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

Article abstract of DOI:10.1016/S0968-0896(99)00086-3

A series of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purines (1-8) and 2-amino-9-(3-alkoxycarbonyl- oxymethyl-4-alkoxycarbonyloxybut-1-yl)purines (9-12) were synthesized as potential prodrugs of penciclovir. Treatment of 6- deoxypenciclovir

Full text of DOI:10.1016/S0968-0896(99)00086-3

Bioorganic & Medicinal Chemistry 7 (1999) 1715±1725
Synthesis and Evaluation of 2-Amino-9-(3-acyloxymethyl-4-
alkoxycarbonyloxybut-1-yl)purines and 2-Amino-9-(3-
alkoxycarbonyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purines
as Potential Prodrugs of Penciclovir
Dae-Kee Kim, a,* Namkyu Lee, a Do Hyun Ryu, a Young-Woo Kim, a Jae-Sun Kim, a
Kieyoung Chang, a Guang-Jin Im, a Won-Son Choi, a Yong-Baik Cho, a Key H. Kim, a
Danni Colledge b and Stephen Locarnini b
aLife Science Research Center, SK Chemicals, 600 Jungja-Dong, Changan-Ku, Suwon-Si, Kyungki-Do 440-745, Korea
bVictorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Victoria 3051, Australia
Received 26 February 1999; accepted 1 April 1999
AbstractÐA series of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purines (1±8) and 2-amino-9-(3-alkoxycarbonyl-
oxymethyl-4-alkoxycarbonyloxybut-1-yl)purines (9±12) were synthesized as potential prodrugs of penciclovir. Treatment of 6-
.
deoxypenciclovir with trimethyl orthoacetate or triethyl orthopropionate (1.2 equiv) in DMF in the presence of p-TsOH H2O (0.1
equiv) followed by quenching with excess H2O gave the corresponding mono-O-acetyl or mono-O-propionyl compound, 17 or 18,
in excellent yields of 95 and 92%, respectively. Reactions of 17 or 18 with an appropriate alkyl (Me, Et, n-Pr, and i-Pr) 4-nitro-
phenyl carbonate (1.2 equiv) in pyridine in the presence of a catalytic amount of DMAP (0.1 equiv) at 80ꢀC a€orded the monoacyl,
monocarbonate derivatives of 6-deoxypenciclovir, 1±8, in 86±94% yields. Similar reactions of 6-deoxypenciclovir with 2.1 equiv of
alkyl 4-nitrophenyl carbonate produced the dicarbonate derivatives 9±12 in 81±83% yields. Of the prodrugs tested in rats, 2-amino-
9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine (4) achieved the highest mean urinary recovery of penciclovir (36%),
followed in order by compounds 2 (35%), 6 (35%), 7 (34%), 10 (34%), 8 (32%), 3 (32%), and famciclovir (31%). The mean urinary
recovery of penciclovir and concentrations of penciclovir in the blood from 4 in mice were also slightly higher than those from
famciclovir. The in vivo antiviral ecacy of 4 in HSV-1-infected normal BALB/c mice was higher than those of famciclovir and
valaciclovir in terms of mortality (100, 80, and 40%) and mean survival time (>21, 13‹5.0 (SEM), and 13‹1.6 days). Compound
4 demonstrated an e€ective anti-hepadnaviral response with intrahepatic viral load being reduced by 90%, the viral supercoiled
DNA levels reduced by 70% and Pre-S expression inhibited by 30% against duck hepatitis B virus (DHBV) in vivo, and did not
cause any signi®cant hepatotoxicity after 4 weeks of treatment. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
triphosphate has a much greater stability than acyclovir
triphosphate within virus-infected cells.2,7 However, like
other acycloguanosine analogues such as acyclovir,8
ganciclovir,9 and buciclovir,10 penciclovir was poorly
absorbed when given orally to rodents.11,12 Therefore,
the search for a prodrug that is orally well absorbed and
then readily converted to penciclovir is of high priority.
Harnden et al. developed famciclovir [2-amino-9-(4-
acetoxy-3-acetoxymethylbut-1-yl)purine], the diacetyl 6-
deoxy analogue of penciclovir, as a prodrug of penci-
clovir.11 In mice, rats, and humans, famciclovir is orally
well absorbed and then extensively converted to penci-
clovir by the enzymatic removal of two O-acetyl groups
followed by oxidation at the 6-position of the purine
ring by xanthine oxidase.11±13 In the US, famciclovir
has recently been approved by the FDA for the treatment
Penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)-
guanine] is a potent and selective inhibitor of members
of the herpesvirus family including herpes simplex virus
type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster
virus (VZV), and Epstein±Barr virus (EBV) both in cell
cultures and in animal models,1±4 and of hepatitis B
virus (HBV) and duck hepatitis B virus (DHBV) in
cell cultures.5,6 The advantage of penciclovir over acy-
clovir is that its antiviral activity in cell culture is
more persistent than that of acyclovir because penciclovir
Key words: Antivirals; nucleosides; chemotherapy; enzyme inhibitors.
* Corresponding author. Tel.: +82-331-258-1709; fax: +82-331-252-
1379; e-mail: dkkim@skchemicals.com
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00086-3

