2-Amino-6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) esters as novel HBV-specific antiviral reagents

Article abstract of DOI:10.1021/jm020036x

Novel 2-amino-6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2, 2,2-trifluoroethyl) esters were synthesized and evaluated for antihepatitis B virus (HBV) activity in vitro using HB611, HuH-6 cell line, stably transfected with the HBV genome. Among the

Full text of DOI:10.1021/jm020036x

3138
J . Med. Chem. 2002, 45, 3138-3142
2-Am in o-6-a r ylth io-9-[2-(p h osp h on om eth oxy)eth yl]p u r in e
Bis(2,2,2-tr iflu or oeth yl) Ester s a s Novel HBV-Sp ecific An tivir a l Rea gen ts
Kouichi Sekiya, Hideaki Takashima, Naoko Ueda, Naohiro Kamiya, Satoshi Yuasa, Yoshiyuki Fujimura, and
Masaru Ubasawa*
Pharmaceuticals Research Division, Mitsubishi Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama,
Kanagawa 227-0033, J apan
Received J anuary 23, 2002
Novel 2-amino-6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) esters
were synthesized and evaluated for antihepatitis B virus (HBV) activity in vitro using HB611,
HuH-6 cell line, stably transfected with the HBV genome. Among the compounds synthesized,
2-amino-6-phenylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (8),
2-amino-6-(4-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl)
ester (16), 2-amino-6-(3-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-
trifluoroethyl) ester (17), and 2-amino-6-(2-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]-
purine bis(2,2,2-trifluoroethyl) ester (18) showed considerably high anti-HBV activity, as
represented by IC50 values of 0.05, 0.03, 0.04, and 0.08 µM, respectively, and exhibited low
cytotoxicity, as represented by CC50 values of more than 1000 µM. It was suggested that these
compounds did not have anti-HIV activity, and compound 8 showed only weak anti-HSV-1
activity. An antiviral agent, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), which was used
as a control in the present study, showed moderate anti-HBV activity, as represented by an
IC50 value of 0.2 µM. Furthermore, compound 16 was administered orally to mice at a dose of
100 mg/kg in order to examine its gastrointestinal absorbability. Consequently, the main active
metabolite was observed in mouse plasma, with especially high concentrations in the liver.
In tr od u ction
analogues have also been investigated. Yokota et al.
have reported that phosphonomethoxyethylpurine ana-
logues, especially 9-[2-(phosphonomethoxy)ethyl]ade-
nine (PMEA) and 9-[2-(phosphonomethoxy)ethyl]-2,6-
diaminopurine (PMEDAP), which are known to have a
broad spectrum of activity against viruses, e.g., HIV5
and HSV,6 also exhibit anti-HBV activity.7 However,
phosphonomethoxyethylpurine analogues are minimally
absorbed from the gastrointestinal tract because of the
negative charge of phosphonic acid. To improve oral
bioavailability, many PMEA prodrugs have been syn-
thesized.8 Among them, the dipivaloyloxymethyl ester
of PMEA, adefovir dipivoxil, shows acceptable oral
bioavailability.9 The present study was conducted to
explore novel 9-[2-(phosphonomethoxy)ethyl]purine ana-
logues that might have more potent anti-HBV activity
and less cytotoxicity. Since PMEDAP is an anti-HBV
agent with higher efficacy compared to PMEA,7 we
synthesized various PMEDAP derivatives that have
alkyl or aryl groups on the 6-amino group.10 Most of the
synthesized compounds showed anti-HBV activity in
vitro but also exhibited toxicological changes, e.g.,
inhibited body weight gain, alopecia, and diarrhea,
when administered to rats. However, 6-(methylphenyl-
amino) analogue retained anti-HBV activity despite
showing less toxicity. Therefore, we planned to introduce
other bulky groups at the 6-position of purine ring. We
describe herein the synthesis of various derivatives with
arylthio groups at the 6-position of 2-amino-9-[2-(phos-
phonomethoxy)ethyl]purine. To increase oral bioavail-
ability, the phosphonates of the relevant analogues were
protected with the bis(2,2,2-trifluoroethyl) group, as is
done with nucleoside phosphate prodrugs.11 The present
Hepatitis B virus (HBV) is the causative agent of both
acute and chronic hepatitis B infections. Treatment of
HBV infection constitutes one of the current therapeutic
challenges in virology. The number of chronic carriers
is estimated to be more than 400 million worldwide,
with roughly 4 million deaths annually from the result-
ing cirrhosis and hepatocellular carcinoma. Only a few
drugs are currently available for the clinical treatment
of hepatitis B. Interferon R appears effective but only
in 10-30% of treated patients.1,2 Another approved
drug, lamivudine,3,4 the 5′-triphosphate derivative of
which inhibits HBV DNA polymerase, has the problem
of inducing drug resistance. Several other nucleoside
* To whom correspondence should be addressed. Phone: +81-
45-963-3371. Fax: +81-45-963-3966. E-mail: Ubasawa.Masaru@
me.m-pharm.co.jp.
