, 2H), 2.58
(dd, J = 13.6, 7.2 Hz, 1H), 1.89 (dd, J = 13.6, 6.6 Hz, 1H),
1.43 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 155.62, 153.32,
145.25, 139.95, 138.21, 135.21, 128.67, 127.23, 69.31,
61.12, 58.56, 42.12, 14.12; Anal calc. for C18H19N5O: C,
67.27; H, 5.96; N, 21.79. Found: C, 67.40; H, 5.88; N, 21.67.
methyl)-4-phenyl-3-methyl-cyclopent-2-ene (8)
To a solution of compound 7β (3.0 g, 9.41 mmol) in anhydrous
pyridine (30 mL) ethyl chloroformate (1.8 mL, 18.83 mmol)
and dimethylaminopyridine (220 mg, 1.8 mmol) were added.
The reaction mixture was stirred overnight at room tempera-
ture. The reaction mixture was quenched using a saturated
NaHCO3 solution (5 mL) and concentrated under vacuum.
The residue was extracted with EtOAc, dried over MgSO4,
filtered, and concentrated. The residue was purified by silica
gel column chromatography (EtOAc/hexane, 1:40) to give
compound 8 (2.94 g, 80%) as a colorless syrup: 1H NMR
(CDCl3, 300 MHz) d 7.34Ϫ7.22 (m, 5H), 5.98 (s, 1H), 5.52
(dd, J = 6.7, 3.0 Hz, 1H), 4.31 (q, J = 7.0 Hz, 2H), 3.88 (d,
J = 9.3 Hz, 1H), 3.81 (d, J = 9.3 Hz, 1H), 2.34 (dd, J = 13.6,
6.4 Hz, 1H), 2.12 (dd, J = 13.6, 3.6 Hz, 1H), 1.56 (s, 3H),
1.43 (t, J = 7.0 Hz, 3H), 0.89 (s, 9H), 0.03 (s, 6H); 13C NMR
(CDCl3, 75 MHz) δ 155.47, 146.12, 138.72, 134.12, 128.23,
126.77, 125.79, 85.67, 71.46, 64.11, 58.21, 40.31, 25.74,
18.34, 14.23, 13.89, Ϫ5.34; Anal calc. for C22H34O4Si: C,
67.65; H, 8.77. Found: C, 67.70; H, 8.62.
(rel)-(1ЈR,4ЈR)-1-[4-(Hydroxymethyl)-4-phenyl-3-methyl-
cyclopent-2-en-1-yl] cytosine (12)
Compound 12 was prepared from compound 10 using the
method described for synthesizing compound 11: yield 81%;
1
mp 174Ϫ176°C; UV (H2O) λmax 272.5 nm; H NMR (DMSO-
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

462 Hong et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 457−463
d6, 300 MHz) d 7.78 (d, J = 7.2 Hz, 1H), 7.30Ϫ7.21 (m, 5H),
7.03 (br d, 2H, D2O exchangeable), 5.81 (d, J = 7.2 Hz, 1H),
5.79 (s, 1H), 5.70 (br s, 1H), 4.70 (t, J = 6.8 Hz, 1H, D2O
exchangeable), 3.71 (dd, J = 13.6, 7.6 Hz, 2H), 2.68 (dd, J =
13.8, 7.6 Hz, 1H), 2.30 (dd, J = 13.8, 6.8 Hz, 1H), 1.56 (s,
3H); 13C NMR (DMSO-d6, 75 MHz) δ 166.31, 155.56, 158.11,
146.98, 140.54, 134.31, 128.98, 128.56, 124.99, 98.87,
83.87, 72.45, 58.69, 42.50, 13.65, Ϫ5.78; Anal calc. for
C17H19N3O2: C, 68.67; H, 6.44; N, 14.13. Found: C, 68.50;
H, 6.38; N, 14.25.
calc. for C25H45N5O2Si2: C, 59.60; H, 9.00; N, 13.90. Found:
C, 59.72; H, 9.11; N, 13.70.
( )-1-[4-Bis-(t-butyldimethylsilyloxymethyl)-2-methyl-cyclo-
pent-2-en-1-yl] cytosine (18)
Compound 18 was prepared from compound 16 using the
method described for synthesizing compound 9: yield 36%;
1H NMR (CDCl3, 300 MHz) δ 8.00 (d, J = 5.7 Hz, 1H), 6.04
(d, J = 5.1 Hz, 1H), 5.74 (dd, J = 7.8, 3.9 Hz, 1H), 5.48 (s,
1H), 3.53-3.43 (m, 4H), 2.23 (dd, J = 8.4, 7.2 Hz, 1H), 1.74
(s, 3H), 1.58 (dd, J = 14.4, 3.6 Hz, 1H), 0.88 (s, 18H), 0.03 (s,
12H); 13C NMR (CDCl3, 75 MHz) δ 165.42, 164.57, 157.59,
140.30, 133.16, 98.92, 83.25, 66.60, 66.05, 55.12, 37.79,
25.93, 18.30, 14.22, Ϫ5.50; Anal calc. for C24H45N3O3Si2: C,
60.08; H, 9.45; N, 8.76. Found: C, 59.81; H, 9.27; N, 8.65.