1716
D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
Chart 1.
of herpes zoster (shingles) and acute recurrent genital
herpes in immunocompetent individuals. Famciclovir
has been shown to inhibit DHBV replication in vivo in
hepatic and nonhepatic tissues of ducklings that had
been infected in ovo with DHBV.14 It also controlled
HBV replication e€ectively in orthotopic liver trans-
plant patients either alone or in combination with a
short course of prostaglandin E15,16 and inhibited HBV
replication in a double-blind, placebo-controlled, pilot
study in patients with chronic HBV infection.17 A large,
multicenter clinical trial of famciclovir against HBV
infection is currently in progress. We have recently
reported the synthesis of amino acid esters of penciclo-
vir and 6-deoxypenciclovir,18,19 esters of 2-amino-6-
¯uoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purines,20
and a series of 2-amino-9-(3-hydroxymethyl-4-alkoxy-
carbonyloxybut-1-yl)purines as potential prodrugs of
penciclovir.21 Amomg them, SK 1875 [2-amino-9-(3-
hydroxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine]
showed oral penciclovir bioavailability comparable to
famciclovir in mice and rats,21 and had desirable physi-
cal properties as a prodrug.21
50%, and viral RNA expression by 31%.22 Unfortu-
nately, after 4 weeks of drug-free follow-up, liver his-
tology showed some signi®cant pathological changes
such as steatosis and nucleoli.22 This hepatotoxicity was
unexpected to us since SK 1875 was readily metabolized
to penciclovir and 6-deoxypenciclovir in rodents,21 the
same metabolites from famciclovir. It was of particular
interest to us to determine whether hepatotoxicity of SK
1875 attributed to a carbonate group or an unprotected
hydroxyl group. In addition to that, we like to examine
if further modi®cation of a hydroxyl group of SK 1875
with an acyl or a carbonate group could increase their
oral bioavailability compared with SK 1875. Therefore,
in this report, we synthesized the monoacyl, mono-
carbonate derivatives of 6-deoxypenciclovir, 1±8 and the
dicarbonate derivatives 9±12, and evaluated for their
potential as prodrugs of penciclovir.
Chemistry
We have recently reported a new method for the
synthesis of the C1±C5 alkyl monocarbonate derivatives
of 6-deoxypenciclovir, where the introduction of cyclic
carbonate group to the acyclic moiety at the early stage
of the synthesis, the successful coupling of cyclic car-
bonate mesylate 13 with 2-amino-6-chloropurine, and
the opening of the cyclic carbonate 14 with an appro-
priate alcohol as a nucleophile in the presence of acti-
vated silica gel as a mild Lewis acid under a mild
On the basis of these results, the extensive preclinical
studies on SK 1875 have been undertaken. Treatment of
ducklings congenitally infected with DHBV with SK
1875 at the dose of 15 mg/kg/day given orally in two
equally divided doses for 4 weeks reduced serum
DHBV-DNA levels below detectable limits, intrahepatic
viral load by 87%, the level of supercoiled DNA by