10.1021/jm020036x CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/04/2002

HBV-Specific Antiviral Reagents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3139
Ta ble 1. Anti-HBV Activity and Cytotoxicity of 6-Arylthio
Analogues in Vitro
Sch em e 1
a
b
compd
R
IC50 (µM)
CC50 (µM)
8
9
SPh
SPh(4-Me)
SPh(3-Me)
SPh(2-Me)
SPh(4-Et)
SPh(4-iPr)
SPh(4-NO2)
SPh(4-Cl)
SPh(4-OMe)
SPh(3-OMe)
SPh(2-OMe)
SPh(4-OEt)
SPh(4-O-nPr)
SPh(4-O-iPr)
SPh(4-O-nBu)
SPh(4-O-iBu)
SPh(4-O-CF3)
S-(2-naphthyl)
0.05
0.06
0.09
0.08
0.4
>1000
110
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
150
340
170
170
380
62
0.98
0.52
0.12
0.03
0.04
0.08
0.64
0.87
0.29
>1.0
0.84
0.06
0.09
0.2
>1000
>1000
>1000
>1000
250
350
260
100
820
198
PMEA
lamivudine
>1000
>1000
2.2
a
Concentrations of compounds achieving 50% inhibition of
b
cytoplasmic HBV-DNA synthesis. Concentrations of compounds
required for 50% extinction of HuH-6 cells.
marized in Table 1. 6-Phenylthio analogue 8 and
6-(methoxyphenylthio) analogues 16-18 showed high
anti-HBV activity and low cytotoxicity. Compound 16
with the highest potency showed about a 10-fold higher
efficacy than PMEA, as represented by the IC50 values
listed in Table 1. The compounds with bulky alkoxy
groups on their phenyl rings, 20-23, showed reduced
anti-HBV activity. The compounds with alkyl groups on
their phenyl rings, 9-11, as well as the 6-(2-naphthyl-
thio) analogue 25, showed high anti-HBV activity but
also exhibited high cytotoxicity. The compounds with
more polar groups on their phenyl rings (14 and 15)
showed low anti-HBV activity.
paper also describes the results of assays on antiviral
activity of newly synthesized compounds and of a
preliminary study on gastrointestinal absorption of a
representative compound among the relevant analogues
in mice.
Resu lts a n d Discu ssion
Ch em istr y. 2-Chloroethyl methyl ether (1) was treated
with tris(2,2,2-trifluoroethyl)phosphine (2) to quantita-
tively afford bis(trifluoroethyl) (2-chloroethoxy)meth-
ylphosphonate (3). Compound 3 was converted to bis-
(trifluoroethyl) (2-iodoethoxy)methylphosphonate (4) with
sodium iodide. Subsequently, 2-amino-6-chloropurine (5)
was treated first with 1,8-diazabicyclo[5,4,0]undec-7-ene
(DBU) and then with compound 4. 2-Amino-6-chloro-9-
[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoro-
ethyl) ester (6) was obtained as the major product, and
2-amino-6-chloro-7-[2-(phosphonomethoxy)ethyl]pu-
rine bis(2,2,2-trifluoroethyl) ester (7) was obtained as
a minor product. 2-Amino-6-arylthio-9-[2-(phosphono-
methoxy)ethyl]purine bis(2,2,2-trifluoroethyl) esters (8-
25) were synthesized by treating compound 6 with
arylthiols, e.g., substituted/nonsubstituted benzenethiol
and naphthalenethiol, in the presence of triethylamine
(Scheme 1).
Subsequently, the 6-arylthio derivatives with high
anti-HBV activity (8, 16-18) were examined for anti-
HIV activity. None of these compounds showed signifi-
cant anti-HIV activity at dose levels of up to 13, 34, 10,
and 22 µM, respectively. Compound 8 showed only slight
anti-HSV-1 activity (IC50 ) 25 µM).
Ga str oin testin a l Absor p tion . Since the above-
mentioned analogues were designed as prodrugs to
allow absorption from the gastrointestinal tract, we
measured concentrations of metabolites in the plasma
and liver of mice given a single oral dose of 100 mg/kg
of compound 16, a representative compound among the
relevant analogues. Blood was collected and the liver
was removed at specified time points, and metabolites
of compound 16 were analyzed by HPLC. A partially
hydrolyzed monoester, 2-amino-6-(4-methoxyphenyl)-
thio-9-[2-(phosphonomethoxy)ethyl]purine 2,2,2-trifluoro-
ethyl ester (26), and a completely hydrolyzed phosphonic
acid, 2-amino-6-(4-methoxyphenyl)thio-9-[2-(phospho-
nomethoxy)ethyl]purine (27), were used as the reference
compounds. Since the main metabolite in the plasma
and liver of mice was monoester 26, which was also
active against HBV in vitro (IC50 ) 0.07 µM), the
concentrations in the plasma and liver of mice were
In Vitr o An tivir a l Assa y. The anti-HBV activity of
the compounds synthesized in the present study was
evaluated according to a modified method of the original
described by Ueda et al., in which HB611, HuH-6 cell
line, stably transfected with the HBV genome, was
used.12 Anti-HBV activity and cytotoxicity are sum-

3140 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Sekiya et al.
to the reaction mixture and subjected to 100 °C for 5 h. After
completion of the reaction, the mixture was cooled to room
temperature and concentrated to dryness. The residue was
dissolved in chloroform, absorbed on a silica gel column, and
eluted with chloroform containing 5% methanol to afford 6
(23.3 g, 49.4 mmoL, yield 56%). Arylthiol (30 mmoL) was added
to a DMF solution (68 mL) of triethylamine (2.1 mL, 15 mmoL)
and compound 6 (7.1 g, 15 mmoL), and the mixture was stirred
at 100 °C for 2 h. The reaction mixture was cooled to room
temperature and concentrated to dryness. The residue was
dissolved in chloroform, absorbed on a silica gel column, and
eluted with chloroform containing 5% methanol to afford the
desired product.
F igu r e 1. Concentrations of 26 in plasma (b) and in the liver
([) after oral administration of 16 to mice.
2-Am in o-6-p h en ylth io-9-[2-(p h osp h on om eth oxy)eth yl]-
p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (8): yield 61%; mp
1
105-106 °C (ethanol); H NMR (CDCl3) δ 3.91-3.95 (m, 4H,
OCH2P, NCH2CH2O), 4.25-4.29 (m, 2H, NCH2CH2O), 4.34-
4.41 (m, 4H, OCH2CF3), 4.77 (b, 2H, NH2), 7.41-7.45 (m, 3H,
Ph), 7.61-7.66 (m, 2H, Ph), 7.72 (s, 1H, 8-H). Anal. (C18H18
F6N5O4PS) C, H, N.
-
2-Am in o-6-(4-m eth ylph en ylth io)-9-[2-(ph osph on om eth -
oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (9): yield
71%; mp 92.5-93 °C (diisopropyl ether); H NMR (CDCl3) δ
2.40 (s, 3H, CH3), 3.89-3.96 (m, 4H, NCH2CH2O, NCH2CH2O),
4.26 (d, J ) 5.1 Hz, 2H, OCH2P), 4.39-4.47 (m, 4H, OCH2-
CF3), 4.79 (br, 2H, NH2), 7.23 (d, J ) 9.8 Hz, 2H, Ph), 7.31 (d,
J ) 9.8 Hz, 2H, Ph), 7.71 (s, 1H, 8-H). Anal. (C19H20F6N5O4-
PS) C, H, N.
2-Am in o-6-(3-m eth ylph en ylth io)-9-[2-(ph osph on om eth -
oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (10): yield
66%; mp 75.5-76.5 °C (diisopropyl ether); 1H NMR (CDCl3) δ
2.39 (s, 3H, CH3), 3.90-3.95 (m, 4H, OCH2P, NCH2CH2O),
4.24-4.29 (m, 2H, NCH2CH2O), 4.34-4.41 (m, 4H, OCH2CF3),
4.77 (b, 2H, NH2), 7.20-7.38 (m, 2H, Ph), 7.42-7.50 (m, 2H,
Ph), 7.72 (s, 1H, 8-H). Anal. (C19H20F6N5O4PS)) C, H, N.
2-Am in o-6-(2-m eth ylph en ylth io)-9-[2-(ph osph on om eth -
oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (11): yield
68%; mp 73-74 °C (diisopropyl ether); 1H NMR (CDCl3) δ 2.42
(s, 3H, CH3), 3.90-3.94 (m, 4H, OCH2P, NCH2CH2O), 4.24-
4.29 (m, 2H, NCH2CH2O), 4.31-4.41 (m, 4H, OCH2CF3), 4.74
(b, 2H, NH2), 7.24-7.40 (m, 3H, Ph), 7.62 (d, J ) 7.9 Hz, 1H,
Ph), 7.72 (s, 1H, 8-H). Anal. (C19H20F6N5O4PS) C, H, N.
1
measured on a time-course basis. The measurements,
shown in Figure 1, indicated that compound 16 was
absorbed from the gastrointestinal tract of mice after
oral administration and active metabolite 26 was de-
tected in the mouse plasma, with high concentrations
in the liver.
Con clu sion s
We synthesized a number of novel 2-amino-6-arylthio-
9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluo-
roethyl) esters. Among them, 6-phenylthio- and 6-(meth-
oxyphenyl)thio derivatives showed potent HBV-specific
antiviral activity in vitro, in contrast to PMEA and
PMEDAP, which have a broad spectrum of activity
against viruses. In a preliminary study, compound 16
was absorbed from the gastrointestinal tract when
administered orally to mice, and active metabolite 26
was highly detected in the liver. These data led us to
consider that some of the analogues examined in the
present study have properties that might be suitable
for hepatitis B chemotherapy.
2-Am in o-6-(4-eth ylp h en ylth io)-9-[2-(p h osp h on om eth -
oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (12): yield
1
72%; mp 82.5-83 °C (diisopropyl ether); H NMR (CDCl3) δ
1.28 (t, J ) 7.6 Hz, 3H, CH3CH2), 2.71 (t, J ) 7.6 Hz, 2H,
CH3CH2), 3.90-3.95 (m, 4H, OCH2P, NCH2CH2O), 4.24-4.29
(m, 2H, NCH2CH2O), 4.34-4.41 (m, 4H, OCH2CF3), 4.77 (b,
2H, NH2), 7.26 (d, J ) 8.1 Hz, 2H, Ph), 7.53 (d, J ) 8.1 Hz,
2H, Ph), 7.72 (s, 1H, 8-H). Anal. (C20H22F6N5O4PS) C, H, N.
Exp er im en ta l Section
Melting points were measured with a Yanagimoto micro-
1
melting-point meter and were not corrected. H NMR spectra
were recorded at 300 MHz on an ARX-300 or a DPX-300
Bruker NMR spectrometer using tetramethylsilane as an
internal standard; chemical shifts (δ) were recorded in parts
per million (ppm). Silica gel column chromatography was
conducted with Wakogel C-300. Lamivudine, extracted from
tablets of Epivir (Glaxo Wellcome), was purified. PMEA was
prepared according to a published method.13
Gen er a l Meth od s To Syn th esize 2-Am in o-6-a r ylth io-
9-[2-(p h osp h on om eth oxy)eth yl]p u r in e Bis(2,2,2-tr iflu o-
r oeth yl) Ester An a logu es. 2-Chloroethyl chloromethyl ether
(87 g, 670 mmoL) and tris(2,2,2-trifluoroethyl)phosphite (200
g, 610 mmoL) were allowed to react at 160 °C for 7 h to
quantitatively afford 3. Compound 3 (206 g, 0.6 moL) and
sodium iodide (270 g, 1.8 mol) were dissolved in methyl ethyl
ketone (2 L), and the resultant solution was heated under
reflux for 8 h. After completion of the reaction, the mixture
was cooled to room temperature and concentrated to dryness.
The residue, dissolved in a solution of chloroform/hexane (1:1
v/v), was absorbed on a silica gel column and was then eluted
with a solution of chloroform/hexane (hexane concentration
was gradually reduced from 50% to 0%) to quantitatively afford
4. Compound 5 (15.0 g, 88 mmoL) was suspended in DMF (360
mL) and treated with DBU (13.9 mL, 93 mmoL) at 80 °C for
1 h. Subsequently, compound 4 (41.6 g, 97 mmoL) was added
2-Am in o-6-(4-isop r op ylp h en ylt h io)-9-[2-(p h osp h on o-
m eth oxy)eth yl]pu r in e Bis(2,2,2-tr iflu or oeth yl) Ester (13):
yield 24%; mp 119.5-120.5 °C (diisopropyl ether); 1H NMR
(CDCl3) δ 1.29 (d, J ) 6.9 Hz, 6H, (CH3)2CH), 2.96 (septet,
J
) 6.9 Hz, 1H, (CH3)2CHO), 3.85-3.98 (m, 4H, OCH2P,
NCH2CH2O), 4.18-4.30 (m, 2H, NCH2CH2O), 4.34-4.44 (m,
4H, OCH2CF3), 4.79 (b, 2H, NH2), 7.28 (d, J ) 8.2 Hz, 2H,
Ph), 7.64 (d, J ) 8.2 Hz, 2H, Ph), 7.75 (s, 1H, 8-H). Anal.
(C21H24F6N5O4PS) C, H, N.
2-Am in o-6-(4-n itr op h en ylth io)-9-[2-(p h osp h on om eth -
oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (14): yield
1
34%; mp 121-122 °C (diisopropyl ether); H NMR (CDCl3) δ
3.91-3.96 (m, 4H, OCH2P, NCH2CH2O), 4.27-4.31 (m, 2H,
NCH2CH2O), 4.34-4.41 (m, 4H, OCH2CF3), 4.83 (b, 2H, NH2),
7.76 (s, 1H, 8-H), 7.80 (d, J ) 8.9 Hz, 2H, Ph), 8.24 (d, J ) 8.9
Hz, 2H, Ph). Anal. (C18H18F6N5O4PS‚1/2H2O) C, H, N.
2-Am in o-6-(4-ch lor op h en ylth io)-9-[2-(p h osp h on om eth -
oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (15): yield
1
53%; mp 126-127.5 °C (diisopropyl ether); H NMR (CDCl3)
δ 3.90-3.94 (m, 4H, OCH2P, NCH2CH2O), 4.24-4.28 (m, 2H,
NCH2CH2O), 4.31-4.44 (m, 4H, OCH2CF3), 4.77 (s, 2H, NH2),
7.39 (d, J ) 8.5 Hz, 2H, Ph), 7.56 (d, J ) 8.5 Hz, 2H, Ph),
7.76(s, 1H, 8-H). Anal. (C18H17ClF6N5O4PS) C, H, N.

HBV-Specific Antiviral Reagents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3141
2-Am in o-6-(4-m et h oxyp h en ylt h io)-9-[2-(p h osp h on o-
m eth oxy)eth yl]pu r in e Bis(2,2,2-tr iflu or oeth yl) Ester (16):
yield 71%; mp 93-95 °C (diisopropyl ether); 1H NMR (CDCl3)
δ 3.86 (s, 3H, OCH3), 3.90-3.94 (m, 4H, OCH2P, NCH2CH2O),
4.24-4.28 (m, 2H, NCH2CH2O), 4.34-4.41 (m, 4H, OCH2CF3),
4.75 (b, 2H, NH2), 6.95 (d, J ) 9.0 Hz, 2H, Ph), 7.53 (d, J )
9.0 Hz, 2H, Ph), 7.71 (s, 1H, 8-H). Anal. (C19H20F6N5O5PS) C,
H, N.
2-Am in o-6-(3-m et h oxyp h en ylt h io)-9-[2-(p h osp h on o-
m eth oxy)eth yl]pu r in e Bis(2,2,2-tr iflu or oeth yl) Ester (17):
yield 87%; mp 62-63.5 °C (diisopropyl ether); 1H NMR (CDCl3)
δ 3.82 (s, 3H, OCH3), 3.90-3.94 (m, 4H, OCH2P, NCH2CH2O),
4.25-4.29 (m, 2H, NCH2CH2O), 4.31-4.44 (m, 4H, OCH2CF3),
4.82 (b, 2H, NH2), 6.92-7.00 (m, 1H, Ph), 7.18-7.40 (m, 4H,
Ph), 7.72 (s, 1H, 8-H). Anal. (C19H20F6N5O5PS) C, H, N.
2-Am in o-6-(2-m et h oxyp h en ylt h io)-9-[2-(p h osp h on o-
m eth oxy)eth yl]pu r in e Bis(2,2,2-tr iflu or oeth yl) Ester (18):
yield 63% (foam); 1H NMR (CDCl3) δ 3.80 (s, 3H, OCH3), 3.89-
3.94 (m, 4H, OCH2P, NCH2CH2O), 4.23-4.27 (m, 2H, NCH2-
CH2O), 4.31-4.45 (m, 4H, OCH2CF3), 4.78 (b, 2H, NH2), 6.96-
7.04 (m, 2H, Ph), 7.43 (dd, J ) 7.7, 1.5 Hz, 1H, Ph), 7.59 (dd,
J ) 7.7, 1.5 Hz, 1H, Ph), 7.69 (s, 1H, 8-H). Anal. (C19H20F6N5O5-
PS) C, H, N.
2-Am in o-6-(4-eth oxyph en ylth io)-9-[2-(ph osph on om eth -
oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (19): yield
32%; mp 61-64 °C (diisopropyl ether); 1H NMR (CDCl3) δ 1.45
(t, J ) 7.0 Hz, 3H, CH3CH2O), 3.91-3.94 (m, 4H, OCH2P,
NCH2CH2O), 4.08 (q, J ) 7.0 Hz, 2H, CH3CH2O), 4.24-4.26
(m, 2H, NCH2CH2O), 4.34-4.41 (m, 4H, OCH2CF3), 4.76 (b,
2H, NH2), 6.94 (d, J ) 8.4 Hz, 2H, Ph), 7.52 (d, J ) 8.4 Hz,
2H, Ph), 7.71 (s, 1H, 8-H). Anal. (C20H22F6N5O5PS) C, H, N.
2-Am in o-6-(4-n -p r op oxyp h en ylt h io)-9-[2(p h osp h on o-
m eth oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester of
(20): yield 29% (foam); 1H NMR (CDCl3) δ 1.05 (t, J ) 7.4 Hz,
3H, OCH2CH2CH3), 1.84 (tq, J ) 6.8, 7.4 Hz, 2H, OCH2CH2-
CH3), 3.82-4.00 (m, 6H, OCH2CH2CH3, OCH2P, NCH2CH2O),
4.24-4.31 (m, 2H, NCH2CH2O), 4.34-4.44 (m, 4H, OCH2CF3),
4.78 (b, 2H, NH2), 6.95 (d, J ) 8.7 Hz, 2H, Ph), 7.51 (d, J )
8.7 Hz, 2H, Ph), 7.71 (s, 1H, 8-H). Anal. (C21H24F6N5O5PS) C,
H, N.
(b, 2H, NH2), 7.27 (d, J ) 8.7 Hz, 2H, Ph), 7.66 (d, J ) 8.7 Hz,
2H, Ph), 7.73 (s, 1H, 8-H). Anal. (C19H17F9N5O5PS) C, H, N.
2-Am in o-6-(2-n a p h th yllth io)-9-[2-(p h osp h on om eth ox-
y)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester (25): yield
1
71%; mp 100-101 °C (diisopropyl ether); H NMR (CDCl3) δ
3.91-4.00 (m, 4H, OCH2P, NCH2CH2O), 4.25-4.32 (m, 2H,
NCH2CH2O), 4.34-4.50 (m, 4H, OCH2CF3), 4.73 (b, 2H, NH2),
7.50-7.62 (m, 2H, Ar), 7.62-7.71 (m, 1H, Ph), 7.74 (s, 1H,
8-H), 7.82-7.94 (m, 3H, Ar), 8.16 (s, 1H, Ar). Anal. (C22H20
F6N5O4PS) C, H, N.