( )-5,5Ј-Bis-(t-butyldimethylsilyloxymethyl)-2-methyl-hepta-
1,6-dien-3-ol (14)
Compound 14 was prepared from compound 13 using iso-
propenylmagnesium bromide instead of vinylmagnesium bro-
mide following the method described for synthesizing com-
1
pound 6: yield: 80%; H NMR (CDCl3, 300 MHz) δ 5.75 (dd,
( )-9-[4,4Ј-Bis-(hydroxymethyl)-2-methyl-cyclopent-2-en-1-
yl]adenine (19)
J = 17.7, 11.1 Hz, 1H), 5.09 (d, J = 11.4 Hz, 1H), 4.98 (d, J =
18.0 Hz, 1H), 4.90 (s, 1H), 4.49 (s, 1H), 4.14 (d, J = 9.6 Hz,
1H), 3.63 (s, 2H), 3.43 (dd, J = 12.9, 9.6 Hz, 2H), 1.65 (s,
3H), 1.48 (m, 2H), 0.83 (s, 18H), 0.03 (s, 12H); 13C NMR
(CDCl3, 75 MHz) δ 148.34, 141.39, 114.72, 109.79, 71.49,
66.86, 65.19, 46.15, 40.40, 25.85, 18.25, Ϫ5.51; Anal calc.
for C22H46O3Si2: C, 63.71; H, 11.18. Found: C, 63.78; H,
11.29.
Compound 18 was prepared from compound 17 using the
method described for synthesizing compound 11: yield 80%;
1H NMR (CDCl3, 300 MHz) δ 8.17 (s, 1H), 8.14 (s, 1H), 7.19
(br s, 2H, D2O exchangeable), 5.61 (s, 1H), 5.53 (t, J = 7.8
Hz, 1H), 4.72 (br s, 2H, D2O exchangeable), 3.52Ϫ3.40 (m,
4H), 2.42 (dd, J = 13.5, 9.3 Hz, 1H), 1.94 (dd, J = 14.4, 6.3
Hz, 1H), 1.47 (s, 1H); 13C NMR (CDCl3, 75 MHz) δ 155.98,
152.32, 149.48, 139.25, 137.83, 134.26, 118.78, 65.40,
65.00, 55.81, 37.36, 13.48; Anal calc. for C13H17N5O2: C,
56.71; H, 6.22; N, 25.44. Found: C, 56.80; H, 6.29; N, 25.31.
( )-4,4Ј-Bis-(t-butyldimethylsilyloxymethyl)-2-methyl-cyclo-
pent-2-enol (15)
Compound 15 was prepared from compound 14 using the
method described for synthesizing compounds 7β and 7␣:
yield: 88%; 1H NMR (CDCl3, 300 MHz) δ 5.19 (s, 1H), 4.21
(dd, J = 11.1, 7.5 Hz, 1H), 3.57 (d, J = 9.3 Hz, 1H), 3.47 (d,
J = 9.3 Hz, 1H), 3.40 (d, J = 9.6 Hz, 1H), 3.34 (d, J = 9.6 Hz,
1H), 1.86 (dd, J = 14.4, 7.5 Hz, 1H), 1.73 (s, 1H), 1.51 (d, J =
14.4 Hz, 1H), 0.82 (s, 18H), 0.03, 0.02 (s, s, 12H); 13C NMR
(CDCl3, 75 MHz) δ 144.54, 130.13, 78.33, 68.25, 66.95,
55.79, 41.56, 41.56, 25.98, 18.54, 13.94, Ϫ5.52; Anal calc.
for C20H42O3Si2: C, 62.12; H, 10.95. Found: C, 62.33; H,
10.83.