D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
1717
condition were the key steps.21 Thus, it was ®rst envi-
sioned that all the target compounds 1±12 could be
prepared from the alkyl monocarbonate derivatives of
6-deoxypenciclovir by acylation reactions using appro-
priate acylating reagents such as acid anhydrides and
alkyl chloroformates, or their equivalents. Unfortu-
nately, this approach was hampered by the fact that our
new method was found not to be practical for the large
scale production of SK 1875 due to the following rea-
sons: (1) the reagent for the formation of cyclic carbon-
ate, 1,10-carbonyldiimidazole, is very expensive, (2) the
coupling reaction of cyclic carbonate mesylate 13 with
2-amino-6-chloropurine was rather low yielding (about
30% in 56 mmol scale), and (3) the product puri®cation
was very dicult. So, it was decided to develop another
practical synthetic method which can be readily scaled
up for the mass production of any preclinical candi-
date. Our works have been mainly focused on those
approaches using 6-deoxy-6-chloropenciclovir or 6-
deoxypenciclovir as the starting materials, since the
potentially practical methods for the preparation of
them have been well documented.23,24 Even though the
direct conversion of 6-deoxypenciclovir to the mono-
alkyl carbonate using alkyl chloroformate has met with
failure, as described in our previous work,21 it occurred
to us that the chlorine atom in the 6-deoxy-6-chloro-
penciclovir could make some di€erence, hopefully due
to its electronic e€ect on the purine ring. Many reac-
tions with 6-deoxy-6-chloropenciclovir and isopropyl
chloroformate were performed using various solvents
(DMF, CH2Cl2, CHCl3, THF, or pyridine), and pyri-
dine was found to be best, a€ording the desired mono-
isopropyl carbonate 15 in a reasonable yield of 60% in a
small scale reaction (0.10 mmol). Unfortunately, it was
observed the yields of this reaction were really variable
and dropped dramatically as the reaction scale
increased. As shown in Scheme 1, the large scale (2 mol)
reaction was a real disaster, yielding less than 10% of
the desired product 15 along with a mess of a dark yel-
low crystalline by-product, whereas the yields were
around 40±45% in the medium size scale (0.5 mol). This
unexpected by-product was assigned as a pyridium salt
16 based on the spectroscopic data (1H NMR, IR, and
FAB±MS) and elemental analysis, which could be formed
via the displacement of chlorine atom by pyridine. For-
mation of the pyridium salt 16 became serious in the large
scale, probably because it required prolonged time to
remove a large amount of pyridine solvent during the
work-up. Therefore, it was concluded that the direct
conversion of the diols, 6-deoxypenciclovir or 6-deoxy-
6-chloropenciclovir, to the corresponding mono-alkyl
carbonate using alkyl chloroformate was not really fea-
sible and the late introduction of carbonate group
would be better.
In the mean time, it was decided to use 6-deoxy-
penciclovir as a starting material and ®nd any practical
ways to di€erentiate the diol functionalilty prior to the
introduction of carbonate group. Literature search
revealed that there are several ecient ways to convert
the diols into their mono-functionalized alcohols with
high selectivities.25,26 Among them, a procedure using
trimethyl orthoacetate and a Lewis acid caught our
attention because it was reported to be very mild and
highly selective.26 This reaction is known to proceed
through the formation of cyclic orthoacetate intermediate
followed by hydrolysis to yield the corresponding mono-
acetate derivative. Gratifyingly, this procedure tuned
out to be suitable for our purpose. As shown in Scheme
2, the starting 6-deoxypenciclovir was ®rst treated with
trimethyl orthoacetate or triethyl orthopropionate (1.2
.
equiv) in DMF in the presence of p-TsOH H2O (0.1
equiv) at room temperature for 2 h, and then the reac-
tion mixture was quenched with excess H2O at ambient
temperature to provide the corresponding mono-O-ace-
tyl or -propionyl compound, 17 or 18, in excellent yields
of 95 and 92%, respectively. Indeed, these reactions
were very mild and highly ecient.
At this stage, it is necessary to seek out an ecient way
to convert the mono-O-acyl derivatives, 17 and 18, to
Scheme 1. (a) Isopropyl chloroformate (1.1 equiv), pyridine, 0ꢀC to rt.

1718
D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
.
Scheme 2. (a) (i) Trimethyl orthoacetate or triethyl orthopropionate (1.2 equiv), p-TsOH H2O (0.1 equiv), DMF, rt, 2 h; (ii) H2O, 1 h; (b) methyl,
ethyl, n-propyl, or isopropyl 4-nitrophenyl carbonate (1.2 equiv), DMAP (0.1 equiv), pyridine, 80ꢀC, 20 h; (c) methyl, ethyl, n-propyl, or isopropyl 4-
nitrophenyl carbonate (2.1 equiv), DMAP (0.1 equiv), pryidine, 80ꢀC, 24 h.
the desired target compounds, 1±8. As was expected
from our previous work,21 the introducton of carbonate
group was not straightforward, although one of the diol
functionality was protected as an ester. A lot of reactions
were carried out with the mono-O-acetyl compound 17
using various carbonate transfer reagents such as alkyl
chloroformate, dialkyl carbonate, dialkyl dicarbonate
(dialkyl pyrocarbonate), and so on. Whereas diethyl
carbonate did not react at all even under very harsh
conditions, isopropyl chloroformate and diisopropyl
dicarbonate27 produced rather complex mixtures under
various conditions. These results indicate that alkyl
chloroformate and dialkyl dicarbonate might be too
much reactive to control and, on the other hand, dialkyl
carbonate is not reactive enough. It is not too much
surprising to observe that the reactive isopropyl chloro-
formate and diisopropyl dicarbonate produced rather
complex mixtures, since the substrate 17 has ®ve nitro-
gen atoms including an amino group which could be
potentially competing reaction sites. Based on these
frustrating results, it was concluded that the search for the
right reagent with intermediate reactivity between alkyl
chloroformate and dialkyl carbonate is a ®rst priority. It
was reasoned by us that the alkyl phenyl carbonate,
where a phenyl ring is substituted with electron with-
drawing groups, might be suitable since various phenols
have been used in the peptide synthesis as an activated
form of esters. We chose 4-nitrophenol because it is very
cheap, and isopropyl 4-nitrophenyl carbonate was pre-
pared in high yield by reacting 4-nitrophenol with iso-
propyl chloroformate in the presence of Et3N in Et2O at
room temperature. We performed several reactions
using isopropyl 4-nitrophenyl carbonate and 17 to ®nd
an optimal condition. Due to the low solubility of 17 in
organic solvents, DMF and pyridine were tested at var-
ious temperature in the presence or absence of a catalytic
amount of 4-dimethylaminopyridine (DMAP). For-
tunately, the reaction worked ®ne in pyridine at 80ꢀC in
the presence of a catalytic amount of DMAP (0.1 equiv)
and was completed in about 12 to 24 h using slight
excess of isopropyl 4-nitrophenyl carbonate (1.2 equiv)
to give the desired product 4 in 89%. It was really slow
in the absence of DMAP at 80±100ꢀC or at room tem-
perature in the presence of DMAP. In contrast, it was
observed that the reaction using DMF was somewhat
complicated probably because DMF itself might be
reacting with isopropyl 4-nitrophenyl carbonate. Other
alkyl (methyl, ethyl, and n-propyl) 4-nitrophenyl carbo-
nates were prepared in the same manner as described for
isopropyl 4-nitrophenyl carbonate, also in high yields.
Reactions of 17 and 18 with each alkyl (Me, Et, n-Pr
and i-Pr) 4-nitrophenyl carbonate were carried out
under the previously mentioned conditions, and the
target compounds, 2-amino-9-(3-acyloxymethyl-4-alk-
oxycarbonyloxybut-1-yl)purines 1±8, were obtained in
high yields of 86±94%. Uneventfully, the di-carbonate
derivatives 9±12 were prepared in 81±83% yields
directly from 6-deoxypenciclovir by reacting with
appropriate alkyl 4-nitrophenyl carbonate (2.1 equiv) in
pyridine at 80ꢀC in the presence of a catalytic amount of
DMAP (0.1 equiv). It should be pointed out that it was
possible to prepare a large quantity of the selected pre-
clinical candidate by adopting this process.
To characterize the metabolites of 4 in vivo, the
unknown, postulated metabolites 9-(3-hydroxymethyl-
4-isopropoxycarbonyloxybut-1-yl)guanine (19) and 9-
(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)-
guanine (20) were prepared from 15 as shown in Scheme
3 as standard compounds for HPLC analysis. First, the
potential metabolite 19 was eciently synthesized in
93% yield from 15 by enzymatic reaction using adeno-
sine deaminase (Sigma) in phosphate bu€er (pH 7.5) at
room temperature. Even though this enzymatic conver-
sion required 5 days at room temperature for comple-
tion, it was very clean, and only the desired product was
isolated. Subsequent acetylation of 19 with acetic anhy-
dride was carried out in anhydrous DMF at room tem-
perature in the presence of a catalytic amount of
DMAP to give the potential metabolite 20 in 83% yield.
Results and Discussion
The bioavailability of penciclovir following a single oral
administration (0.2 mmol/kg) of the prodrugs 1±12 to
rats was evaluated and compared with those from pen-
ciclovir, famciclovir and SK 1875. The total amount of
penciclovir recovered in the urine over a 48-h period
was determined by HPLC (Table 1). The mean urinary