-
P r ep a r a tion of 2-Am in o-6-(4-m eth oxyp h en ylth io)-9-[2-
(p h osp h on om eth oxy)eth yl]p u r in e 2,2,2-Tr iflu or oeth yl
Ester Sod iu m Sa lt (26). Compound 16 (5.754 g, 10 mmoL)
was dissolved in THF (30 mL). A 1 N NaOH aqueous solution
(9.9 mL) was added to this solution, and the mixture was left
at ambient temperature for 16 h. After THF was removed by
evaporation, the residue was diluted with water (50 mL) and
the solution was extracted with ethyl ether (30 mL, three
times) to remove any remaining compound 16. The water layer
was freeze-dried to afford the desired material: yield 5.04 g
(9.8 mmoL, 98%); 1H NMR (CDCl3) δ 3.30-3.39 (m, 2H,
OCH2P), 3.75-3.80 (m, 2H, NCH2CH2O), 3.80 (s, 3H, OCH3),
4.01-4.07 (m, 2H, OCH2CF3), 4.09-4.18 (m, 2H, NCH2CH2O),
6.23 (b, 2H, NH2), 7.01 (d, J ) 8.70 Hz, 2H, Ph), 7.50 (d, J )
8.7 Hz, 2H, Ph), 8.01 (s, 1H, 8-H). Anal. (C17H18F3N5NaO5PS‚
H2O) C, N. H calcd, 3.78; found, 3.31.
P r ep a r a tion of 2-Am in o-6-(4-m eth oxyp h en ylth io)-9-[2-
(p h osp h on om eth oxy)eth yl]p u r in e (27). 2-Amino-6-chloro-
9-[2-(phosphonomethoxy)ethyl]purine (2.1 g, 6.83 mmoL),
which was obtained according to a published method,14 was
dissolved in DMF (30 mL). 4-Methoxybenzenethiol (1 mL, 8.2
mmoL) and pyridine (2.7 mL, 32.8 mmoL) were added to this
solution, and the solution was heated at 80 °C for 3 h. The
solvent was removed by evaporation under reduced pressure.
The residue was dissolved in water (10 mL), and acetone (60
mL) was added to afford crystals of the desired compound:
yield 1.68 g (4.1 mmoL, 60%); mp 228-231.5 °C; 1H NMR (Me2-
SO-d6) δ 3.19 (d, 2H, J ) 8.70 Hz, OCH2P), 3.80 (s, 3H, OCH3),
3.80-3.85 (m, 2H, NCH2CH2O), 4.17-4.21 (m, 2H, NCH2-
CH2O), 7.02 (d, J ) 8.4 Hz, 2H, Ph), 7.50 (d, J ) 8.4 Hz, 2H,
Ph), 7.95 (s, 1 H, 8-H). Anal. (C15H18N5O5PS) C, H, N.
P r oced u r e To Assess An ti-HBV Activity. The anti-HBV
activity of the above-mentioned compounds was evaluated
according to a modified method of the original reported by K.
Ueda et al.12 HB611, an HBV-producing cell line, was main-
tained in Dulbecco’s modified eagle’s medium (DMEM) that
was supplemented with 10% fetal bovine serum, 100 µg/mL
streptomycin, 100 IU/mL penicillin, and 0.2 mg/mL Geneteicin
(Life Technologies) at 37 °C in the presence of 5% CO2. HB611
cells (2 × 104 cells/well) were cultured in a 24-well plate, and
the medium was replaced with fresh medium on days 2 and 5
of culture. On day 8 of culture, the medium was replaced with
another medium containing a test compound at final concen-
trations of 0.001-10 µM. Cells in three wells were similarly
treated with a test compound of the same concentration. The
cells were cultured for 9 more days, and then all DNA was
collected from the cells. The amounts of HBV replication
intermediates were determined by Southern blot analysis. The
mean amount of HBV replication intermediates at each
concentration was calculated, and the 50% inhibitory concen-
tration (IC50) of the test compound was determined. The
experiment was repeated at least twice in the same manner.
The 50% cytotoxic concentration (CC50) of each compound was
determined in HuH-6 cell line, the original for HB611 cell line.
HuH-6 cells, applied to a 96-well plate at a density of 1 × 105
cells/mL, were cultured in DMEM, which was supplemented
with 10% fetal bovine serum and each test compound, at 37
°C for 3 days in the presence of 5% CO2. At the end of the
culture, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphen-
yl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added to the
medium, and the plate was further incubated at 37 °C for 2 h.
To determine cell viability, the absorbance, at 490 nm, of each
well was measured with a microplate reader (NJ -2000: IN-
TERMED).
2-Am in o-6-(4-isop r op oxyp h en ylt h io)-9-[2-(p h osp h o-
n om eth oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Ester
1
(21): yield 51%; mp 68.5-70 °C (diisopropyl ether); H NMR
(CDCl3) δ 1.37 (d, J ) 6.0 Hz, 6H, (CH3)2CHO), 3.85-3.98 (m,
4H, OCH2P, NCH2CH2O), 4.18-4.30 (m, 2H, NCH2CH2O),
4.34-4.44 (m, 4H, OCH2CF3), 4.60 (septet, J ) 6.0 Hz, 1H,
(CH3)2CHO), 4.77 (b, 2H, NH2), 6.93 (d, J ) 8.7 Hz, 2H, Ph),
7.51 (d, J ) 8.7 Hz, 2H, Ph), 7.71 (s, 1H, 8-H). Anal.
(C21H24F6N5O5PS) C, H, N.
2-Am in o-6-(4-n -b u t oxyp h en ylt h io)-9-[2-(p h osp h on o-
m eth oxy)eth yl]pu r in e Bis(2,2,2-tr iflu or oeth yl) Ester (22):
yield 80%; mp 89-92 °C (diisopropyl ether); 1H NMR (CDCl3)
δ 0.99 (t, J ) 7.5 Hz, 3H, OCH2CH2CH2CH3), 1.51 (tq, J )
8.1, 7.5 Hz, 2H, OCH2CH2CH2CH3), 1.79 (tt, J ) 6.4, 8.1 Hz,
2H, OCH2CH2CH2CH3), 3.84-3.98 (m, 4H, OCH2P, NCH2CH2-
O), 4.00 (t, J ) 6.4 Hz, 2H, OCH2CH2CH2CH3), 4.20-4.28 (m,
2H, NCH2CH2O), 4.34-4.40 (m, 4H, OCH2CF3), 4.76 (b, 2H,
NH2), 6.94 (d, J ) 8.8 Hz, 2H, Ph), 7.52 (d, J ) 8.8 Hz, 2H,
Ph), 7.71 (s, 1H, 8-H). Anal. (C22H26F6N5O5PS) C, H, N.
2-Am in o-6-(4-isob u t oxyp h en ylt h io)-9-[2-(p h osp h on o-
m eth oxy)eth yl]pu r in e Bis(2,2,2-tr iflu or oeth yl) Ester (23):
yield 69%; mp 92.5-93 °C (diisopropyl ether); 1H NMR (CDCl3)
δ 1.05 (d, J ) 6.8 Hz, 6H, (CH3)2CHCH2O), 2.14 (d and septet,
J ) 6.5 and 6.8 Hz, 1H, (CH3)2CH CH2O), 3.76 (d, J ) 6.5 Hz,
2H, (CH3)2CH CH2O), 3.85-3.98 (m, 4H, OCH2P, NCH2CH2O),
4.18-4.30 (m, 2H, NCH2CH2O), 4.34-4.44 (m, 4H, OCH2CF3),
4.77 (b, 2H, NH2), 6.94 (d, J ) 8.7 Hz, 2H, Ph), 7.51 (d, J )
8.7 Hz, 2H, Ph), 7.71 (s, 1H, 8-H). Anal. (C22H26F6N5O5PS) C,
H, N.
2-Am in o-6-(4-tr iflu or om eth oxyp h en ylth io)-9-[2(p h os-
p h on om eth oxy)eth yl]p u r in e Bis(2,2,2-tr iflu or oeth yl) Es-
1
ter (24): yield 25%; mp 127-127.5 °C (diisopropyl ether); H
NMR (CDCl3) δ 3.86-3.98 (m, 4H, OCH2P, NCH2CH2O), 4.22-
4.30 (m, 2H, NCH2CH2O), 4.34-4.44 (m, 4H, OCH2CF3), 4.78

3142 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Sekiya et al.
P r oced u r e for An ti-HIV-1 Assa y. MT-4 cells (1 × 105
cells/mL) were infected with HIV-1 (HTLV-IIIB strain) at a
multiplicity of infection (MOI) of 0.02. The cells were cultured
with various concentrations of test compounds; 3′-azido-3′-
deoxythymidine (AZT) was used as a control. After a 4-day
incubation at 37 °C, the number of viable cells was counted
according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) method, and the inhibitory con-
centration at which cell viability is inhibited by 50% (IC50) was
determined. The experiment was repeated twice in the same
manner. The concentration required for 50% extinction of MT-4
cells (CC50) was also determined.
P r oced u r e for An ti-HSV-1 Assa y. RPMI8226 cells (3 ×
104 cells) were infected with HSV-1. The cells were cultured
with various concentrations of test compounds; aciclovir was
used as a control. After a 4-day incubation at 37 °C, the
number of viable cells was counted according to the MTT
method, and the inhibitory concentration at which cell viability
is inhibited by 40% (IC40) was determined. The concentration
required for 50% extinction of RPMI8226 cells (CC50) was also
determined.
(2) Thomas, H. C. Treatment of hepatitis B viral infection. In Viral
Hepatitis and Liver Disease; Zuckerman, A. J ., Ed.; Alan R. Liss,
Inc.: New York, 1988; pp 817-822.
(3) Dienstag, J . L.; Schiff, E. R.; Wright, T. L.; Perrillo, R. P.; Hann,
H. L.; Goodman, Z.; Crowther, L.; Condreay, L. D.; Woessner,
M.; Rubin, M.; Brown, N. A. Lamivudine as initial treatment
for chronic hepatitis B in the United States. N. Engl. J . Med.
1999, 341, 1256-1263.
(4) Lai, C. L.; Chien, R. N.; Leung, N. W. Y.; Chang, T. T.; Guan,
R.; Tai, D. I.; Ng, K. Y.; Wu, P. C.; Dent, J . C.; Barber, J .;
Stephenson, S. L.; Gray, D. F. A One-Year Trial of Lamivudine
for Chronic Hepatitis B. N. Engl. J . Med. 1998, 339, 61-68.
(5) Pauwels, R.; Balzarini, J .; Schols, D.; Baba, M.; Desmyter, J .;
Rosenberg, I.; Holy, A.; De Clercq, E. Phsphonylmethoxyethyl
purine derivatives, a new class of anti-human immunodeficiency
virus agents. Antimicrob. Agents Chemother. 1988, 32, 1025-
1030.
(6) De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J .;
Rosenberg, I.; Holy, A. Antiviral activity of phosphonylmethoxy-
alkyl derivatives of purines and pyrimidines. Antiviral Res. 1987,
8, 261-272.
(7) Yokota, T.; Konno, K.; Shigeta, S.; Holy, A.; Balzarini, J .; De
Clercq, E. Inhibitory effects of acyclic nucleoside phosphonate
analogues on hepatitis B virus DNA synthesis in HB611 cells.
Antiviral Chem. Chemother. 1994, 5, 57-63.
Estim a tion of Ga str oin testin a l Absor p tion in Mice.
Compound 16 was administered by oral gavage to male C57B/
6J mice as a single 100 mg/kg dose in 10 mL of 0.5%
tragacanth in water. Blood, collected from two mice at given
time points, was centrifuged (10000g for 3 min) to obtain
plasma. Acetonitrile (0.1 mL) was added to the plasma (0.05
mL), the mixture was centrifuged (10000g for 3 min), and 0.05
mL of the supernatant was analyzed by HPLC. The liver was
removed from two mice previously given compound 16 in the
same manner as described above. Each liver was immersed
in 2-fold (W/V) saline and was then homogenized with a
homogenizer (Hitachi). Acetonitrile (0.1 mL) was added to the
homogenate (0.05 mL), and the mixture was centrifuged
(10000g for 3 min), and then 0.05 mL of the supernatant was
analyzed by HPLC. HPLC was conducted using a D-7000
system (Hitachi) equipped with a Capcellpack C18 UD column
(Shiseido, 4.6 mm × 250 mm). The detector wavelength, flow
rate, and column temperature were set at 311 nm, 0.8 mL/
min, and 30 °C, respectively. The mobile phase consisted of
35% acetonitrile in water containing 1.25 mM Pic A reagent
(Waters).
(8) Starrett, J . E., J r.; Tortolani, D. R.; Russell, J .; Hitchcock, M. J .
M.; Whiterock, V.; Martin, J . C.; Mansuri, M. M. Synthesis, oral
bioavailability determination, and in vitro evaluation of prodrugs
of the antiviral agent 9-[2-(phosphonomethoxy)ethyl]adenine
(PMEA). J . Med. Chem. 1994, 37, 1857-1864.
(9) Naesens, L.; Balzarini, J .; Bischofberger, N.; De Clercq, E.
Antiretroviral activity and pharmacokinetics in mice of oral bis-
(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine, the
bis(pivaloyloxymethyl) ester prodrug of 9-(2-phosphonylmeth-
oxyethyl)adenine. Antimicrob. Agents Chemother. 1996, 40, 22-
28.
(10) Ubasawa, M.; Takashima, H.; Sekiya, K.; Inoue, N.; Yuasa, S.;
Kamiya, N. Preparation of phosphonate acyclic nucleotide
derivatives as antiviral agent. Patent PCT Int. Appl. WO
9700262, 1997; Chem. Abstr. 1997, 126, 131752. During our
research, Holy et al. reported antiviral activity on the same type
of compounds but no data on antiviral activity against HBV.
Holy, A.; Zidek, Z.; Votruba, I. Inhibition of murine lymphocyte
proliferation by N6-substituted acyclic purine nucleoside phos-
phonates. Collect. Czech. Chem. Commun. 1996, 61 (Special
Issue), S182-S187.
(11) McGuigan, C.; O’Connor, T. J .; Nicholls, S. R.; Nickson, C.;
Kinchington, D. Synthesis and anti-HIV activity of some novel
substituted dialkyl phosphate derivatives of AZT and ddCyd.
Antiviral Chem. Chemother. 1990, 1, 355-360.
(12) Ueda, K.; Tsurimoto, T.; Nagahata, T.; Chisaka, O.; Matsubara,
K. An in vitro system for screening anti-hepatitis B virus drugs.
Virology 1989, 169, 213-216.
(13) Holy´, A.; Rosenberg, I. Synthesis of 9-(2-phosphonylmethoxy-
ethyl)adenine and related compounds. Collect. Czech. Chem.
Commun. 1987, 52, 2801-2809.
(14) Holy´, A.; Gu¨nter, J .; Dvora´kova´, H.; Masoj´ıdkova´, M.; Andrei,
G.; Snoeck, R.; Balzarini, J .; De Clercq, E. Structure-antiviral
activity relationship in the series of pyrimidine and purine N-[2-
(2-phosphonomethoxy)ethyl] nucleotide anlogues. 1. Derivatives
substituted at the carbon atoms of the base. J . Med. Chem. 1999,
42, 2064-2086.
Ack n ow led gm en t. We thank Dr. Kenichi Matsu-
bara, Professor of Nara Institute of Science and Tech-
nology, for providing HB611 cells, Dr. Shiro Shigeta,
Professor of Fukushima Medical College, for anti-HSV-1
assay, and Dr. Masanori Baba, Professor of Kagoshima
University, for anti-HIV-1 assay.
Refer en ces
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Realdi, G. Long-term follow-up of anti-HBe-positive chronic
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