( )-1-[4,4Ј-Bis-(hydroxymethyl)-2-methyl-cyclopent-2-en-1-
yl]cytosine (20)
Compound 20 was prepared from compound 18 using the
method described for synthesizing compound 11: yield 83%;
1H NMR (DMSO-d6, 300 MHz) δ 7.94 (d, J = 6.0 Hz, 1H),
7.20 (br s, 1H, D2O exchangeable), 6.98 (br s, 1H, D2O ex-
changeable), 5.87 (d, J = 5.9 Hz, 1H), 5.70 (dd, J = 7.6, 4.0
Hz, 1H), 5.45 (s, 1H), 4.71 (br s, 2H, D2O exchangeable),
3.50Ϫ3.39 (m, 4H), 2.22 (dd, J = 13.8, 6.8 Hz, 1H), 1.72 (s,
3H), 1.52 (dd, J = 13.8, 3.8 Hz, 1H); 13C NMR (DMSO-d6,
75 MHz) δ 166.17, 165.27, 157.78, 141.37, 133.88, 97.45,
82.43, 66.12, 65.78, 54.23, 38.46, 13.45; Anal calc. for
( )-1-Ethoxycarbonyloxy-4,4Ј-bis-(t-butyldimethylsilyloxy-
methyl)-2-methyl-cyclopent-2-ene (16)
C12H17N3O3: C, 57.36; H, 6.82; N, 16.72. Found: C, 57.46;
Compound 16 was prepared from compound 15 using the
method described for synthesizing compound 8: yield 82%;
1H NMR (CDCl3, 300 MHz) d 5.50 (s, 1H), 5.45 (dd, J = 7.5,
4.2 Hz, 1H), 4.19 (q, J = 7.8 Hz, 2H), 3.52Ϫ3.41 (m, 4H),
2.17 (dd, J = 13.8, 7.5 Hz, 1H), 1.72 (s, 3H), 1.60 (dd, J =
14.1, 3.6 Hz, 1H), 1.30 (t, J = 7.8 Hz, 3H), 0.86 (s, 18H), 0.01
(s, 12H); 13C NMR (CDCl3, 75 MHz) δ 155.28, 139.01,
134.51, 85.05, 66.36, 65.93, 63.72, 55.41, 37.28, 25.87,
18.26, 14.30, 13.92, Ϫ5.50; Anal calc. for C23H46O5Si2: C,
60.21; H, 10.11. Found: C, 60.02; H, 10.20.
H, 6.79; N, 16.98.
Evaluation of anti-HCMV activity
Standard CPE inhibition assay was used [25]. HEL cells in
stationary phase were infected with the virus at a multiplicity
of infection of 2Ϫ4 CCID50 per well of 96%-well plates. After
2 h adsorption at 37°C, the liquid was aspirated off to remove
the unadsorped viruses and 100 µL of MEM/2% FBS contain-
ing 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
microscopical observation the cells were fixed with 70% etha-
nol, stained with 2.5% Giemsa solution for 2 h, rinsed with
distilled water, and air-dried. Antiviral activity was expresses
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 fluoro-
metric assay [26], 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 solu-
(
)-9-[4-Bis-(t-butyldimethylsilyloxymethyl)-2-methyl-
cyclopent-2-en-1-yl] adenine (17)
Compound 17 was prepared from compound 16 using the
method described for synthesizing compound 9: yield 40%;
1H NMR (CDCl3, 300 MHz) δ 8.32 (s, 1H), 7.85 (s, 1H), 5.67
(s, 1H), 5.55 (dd, J = 15.0, 7.2 Hz, 1H), 3.64 (d, J = 9.9 Hz,
1H), 3.57 (d, J = 9.9 Hz, 1H), 3.44 (s, 2H), 2.44 (dd, J = 14.4,
9.6 Hz, 1H), 1.85 (dd, J = 14.2, 5.4 Hz, 1H), 1.51 (s, 3H),
0.85 (s, 18H), 0.02 (s, 16H); 13C NMR (CDCl3, 75 MHz) δ
155.37, 152.90, 150.27, 139.28, 138.59, 134.77, 119.65,
66.66, 61.63, 56.06, 38.20, 25.99, 18.46, 13.79, Ϫ5.40; Anal
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 457−463
Novel Doubly Branched Carbocyclic Nucleosides 463
tion was removed by aspiration and each well was washed
with 100 µL of PBS. The fluorescence intensity (as absolute
fluorescent units, AFU) in each well was measured with a
fluorescent microplate reader equipped with a 485-nm exci-
tation filter and a 538-nm emission filter.
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USA 1989, 86, 8541Ϫ8544.
Cytotoxicity assay
[9] S. Hayashi, D. W. Norbeck, W. Rosenbrook, R. L. Fine,
M. Matsukura, J. J. Plattner, S. Broder, H. Mitsuya, Anti-
microb. Agents Chemother. 1990, 34, 287Ϫ294.
The effect of the test compounds on host cell growth and on
viability was assayed microscopically and fluorometrically by
using propidium iodide (PI; Sigma, St. Louse, MI, USA). To
measure cytostatic effects, HEL cells were seeded at 3000
cell/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. 100 µL of 40 µg/mL PI diluted with medium was added
each well following incubation at room temperature for 60 min
in the dark to allow dye penetration. Fluorescence was meas-
ured using a fluorescence microplate reader (544-nm exci-
tation; 620-nm emission), the concentration of compounds re-
sponsible for 50% inhibition of cell growth was calculated and
expressed as CS50 (50% cytostatic effect).
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haeghe, J. Med. Chem. 1985, 28, 550Ϫ555.
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1130Ϫ1136.
Cytocidal assay was performed as a control experiment for
the 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 compounds responsible
for 50% reduction of cell viability was calculated and ex-
pressed as CC50 (50% cytocidal effect).
[16] J. H. Hong, O. H. Ko, Bull. Kor. Chem. Soc. 2003, 24,
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© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim