Synthesis and evaluation of 2-amino-9-(3-hydroxymethyl-4- alkoxycarbonyloxybut-1-yl)purines as potential prodrugs of penciclovir

Article abstract of DOI:10.1021/jm980138g

A series of 2-amino-9-(3-hydroxymethyl-4-alkoxycarbonyloxybut-1- yl)purines (4-10) and 2-amino-9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (1) were synthesized as potential prodrugs of penciclovir and evaluated for their oral penciclovir bioavailability in m

Full text of DOI:10.1021/jm980138g

J . Med. Chem. 1998, 41, 3435-3441
3435
Syn th esis a n d Eva lu a tion of
2-Am in o-9-(3-h yd r oxym eth yl-4-a lk oxyca r bon yloxybu t-1-yl)p u r in es a s P oten tia l
P r od r u gs of P en ciclovir
Dae-Kee Kim,* Namkyu Lee, Young-Woo Kim, Kieyoung Chang, J ae-Sun Kim, Guang-J in Im, Won-Son Choi,
Inho J ung, Taek-Soo Kim, Yong-Youn Hwang, Dong-Sun Min, Key An Um, Yong-Baik Cho, and Key H. Kim
Life Science Research Center, SK Chemicals, 600 J ungja-Dong, Changan-Ku, Suwon-Si, Kyungki-Do 440-745, Korea
Received March 5, 1998
A series of 2-amino-9-(3-hydroxymethyl-4-alkoxycarbonyloxybut-1-yl)purines (4-10) and 2-amino-
9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (1) were synthesized as potential prodrugs of penciclovir
and evaluated for their oral penciclovir bioavailability in mice and rats. Treatment of 2-(2-
benzyloxyethyl)propane-1,3-diol (11) with 1,1′-carbonyldiimidazole in THF followed by hydro-
genolytic removal of the benzyl group of the resulting cyclic carbonate 12 gave 5-(2-
hydroxyethyl)-1,3-dioxan-2-one (13). Mesylation of the alcohol 13 and then a coupling reaction
of the resulting mesylate 14 with 2-amino-6-chloropurine using anhydrous Cs2CO3 in DMF
afforded 2-amino-6-chloro-9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (16) after purification by flash
column chromatography on silica gel using EtOAc/MeCN/Et3N as eluent. Hydrogenation of
the 6-chloro cyclic carbonate 16 followed by a ring-opening reaction of the 6-deoxy cyclic
carbonate 1 in a mixture of an appropriate alcohol and CHCl3 using activated SiO2 as a Lewis
acid afforded the corresponding alkyl monocarbonate derivatives 3-10 in fair to good yields.
Of the prodrugs tested in mice, the isopropyl monocarbonate 6 achieved the highest mean
urinary recovery of penciclovir (53%), followed in order by the propyl monocarbonate 5 (51%),
the isopentyl monocarbonate 10 (51%), the ethyl monocarbonate 4 (50%), and famciclovir (48%).
In rats, the methyl monocarbonate 3, 4, 6, the n-butyl monocarbonate 7, and 10 (39-41%)
showed levels of mean urinary recovery of penciclovir similar to that from famciclovir (40%).
The alkyl monocarbonates 4-10 were found to be quite stable in the aqueous buffer solutions,
and among them, 6 was the most stable with the half-lives (t1/2) of 88, >200, 61, and 26 days
at pH 1.2, 6.0, 7.4, and 8.0, respectively. In addition, 6 was highly soluble in H2O (138.8 mg/
mL, 20 °C).
In tr od u ction
converted to penciclovir by the enzymatic removal of two
O-acetyl groups, followed by oxidation at the 6-position
of the purine ring by xanthine oxidase in mice,12 rats,13
and humans.14 Famciclovir has recently been approved
by FDA for the treatment of herpes zoster (shingles) and
acute recurrent genital herpes. Famciclovir has been
reported to inhibit DHBV replication in chronically
infected ducks,15 to control HBV replication effectively
in liver transplant patients,16,17 and to inhibit HBV
replication in a double-blind, placebo-controlled, single-
center clinical trial in patients with chronic HBV
infection.18 A large, multicenter trial of famciclovir
against chronic HBV infection is currently in progress.
An acyclonucleoside 9-(4-hydroxy-3-hydroxymethyl-
but-1-yl)guanine (penciclovir) is a potent and highly
selective inhibitor of the replication of herpesviruses
including herpes simplex virus types 1 and 2 (HSV-1
and HSV-2), varicella-zoster virus (VZV), and Epstein-
Barr virus (EBV) in cell cultures and in animals,1-4 and
of hepatitis B virus (HBV) and duck hepatitis B virus
(DHBV) in cell cultures.5,6 The antiviral spectrum of
penciclovir against human herpesviruses is similar to
that of 9-(2-hydroxyethoxymethyl)guanine (acyclovir),
and both compounds have comparable activity against
these viruses.7 The advantage of penciclovir over acy-
clovir is that its antiviral activity in cell culture is more
pesistent than that of acyclovir since penciclovir tri-
phosphate is much more stable than acyclovir triphos-
phate within infected cells.2,8
However, like other acyclic nucleoside analogues such
as acyclovir,9 ganciclovir,10 and buciclovir,11 penciclovir
has poor oral bioavailability in mice and rats.12,13 To
overcome this inadequate oral bioavailability, 2-amino-
9-(4-acetoxy-3-acetoxymethylbut-1-yl)purine (famciclo-
vir), the diacetyl 6-deoxy analogue of penciclovir, has
been developed as a prodrug of penciclovir.12 Famci-
clovir is orally well absorbed and then extensively
In a previous report, Harnden et al. attempted to
synthesize the cyclic carbonate 1 as a potential prodrug
of penciclovir.12 However, treatment of 6-deoxypenci-
clovir 2 with tetramethyl orthocarbonate gave the
methyl monocarbonate 3 in a low yield (22%) instead of
the desired cyclic carbonate 1.12 When 3 was evaluated
for its bioavailability in mice, it achieved maximum
concentration of penciclovir in the blood that was
comparatively lower (73%) than that from famciclovir,
but 11 times higher than that from penciclovir when
assayed after administration of the equivalent oral doses
of each compound.12 On the basis of these findings, it
was of particular interest to us to determine whether
introduction of a higher alkyl carbonate group rather
* Author to whom correspondence should be addressed.
S0022-2623(98)00138-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/07/1998

3436 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Kim et al.
Sch em e 1a
than that of a methyl carbonate group into one of the
two hydroxyl groups of 2 could increase its oral bio-
availability. These alkyl monocarbonates are expected
to be more efficiently converted to penciclovir as com-
pared with famciclovir after gastrointestinal absorption
since they have only one carbonate group to be hydro-
lyzed in an acyclic moiety of the molecule.
a
(a) 1,1′-carbonyldiimidazole, THF, reflux, 8 h; (b) H2 (1 atm),
10% Pd/C, THF, room temperature, 3 h; (c) MsCl, Et3N, CH2Cl2,
0 °C, 1 h; (d) 2-amino-6-chloropurine, Cs2CO3, DMF, room tem-
perature, 16 h; (e) H2 (1 atm), 10% Pd/C, Et3N, MeCN/DMF, room
temperature, 3 h; (f) ROH, SiO2, CHCl3, 70 °C, 4 h (for 3-5 and
7-10) or 24 h (for 6).
In this report, we describe the synthesis of the cyclic
carbonate 1 and the C2-C5 alkyl monocarbonates 4-10
as potential prodrugs of penciclovir and their physical
properties and oral bioavailability in mice and rats.
various reaction conditions. We performed several
reactions on a small scale using 1,1′-carbonyldiimidazole
or triphosgene with 2,6-lutidine as a base in THF or
benzene as solvents. Both reagents (1,1′-carbonyldi-
imidazole and triphosgene) showed comparable results;
fairly good yields of 80% and 85%, respectively, were
obtained in THF, and lower yields of 64% and 59%,
respectively, were obtained in benzene. However, the
large-scale reaction with 1,1′-carbonyldiimidazole was
more reproducible in yields (79-80%) than that with
triphosgene (61%). Therefore, 1,1′-carbonyldiimidazole
was selected as the choice of reagent for the carbony-
lation of the diol 11, and the cyclic carbonate 12 was
prepared in 79% yield. Removal of the benzyl group in
the cyclic carbonate 12 proceeded smoothly under hy-
drogenolytic condition (1 atm of H2, THF, room tem-
perature) to give the corresponding alcohol 13 in almost
quantitative yield. Treatment of the alcohol 13 with
methanesulfonyl chloride (MsCl) using Et3N as a base
in CH2Cl2 at 0 °C afforded the mesylate 14 in a good
yield of 84%. With the mesylate 14 in hand, the
coupling reaction of 2-amino-6-chloropurine with the
mesylate 14 using anhydrous cesium carbonate (Cs2-
CO3) in DMF was first carried out to see if the cyclic
carbonate group would survive under the basic reaction
conditions. Gratifyingly, this reaction worked well (TLC
analysis), and the methyl monocarbonate 15, instead
of the desired cyclic carbonate 16, was isolated in 50%
Ch em istr y
First, we have investigated the possibility of convert-
ing the diol 2 into the desired monocarbonate deriva-
tives, which seemed to be the most obvious approach
since the starting 2 can be readily prepared by using
well-documented procedures.1,12 Several reactions of 2
with ethyl chloroformate (1 equiv) were carried out by
using an amine base (Et3N or 2,6-lutidine) in DMF
solvent or using pyridine as a base and solvent. These
reactions were rather complex and unfruitful. Thus it
was conceived that the carbonate group must be intro-
duced to the acyclic portion prior to being coupled with
the purine moiety. Cyclic carbonate was chosen because
it can act as a protecting group for the diol functionality,
and it can also undergo ring-opening reactions to afford
the target monocarbonate compounds.
As shown in Scheme 1, the synthesis of the target
carbonate compounds 1 and 4-10 began with the
known diol 1119 which was prepared from diethyl
malonate and O-benzyl-2-bromoethanol using a known
procedure. It has been reported that the formation of
cyclic carbonate from a diol can be easily accomplished
by using 1,1′-carbonyldiimidazole or triphosgene under

Potential Prodrugs of Penciclovir
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3437
Ta ble 1. Solubility and Stability in Aqueous Solution, In Vitro Anti-HSV-1 Activity, and Oral Bioavailability of
2-Amino-9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (1) and 2-Amino-9-(3-hydroxymethyl-4-alkoxycarbonyloxybut-1-yl)purines 3-10
half-life (day, 37 °C)b
urinary recovery of
penciclovirg (% dose)h
solubilitya
(mg/mL, 20 °C)
pH
pH
pH
pH
EC50 (µM)e
HSV-1 (KOS)
CC50 (µM)f
compd
R
1.2c
6.0d
7.4d
8.0d
Vero
mouse
rat
1
4
5
6
7
8
9
10
3
9.9
20.0
68.0
138.8
15.0
1.5
4.2
5.6
14.1
0
35
29
88
29
28
25
24
23
2.6
112
89
0.12
7
10
61
11
17
7
11
1
0.07
3
4
26
5
8
5
6
0.5
>125i
>125
>125
>125
>125
>125
>125
>125
>125
>125
8.65
>500i
>500
>500
>500
>500
>500
>500
357
27
50
51
53
38
38
47
51
45
48
3
15
40
34
40
41
37
38
40
39
40
9
Et
n-Pr
i-Pr
n-Bu
>200
74
87
17
65
i-Bu
n-pentyl
isopentyl
Me
18
>500
>500
>500
famciclovir
penciclovir
a
Determined by the comparison of the UV absorbance of the saturated solution of each compound at 290 nm with that of the standard
curve. Determined by HPLC using a C18 reversed-phase column. c HCl/NaCl buffer. Sodium phosphate buffer. e Concentration required
b
d
to reduce the OD value by 50% of the virus-infected control. f Concentration required to reduce the OD value by 50% of the control.
A
g
single oral dose of the test compound (0.2 mmol/kg) was administered to six male ICR mice or to six male Sprague-Dawley rats. The total
h
amount of penciclovir recovered in the urine over a 48-h period was determined by HPLC using a C18 reversed-phase column. Mean
i
values of six animals. Mean values of two independent experiments run in quadruplicate.
yield after flash column chromatography on silica gel
using 10% MeOH in CHCl3. This unexpected product
could have resulted from the ring opening of the desired
cyclic carbonate product 16 by MeOH of the eluent. This
accidental opening of the cyclic carbonate 16 could be
avoided by using a different solvent system as eluent.
Thus, the alkylation of 2-amino-6-chloropurine with
mesylate 14 in DMF at room temperature using anhy-
drous Cs2CO3 and purification by flash column chro-
matography on silica gel using EtOAc/MeCN/Et3N as
eluent afforded the desired cyclic carbonate product 16
in 48% yield. It occurred to us that the cyclic carbonate
group in 16 might be opened up to produce a variety of
monocarbonate derivatives directly by using an ap-
propriate alcohol and activated silica gel as a mild Lewis
acid since the 6-chloro cyclic carbonate 16 was quanti-
tatively converted to the methyl monocarbonate 15
reaction proceeded fairly well even with i-PrOH at 70
°C although a small amount of 6-chloropenciclovir 17
was also formed. On the basis of this promising result,
we felt confident it would be feasible to prepare all the
target monocarbonate compounds in a single step by
opening a common intermediate, 6-deoxy cyclic carbon-
ate 1 with an appropriate alcohol under the previously
mentioned reaction condition. As a first step, the chloro
atom in the compound 16 was removed by hydrogena-
tion (1 atm of H2, Et3N, MeCN/DMF, room temperature)
to afford the 6-deoxy product 1 in 90% yield. Ring-
opening reactions of the 6-deoxy cyclic carbonate 1 were
performed in a 2:1 mixture of an appropriate alcohol
(MeOH, EtOH, n-PrOH, i-PrOH, n-BuOH, i-BuOH,
n-pentanol, and isopentanol) and CHCl3 (free from
EtOH stabilizer) at 70 °C using activated silica gel (10-
fold excess by weight, activated by heating overnight
at 80 °C under vacuum), and the corresponding mono-
carbonate derivatives 3-10 were obtained as racemates
in fair to good yields (64-89%). It should be pointed
out that the activated silica gel is essential for the ring
opening since even the reaction with MeOH under
otherwise identical conditions did not proceed at all
without activated silica gel.
Resu lts a n d Discu ssion
The in vitro antiviral activity and cytotoxicity of the
prodrugs 1 and 4-10 along with the methyl monocar-
bonate 3, famciclovir, and penciclovir against HSV-1
(KOS strain) in Vero cells were evaluated (Table 1). As
was expected, all the prodrugs showed no significant
antiviral activity at concentrations up to 125 µM, while
penciclovir was active against HSV-1 replication with
an EC50 value of 8.65 µM. With the exception of the
while it was being purified by flash column chromatog-
raphy on silica gel using 10% MeOH in CHCl3. This
approach was tested with the 6-chloro cyclic carbonate
16 on a small scale using activated silica gel in a 2:1
mixture of an alcohol (MeOH, EtOH, i-PrOH, and
isopentanol) and CHCl3 (a spectroscopic grade free from
EtOH stabilizer), and TLC analysis revealed that the

3438 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Kim et al.
isopentyl monocarbonate 10 (CC50 ) 357 µM), none of
the prodrugs showed cytotoxicity to Vero cells even at
the maximum concentration of 500 µM. We evaluated
the bioavailability of penciclovir after a single oral
administration (0.2 mmol/kg) of the prodrugs in mice
and rats and compared them with that from famciclovir.
The total amount of penciclovir recovered in the urine
over a 48-h period was determined by HPLC. Of the
prodrugs tested in mice, the isopropyl monocarbonate
6 achieved the highest mean urinary recovery of pen-
ciclovir (53%), followed in order by the propyl monocar-
bonate 5 (51%), the isopentyl monocarbonate 10 (51%),
the ethyl monocarbonate 4 (50%), famciclovir (48%), the
n-pentyl monocarbonate 9 (47%), and the methyl mono-
carbonate 3 (45%). The mean urinary recoveries of
penciclovir from the n-butyl monocarbonate 7 (38%), the
isobutyl monocarbonate 8 (38%), and the cyclic carbon-
ate 1 (27%) were comparatively lower than that from
famciclovir but significantly higher than that from
penciclovir (3%). In rats, compounds 3, 4, 6, 7, and 10
(39-41%) showed levels of mean urinary recovery of
penciclovir similar to that from famciclovir (40%), while
compounds 5 (34%), 8 (37%), and 9 (38%) were slightly
less bioavailable. Once again, 1 (15%) showed the
lowest mean urinary recovery of penciclovir in this
series of compounds, which was consistent with the
result in mice. The low oral bioavailability of 1 could
be due to its extreme instability in acidic aqueous
solution since 1 was immediately converted to the diol
2 in the artificial gastric juice (HCl/NaCl buffer, pH 1.2).
However, the alkyl monocarbonates 4-10 were found
to be quite stable in the examined aqueous buffer
solutions (pH 1.2, 6.0, 7.4, and 8.0), and they were much
more stable at pH 1.2 and 6.0 than at pH 7.4 and 8.0.
The isopropyl monocarbonate 6 was the most stable
with the half-lives (t1/2) of 88, >200, 61, and 26 days at
pH 1.2, 6.0, 7.4, and 8.0, respectively, but the methyl
monocarbonate 3, having the smallest alkyl group in
this series, had relatively short half-lives at pH 7.0 (24
h) and 8.4 (12 h). In addition, compounds 5 (68.0
mg/mL) and 6 (138.8 mg/mL) were highly soluble in H2O
at 20 °C, showing a remarkable increase in aqueous
solubility compared with that of penciclovir (3.2 mg/mL).
will be undertaken when a new asymmetric synthesis
of each enantiomer is developed.
In conclusion, 2-amino-9-(3-hydroxymethyl-4-iso-
propoxycarbonyloxybut-1-yl)purine (6) showed the high-
est oral mean urinary recovery of penciclovir in rodents
in a series of 2-amino-9-(3-hydroxymethyl-4-alkoxycar-
bonyloxybut-1-yl)purines that was comparable to or
slightly higher than that from famciclovir, and it was
found to be quite stable and soluble in the aqueous
solution. On the basis of these results, the extensive
studies on pharmacokinetics, metabolism, disposition,
and toxicology of the racemic 6 are presently under way
in our laboratory.
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover melt-
ing point apparatus and are uncorrected. Infrared spectra
were recorded on a Perkin-Elmer 1600 FTIR spectrophotom-
eter. 1H NMR spectra were recorded on a Varian Unity 300
spectrometer. The chemical shifts are reported in parts per
million (ppm) relative to internal tetramethylsilane in CDCl3
or DMSO-d6. 1H noise-decoupled 13C NMR spectra were
recorded on a Varian Unity 300 spectrometer at 75.4 MHz.
When CDCl3 or DMSO-d6 was used as solvent, it served as
the internal standard at δ 77.0 or 39.5, respectively. Electron
impact mass spectra (EI-MS) were obtained on a VG Quattro
mass spectrometer. Analytical thin-layer chromatography
(TLC) was performed on Merck silica gel 60F-254 glass plates.
Flash column chromatography was performed using Merck
silica gel 60 (230-400 mesh). Elemental analyses were
performed on a Carlo Erba 1106 elemental analyzer. Where
indicated by the symbols of the elements, analyses were within
(0.4% of theoretical values.
5-(2-Ben zyloxyeth yl)-1,3-d ioxa n -2-on e (12). To a stirred
solution of 2-(2-benzyloxyethyl)propane-1,3-diol (11) (15.30 g,
72.9 mmol) in anhydrous THF (1 L) was added 1,1′-carbonyl-
diimidazole (14.18 g, 87.4 mmol) in one portion, and the
mixture was refluxed for 4 h. A second portion of 1,1′-
carbonyldiimidazole (3.54 g, 21.9 mmol) was added to the
reaction mixture, and it was refluxed for an additional 2 h.
Another 0.3 equiv of 1,1′-carbonyldiimidazole (3.54 g, 21.9
mmol) was added, and refluxing was continued for 2 h to
complete the reaction. The solvent was evaporated to dryness,
and the oily residue was dissolved in EtOAc (600 mL). The
EtOAc solution was washed with 1 N aqueous HCl solution
(600 mL). The separated aqueous phase was saturated with
NaCl and extracted with EtOAc (600 mL × 3). The combined
EtOAc solution was dried over anhydrous MgSO4, filtered, and
evaporated to dryness under reduced pressure. The oily
residue was purified by MPLC over SiO2 with a mixture of
EtOAc/hexane (2:1, v/v) as eluent to give 13.50 g (79%) of 12
as a white solid, which was crystallized from Et2O: mp 38.7-
39.5 °C; IR (KBr) 1746 (CdO) cm-1; 1H NMR (CDCl3) δ 1.60-
1.75 (m, 2 H, CHCH2CH2), 2.36-2.48 (m, 1 H, CH), 3.54 (t, J
) 5.7 Hz, 2 H, CHCH2CH2), 4.12 (dd, J ) 11.1 Hz, J ) 9.3 Hz,
2 H, (CdO)OCHax), 4.41 (dd, J ) 11.1 Hz, J ) 4.8 Hz, 2 H,
(CdO)OCHeq), 4.49 (s, 2 H, OCH2Ph), 7.28-7.35 (m, 5 H, Ar
H); 13C NMR (CDCl3) δ 27.57, 29.41, 66.96, 71.93, 73.16,
127.60, 127.82, 128.45, 137.71, 148.44; MS (EI) m/ z 236 (M+).
Anal. (C13H16O4) C, H.
As previously shown in the amino acid ester prodrugs
of acyclovir, the absolute stereochemistry of the pro-
drugs might affect hydrolytic cleavage and absorption.20
Thus, it seemed necessary to separate the enantiomers
of 6 since it showed the best bioavailability and physical
properties among the alkyl monocarbonates 3-10 tested
and to subject each enantiomer to biological evaluation
to investigate the effect of the stereochemistry. Initially,
HPLC separation of the racemic alcohol 6 using chiral
columns, Chiralcel-OD (Daicel) and Whelk-OI (Regis),
was attempted. Unfortunately, all of the efforts with
various eluents proved unsuccessful. We then prepared
Mosher’s ester of the racemic alcohol 6 by coupling it
with (S)-(-)-R-methoxy-R-(trifluoromethyl)phenylacetic
acid using DCC, hoping to obtain one pure diastereomer
by conventional purification methods. Unfortunately,
various attempts to purify Mosher’s ester of racemic 6
either by repeated recrystallization or by HPLC were
unfruitful. We concluded that the separation of enan-
tiomers at this stage was too difficult. The study of the
stereochemical effect of the monocarbonate prodrugs
5-(2-Hyd r oxyeth yl)-1,3-d ioxa n -2-on e (13). A stirred so-
lution of 12 (12.50 g, 53.0 mmol) in anhydrous THF (300 mL)
was treated with 10% Pd/C (1.00 g) and purged with H2 gas
three times. The mixture was stirred under H2 atmosphere
at room temperature for 3 h with a hydrogen-filled balloon
and filtered. The organic solvent was evaporated to dryness
1
under reduced pressure to give the almost pure (>95% by H
NMR analysis) 7.73 g (100%) of 13 as a pale yellow oil, which
was used in the next step without further purification. An
aliquot was purified for analysis by MPLC over SiO2 with a
mixture of MeOH/CHCl3 (1:9, v/v) as eluent: IR (neat) 3433
(OH), 1730 (CdO) cm-1 1H NMR (CDCl3) δ 1.62 (m, 2 H,
;

Potential Prodrugs of Penciclovir
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3439
CHCH2CH2), 2.40-2.55 (m, 1 H, CH), 2.66 (br s, 1 H, OH),
3.74 (t, J ) 5.7 Hz, 2 H, CH2OH), 4.19 (dd, J ) 10.8 Hz, J )
9.0 Hz, 2 H, (CdO)OCHax), 4.51 (dd, J ) 10.8 Hz, J ) 4.8 Hz,
2 H, (CdO)OCHeq); 13C NMR (CDCl3) δ 28.94, 29.80, 59.45,
72.18, 149.03; MS (EI) m/ z 147 (M + H)+. Anal. (C6H10O4)
C, H.
from a mixture of MeCN/THF/hexane (1:4:10, v/v/v): mp
183.0-183.5 °C (dec, brown from 175.0 °C); IR (KBr) 3365,
3326, 3189 (NH2), 1749 (CdO) cm-1 1H NMR (DMSO-d6) δ
;
1.84 (m, 2 H, CHCH2CH2), 2.07-2.17 (m, 1H, CH), 4.14 (t, J
) 7.1 Hz, 2 H, NCH2), 4.22 (dd, J ) 10.8 Hz, J ) 8.1 Hz, 2 H,
(CdO)OCHax), 4.46 (dd, J ) 10.8 Hz, J ) 4.5 Hz, 2 H, (CdO)-
OCHeq), 6.51 (br s, 2 H, NH2), 8.11 (s, 1 H, H-8), 8.57 (s, 1 H,
H-6); 13C NMR (DMSO-d6) δ 26.90, 28.11, 40.05, 71.03, 126.79,
142.57, 147.83, 148.95, 152.98, 160.42; MS (EI) m/ z 263 (M+).
Anal. (C11H13N5O3) C, H, N.
5-(2-Meth a n esu lfon yloxyeth yl)-1,3-d ioxa n -2-on e (14).
To a stirred solution of 13 (4.50 g, 30.8 mmol) in anhydrous
CH2Cl2 (40 mL) cooled at 0 °C under N2 atmosphere was slowly
added Et3N (9.36 g, 92.5 mmol) followed by dropwise addition
of MsCl (3.88 g, 33.9 mmol) via a syringe, and the mixture
was stirred at 0 °C for 30 min. Additional MsCl (1.77 g, 15.4
mmol) was added dropwise to the reaction mixture at 0 °C,
and it was stirred for another 30 min. The reaction mixture
was filtered, and the filtrate was evaporated to dryness under
reduced pressure. The residue was purified by MPLC over
SiO2 with a mixture of EtOAc/hexane (9:1, v/v) followed by
EtOAc as eluent to give 5.77 g (84%) of 14 as a white solid
that should be used as soon as possible: mp 71.8-74.1 °C; IR
(KBr) 1751 (CdO), 1358, 1182 (OSO2) cm-1; 1H NMR (CDCl3)
δ 1.91 (m, 2 H, CHCH2CH2), 2.40-2.51 (m, 1 H, CH), 3.06 (s,
3 H, CH3), 4.21 (dd, J ) 11.1 Hz, J ) 7.8 Hz, 2 H, (CdO)-
OCHax), 4.34 (t, J ) 6.3 Hz, 2 H, CHCH2CH2), 4.52 (dd, J )
11.1 Hz, J ) 3.9 Hz, 2 H, (CdO)OCHeq); 13C NMR (CDCl3) δ
27.35, 28.37, 37.62, 66.22, 71.15, 147.89. Anal. (C7H12O6S)
C, H.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Am in o-
9-(3-h y d r o x y m e t h y l-4-a lk o x y c a r b o n y lo x y b u t -1-y l)-
p u r in es 3-10. A mixture of 1 (395 mg, 1.50 mmol) and dried
SiO2 (4.0 g) in CHCl3 (20 mL) was treated with an appropriate
alcohol (40 mL) and heated at 70 °C for 4 h (for 3-5 and 7-10)
or 24 h (for 6) under N2 atmosphere. After being cooled, the
reaction mixture was filtered, and the filtrate was evaporated
to dryness under reduced pressure. The residue was purified
by MPLC over SiO2 using a mixture of MeOH/CHCl3 (1:9, v/v)
as eluent to give the titled compound, which was crystallized
from a suitable solvent.
2-Am in o-9-(3-h yd r oxym eth yl-4-m eth oxyca r bon yloxy-
bu t-1-yl)p u r in e (3): yield 91%; mp 129.0-132.0 ° (lit.12 129-
132 °C); spectral data were identical with those reported.12
2-Am in o-9-(3-h ydr oxym eth yl-4-eth oxycar bon yloxybu t-
1-yl)p u r in e (4): yield 87%; mp 107.5-109.5 °C (EtOAc/
hexane); IR (KBr) 3487, 3307, 3187 (NH2 and OH), 1731 (Cd
O) cm-1; 1H NMR (CDCl3) δ 1.29 (t, J ) 7.2 Hz, 3 H, CH2CH3),
1.85-2.02 (m, 2 H, CHCH2CH2), 1.98-2.06 (m, 1 H, CH),
3.64-3.76 (m, 2 H, CHCH2OH), 4.12-4.28 (m, 6 H, NCH2 and
2 OCH2), 5.53 (br s, 2 H, NH2), 7.79 (s, 1 H, H-8), 8.64 (s, 1 H,
H-6); 13C NMR (CDCl3) δ 14.15, 28.65, 37.87, 41.09, 61.64,
64.23, 67.66, 127.87, 142.39, 149.70, 152.98, 155.26, 159.79;
MS (EI) m/ z 309 (M+). Anal. (C13H19N5O4) C, H, N.
2-Am in o-9-(3-h yd r oxym eth yl-4-p r op oxyca r bon yloxy-
bu t-1-yl)p u r in e (5): yield 89%; mp 67.0-68.7 °C (EtOAc/
2-Am in o-6-ch lor o-9-(3-h yd r oxym eth yl-4-m eth oxyca r -
bon yloxybu t-1-yl)p u r in e (15). A mixture of 2-amino-6-
chloropurine (9.49 g, 56.0 mmol), 14 (12.54 g, 56.0 mmol), and
Cs2CO3 (22.8 g, 70.0 mmol) in anhydrous DMF (150 mL) was
stirred at room temperature for 16 h under N2 atmosphere.
The reaction mixture was filtered, and the filtrate was
evaporated to dryness in vacuo. The residue adsorbed to SiO2
using DMF as a solvent was placed on the top of the SiO2
column and purified by MPLC with a mixture of MeOH/CHCl3
(1:9, v/v) as eluent to give 9.25 g (50%) of 15 as a white solid,
which was crystallized from EtOH: mp 140.2-141.2 °C dec;
hexane); IR (KBr) 3350, 3221 (NH2 and OH), 1750 (CdO) cm-1
;
IR (KBr) 3379, 3310, 3212 (NH2 and OH), 1733 (CdO) cm-1
;
1H NMR (CDCl3) δ 0.96 (t, J ) 7.5 Hz, 3 H, CH2CH3), 1.62-
1.76 (m, 2 H, CH2CH3), 1.84-2.01 (m, 2 H, CHCH2CH2), 1.97-
2.06 (m, 1 H, CH), 3.72 (m, 2 H, CHCH2OH), 4.13 (t, J ) 6.9
Hz, 2 H, OCH2CH2), 4.18-4.30 (m, 4 H, NCH2 and OCH2CH),
1H NMR (DMSO-d6) δ 1.66-1.92 (m, 3 H, CHCH2CH2), 3.41
(t, J ) 5.0 Hz, 2 H, CHCH2OH), 3.68 (s, 3 H, OCH3), 4.06-
4.18 (m, 4 H, NCH2 and OCH2CH), 4.67 (t, J ) 5.3 Hz, 1 H,
OH), 6.88 (br s, 2 H, NH2), 8.14 (s, 1 H, H-8); 13C NMR (DMSO-
d6) δ 27.73, 37.57, 41.05, 54.51, 60.20, 67.55, 123.34, 143.10,
149.27, 154.01, 155.10, 159.69. Anal. (C12H16ClN5O4) C, H,
N.
5.18 (br s, 2 H, NH2), 7.81 (s, 1 H, H-8), 8.69 (s, 1 H, H-6); 13
C
NMR (CDCl3) δ 10.07, 21.90, 28.66, 37.88, 41.09, 61.66, 67.66,
69.83, 127.89, 142.38, 149.72, 152.98, 155.41, 159.80; MS (EI)
m/ z 323 (M+). Anal. (C14H21N5O4) C, H, N.
2-Am in o-9-(3-h yd r oxym et h yl-4-isop r op oxyca r b on yl-
oxybu t-1-yl)p u r in e (6): yield 64%; mp 75.0-78.0 °C (EtOAc);
IR (KBr) 3350, 3221 (NH2 and OH), 1747 (CdO); 1H NMR
(CDCl3) δ 1.29 (d, J ) 6.3 Hz, 6 H, CH(CH3)2), 1.85-2.01 (m,
2 H, CHCH2CH2), 1.98-2.10 (m, 1 H, CH) 3.71 (m, 2 H,
CHCH2OH), 4.15-4.28 (m, 4 H, NCH2 and OCH2CH), 4.87
(septet, J ) 6.3 Hz, 1 H, CH(CH3)2), 5.47 (br s, 2 H, NH2),
7.79 (s, 1 H, H-8), 8.66 (s, 1 H, H-6); 13C NMR (CDCl3) δ 21.67,
28.68, 37.89, 41.09, 61.71, 67.44, 72.30, 127.92, 142.38, 149.73,
152.99, 154.81, 159.78; MS (EI) m/ z 323 (M+). Anal.
(C14H21N5O4) C, H, N.
2-Am in o-9-(3-h ydr oxym eth yl-4-bu toxycar bon yloxybu t-
1-yl)p u r in e (7): yield 69%; mp 76.9-78.1 °C (EtOAc/hexane);
IR (KBr) 3346, 3219 (NH2 and OH), 1746 (CdO) cm-1; 1H NMR
(CDCl3) δ 0.94 (t, J ) 7.4 Hz, 3 H, CH2CH3), 1.40 (m, 2 H,
CH2CH3), 1.66 (m, 2 H, CH2CH2CH3), 1.84-2.02 (m, 2 H,
CHCH2CH2), 1.99-2.06 (m, 1 H, CH), 3.72 (m, 2 H, CHCH2-
OH), 4.15 (t, J ) 6.8 Hz, 2 H, OCH2CH2CH2), 4.18-4.34 (m, 4
H, NCH2 and OCH2CH), 5.19 (br s, 2 H, NH2), 7.78 (s, 1 H,
H-8), 8.69 (s, 1 H, H-6); 13C NMR (CDCl3) δ 13.55, 18.81, 28.66,
30.54, 37.88, 41.09, 61.65, 67.66, 68.14, 127.88, 142.38, 149.71,
152.97, 155.41, 159.80; MS (EI) m/ z 337 (M+). Anal.
(C15H23N5O4) C, H, N.
2-Am in o-6-ch lor o-9-(2-(2-oxo-1,3-d ioxa n -5-yl)eth yl)p u -
r in e (16). A mixture of 2-amino-6-chloropurine (9.49 g, 56.0
mmol), 14 (12.54 g, 56.0 mmol), and Cs2CO3 (22.8 g, 70.0 mmol)
in anhydrous DMF (150 mL) was stirred at room temperature
for 16 h under N2 atmosphere. The reaction mixture was
filtered, and the filtrate was evaporated to dryness in vacuo.
The residue adsorbed to SiO2 using DMF as a solvent was
placed on the top of the SiO2 column and purified by MPLC
with a mixture of EtOAc/MeCN/Et3N (49:49:2, v/v/v) as eluent
to give 8.02 g (48%) of 16 as a white solid, which was
crystallized from a mixture of MeCN/THF/hexane (1:4:10, v/v/
v): mp 185.2-186.0 °C dec; IR (KBr) 3347, 3322, 3199 (NH2),
1733 (CdO) cm-1; 1H NMR (DMSO-d6) δ 1.86 (m, 2 H, CHCH2-
CH2), 2.07-2.19 (m, 1 H, CH), 4.13 (t, J ) 7.2 Hz, 2 H, NCH2),
4.21 (dd, J ) 10.5 Hz, J ) 7.8 Hz, 2 H, (CdO)OCHax), 4.46
(dd, J ) 10.5 Hz, J ) 4.2 Hz, 2 H, (CdO)OCHeq), 6.90 (br s, 2
H, NH2), 8.17 (s, 1 H, H-8); 13C NMR (DMSO-d6) δ 26.78, 27.97,
40.27, 71.01, 123.30, 143.04, 147.83, 149.35, 154.06, 159.72;
MS (EI) m/ z 297 (M+). Anal. (C11H12ClN5O3) C, H, N.
2-Am in o-9-(2-(2-oxo-1,3-d ioxa n -5-yl)eth yl)p u r in e (1). A
stirred solution of 16 (534 mg, 1.79 mmol) in a mixture of
anhydrous MeCN (25 mL) and anhydrous DMF (10 mL) was
treated with Et3N (543 mg, 5.37 mmol) and 10% Pd/C (60 mg)
and purged with H2 gas three times. The mixture was stirred
under H2 atmosphere at room temperature for 3 h with a
hydrogen-filled balloon and filtered. The filtrate was evapo-
rated to dryness under reduced pressure, and the residue was
triturated with a small volume of absolute EtOH (2 mL) to
give 427 mg (90%) of 1 as a white solid, which was crystallized
2-Am in o-9-(3-h yd r oxym eth yl-4-isobu toxyca r bon yloxy-
bu t-1-yl)p u r in e (8): yield 87%; mp 117.5-118.7 °C (EtOAc);
IR (KBr) 3411, 3312, 3175 (NH2 and OH), 1735 (CdO) cm-1
;
1H NMR (CDCl3) δ 0.95 (d, J ) 6.6 Hz, 6 H, CH(CH3)2), 1.85-
2.12 (m, 4 H, CHCH2CH2 and CH(CH3)2), 3.73 (m, 2 H, CHCH2-
OH), 3.92 (d, J ) 6.9 Hz, 2 H, OCH2CH), 4.18-4.29 (m, 4 H,

3440 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Kim et al.
NCH2 and OCH2), 5.25 (br s, 2 H, NH2), 7.78 (s, 1 H, H-8),
8.69 (s, 1 H, H-6); 13C NMR (CDCl3) δ 18.85, 27.74, 28.74,
37.87, 41.04, 62.00, 67.62, 74.34, 128.17, 142.34, 149.86,
153.12, 155.54, 159.78; MS (EI) m/ z 337 (M+). Anal.
(C15H23N5O4) C, H, N.
ELISA reader (Dynatech, MR 5000). Each experiment was
performed in quadruplicate and repeated twice.
In Vitr o An tivir a l Activity. The in vitro antiviral activity
was tested by dye-uptake assay.21 Vero cells were grown in
EMEM containing 5% fetal bovine serum (FBS) (Gibco).
Herpes simplex virus type 1 (HSV-1) (KOS strain) was
obtained from ATCC, and stocks were prepared in Vero cell
suspensions and stored at -70 °C. One hundred microliters
of Vero cell suspensions (105 cells/mL) was plated in 96-well
plates. After a 1-h plating period at 37 °C in an incubator
containing 5% CO2, 50 µL of 100 PFU/well of virus in EMEM
with 5% FBS was added into each well. One hundred
microliters of the test compound (serial 3-fold dilutions in
EMEM from maximum concentration of 125 µM) was added
into 96-well plates in quadruplicate following a 1-h virus
adsorption period. After 48 h of incubation at 37 °C in an
incubator containing 5% CO2, 100 µL of neutral red dye (0.15%
in PBS, pH 5.5) was placed into each well. The plates were
then incubated for an additional 30 min at 37 °C, and then
the medium and residual stain were removed, and the wells
were rinsed twice with PBS (pH 6.5). Thereafter, 200 µL of
phosphate ethanol buffer (PBS/EtOH ) 1:1, v/v, pH 4.2) was
distributed into each well for elution of the dye incorporated
by supposedly viable cells. The OD of the solutions was read
at 550 nm using an ELISA reader. The drug concentration
exhibiting a dye uptake of 50% (EC50) in comparison to that
of the virus control () 0%) and that of the cell control () 100%)
was calculated by quantal probit analysis of pharmacologic
calculations with a computer program.22
2-Am in o-9-(3-h ydr oxym eth yl-4-pen toxycar bon yloxybu t-
1-yl)p u r in e (9): yield 74%; mp 66.5-68.5 °C; IR (KBr) 3374,
3341, 3211 (NH2 and OH), 1747 (CdO) cm-1; 1H NMR (CDCl3)
δ 0.91 (t, J ) 7.1 Hz, 3 H, CH2CH3), 1.34 (m, 4 H, CH2CH2-
CH3), 1.67 (m, 2 H, CH2CH2CH2CH3), 1.85-2.10 (m, 3 H,
CHCH2CH2), 3.72 (m, 2 H, CHCH2OH), 4.13 (t, J ) 6.9 Hz, 2
H, OCH2CH2), 4.18-4.31 (m, 4 H, NCH2 and OCH2), 5.15 (br
s, 2 H, NH2), 7.78 (s, 1 H, H-8), 8.69 (s, 1 H, H-6); 13C NMR
(CDCl3) δ 13.88, 22.24, 27.77, 28.30, 28.74, 37.85, 41.01, 62.14,
67.57, 68.52, 128.29, 142.32, 149.97, 153.13, 155.53, 159.74;
MS (EI) m/ z 351 (M+). Anal. (C16H25N5O4) C, H, N.
2-Am in o-9-(3-h yd r oxym et h yl-4-isop en t oxyca r b on yl-
oxybu t-1-yl)p u r in e (10): yield 84%; mp 81.3-83.1 °C (EtOAc/
hexane); IR (KBr) 3377, 3340, 3211 (NH2 and OH), 1750 (Cd
O) cm-1 1H NMR (CDCl3) δ 0.93 (d, J ) 6.9 Hz, 6 H,
;
CH(CH3)2), 1.56 (m, 2 H, CHCH2CH2), 1.71 (septet, J ) 6.9
Hz, 1 H, CH(CH3)2), 1.88-2.11 (m, 3 H, CHCH2CH2), 3.73 (m,
2 H, CHCH2OH), 4.17 (t, J ) 6.9 Hz, 2 H, OCH2CH2), 4.18-
4.28 (m, 4 H, NCH2 and OCH2), 5.28 (br s, 2 H, NH2), 7.78 (s,
1 H, H-8), 8.69 (s, 1 H, H-6); 13C NMR (CDCl3) δ 22.36, 24.79,
28.74, 37.25, 37.87, 41.04, 62.02, 67.00, 67.60, 128.17, 142.32,
149.88, 153.10, 155.47, 159.78; MS (EI) m/ z 351 (M+). Anal.
(C16H25N5O4) C, H, N.
Wa ter Solu bility. A standard solution was prepared by
dissolving 1 mg of the test compound in 10 mL of H2O at 20
°C. The standard solution was diluted with H2O as necessary,
and the diluted standard solutions were scanned by UV at 290
nm to obtain a standard curve. A saturated solution was
prepared by vortex-mixing in H2O for 1 min, ultrasonification
for 1 min, vortex-mixing for 3 min, ultrasonification for 1 min,
and finally vortex-mixing for 5 min in the presence of excess
compound. After the saturated solution was filtered to remove
excess compound using 0.45-µm Millipore filters, the solution
was diluted with H2O and then scanned by UV at 290 nm.
Total solubility was then determined by the comparison of the
absorbance of the saturated solution with that of the standard
curve.
Or a l Bioa va ila bility. The bioavilability of the test com-
pound was estimated by determining the total amount of
penciclovir in the urine using HPLC. Urine was collected for
48 h in a metabolic cage after oral administration of a single
0.2 mmol/kg dose of the test compound to six male ICR mice
(25-30 g) or six male Sprague-Dawley rats (200-250 g). A
5% solution of sodium azide (0.4 mL per estimated 100 mL of
urine) was added to each urine receptacle before collection to
prevent bacterial growth. The collected urine was filtered
(0.45-µm), and the penciclovir concentration was analyzed by
HPLC as follows. A C18 symmetry column equipped with a
compatible guard column was eluted at a flow rate of 1 mL/
min with the following three-step gradient: (step 1) a 10-min
isocratic elution with 100% buffer A (0.1% phosphoric acid),
(step 2) a 25-min linear gradient from 100% buffer A to 55%
buffer A and 45% buffer B (80% MeCN in 0.1% phosphoric
acid), and (step 3) a 4-min isocratic elution with 55% buffer A
and 45% buffer B. The column was equilibrated with 100%
buffer A for 10 min before each sample injection. The UV
absorbance of the column effluent was monitored at 248 nm.
Aqu eou s Sta bility. Fifty microliters of 2.5 mM stock
solution of the test compound was added to sodium phosphate
buffer (pH 6.0, pH 7.4, or pH 8.0) or HCl/NaCl buffer (pH 1.2)
to give a final concentration of 125 µM. Immediately after the
compound was mixed with the buffer, incubation of the
solution was initiated at 37 °C. At various intervals, 100 µL
of the sample was taken throughout the indicated incubation
times and immediately mixed with 900 µL of 0.1 M phosphate
buffer (pH 7.0). The sample was then isocratically eluted at
a flow rate of 1 mL/min with buffer A (0.1% phosphoric acid)
and buffer B (80% MeCN in 0.1% phosphoric acid) with various
ratios using a C18 symmetry column. The half-life of the
compound was calculated from the concentration of the parent
compound.
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