D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
1719
Scheme 3. (a) Adenosine deaminase (10% by wt), phosphate bu€er (pH 7.5), rt, 5 days; (b) acetic anhydride (2 equiv), DMAP (0.1 equiv), rt, 1 h.
Table 1. Urinary recovery of penciclovir and 6-deoxypenciclovir fol-
lowing oral administration of 2-amino-9-(3-acyloxymethyl-4-alkoxy-
carbonyloxybut-1-yl)purines 1±8 and 2-amino-9-(3-alkoxycarbonyl-
oxymethyl-4-alkoxycarbonyloxybut-1-yl)purines 9±12 to rats
6-deoxypenciclovir (16±28%) was also found in the
urine from all of the prodrugs (Table 1), indicating that
the rate-determining step in the conversion of these
prodrugs to penciclovir was oxidation at the 6-position
of the purine ring.
Since compound 4 showed the highest mean urinary
recovery of penciclovir and total mean urinary recovery
of penciclovir and 6-deoxypenciclovir (63%) among
these prodrugs, it was selected for further evaluation.
Following a single oral administration (0.2 mmol/kg) of
4 to mice, concentrations of penciclovir and 6-deoxy-
penciclovir in the blood were determined by HPLC and
compared with those obtained after oral administration
of the equivalent dose of famciclovir (Table 2). The
mean values (‹SD) for maximum concentrations of
penciclovir and 6-deoxypenciclovir in the blood were
63.5‹14.9 and 29.4‹9.0 mM for 4 and 62.6‹2.5 and
25.9‹5.7 mM for famciclovir, respectively, and these
were achieved 15 min after administration. The values
of concentrations of penciclovir and 6-deoxypenciclovir
from 4 were similar to those from famciclovir at all time
points tested with the exception of that concentration of
penciclovir from 4 (54.4‹7.2 mM) was much higher
than that from famciclovir (41.9‹8.3 mM) 30 min after
administraton.
Urinary recovery of penciclovir
and 6-deoxypenciclovira
(% dose)b
Compd
R
R1
Penciclovir
6-Deoxypenciclovir
1
2
3
4
5
6
7
8
9
10
11
12
Me
Me
Me
Me
Et
Et
Et
Et
MeO
EtO
n-PrO
i-PrO
Me
Et
n-Pr
i-Pr
Me
Et
n-Pr
i-Pr
Me
Et
n-Pr
i-Pr
30
35
32
36
28
35
34
32
30
34
32
29
31
31
6
23
26
22
27
22
24
24
28
17
23
20
16
28
27
SK 1875
Famciclovir
Penciclovir
a
A single oral dose of the test compound (0.2 mmol/kg) was adminis-
tered to two male Sprague±Dawley rats. The total amount of penciclovir
and 6-deoxypenciclovir recovered in the urine over a 48-h period was
determined by HPLC using a C18 reversed-phase column.
b
Mean values of two independent experiments.
recovery of penciclovir from all of the prodrugs (28±
36%) was 4.7±6.0-fold higher than that from penciclovir
(6%). Compound 4 (RˆMe, R1ˆi-Pr in Table 1)
achieved the highest penciclovir bioavailability (36%),
followed in order by compounds 2 (35%; RˆMe,
R1ˆEt), 6 (35%; RˆR1ˆEt), 7 (34%; RˆEt, R1ˆn-Pr),
and 10 (34%; RˆOEt, R1=Et). The mean urinary
recovery of penciclovir from compounds 8 (32%; RˆEt,
R1ˆi-Pr), 3 (32%; RˆMe, R1ˆn-Pr), and 9 (30%;
RˆOMe, R1ˆMe) was comparable to those from fam-
ciclovir (31%) and SK 1875 (31%), while compounds 12
(29%; Rˆi-PrO, R1ˆi-Pr) and 5 (28%; RˆEt, R1ˆMe)
were slightly less bioavailable. A substantial amount of
The parent prodrug 4 and all the other postulated
metabolites SK 1875, 17, 19, 20, and 9-(3-acetoxy-
methyl-4-hydroxybut-1-yl)guanine (21)12 were also
checked in the blood, but they were not detected at any
time points in any mice, demonstrating very rapid
hydrolysis of acyl group and carbonate group after oral
administration.
The urinary recovery of penciclovir and 6-deoxypenciclovir
following a single oral administration (0.2 mmol/kg) of
4 to mice was determined and compared with that from

1720
D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
Table 2. Concentrations of penciclovir and 6-deoxypenciclovir in the blood following oral administration of 2-amino-9-(3-acetoxymethyl-4-
isopropoxycarbonyloxybut-1-yl)purine (4) and famciclovir to mice
Concn (mM) in blooda,b
4
Famciclovir
6-Deoxypenciclovir
Time after dosing (h)
Penciclovir
6-Deoxypenciclovir
Penciclovir
0.25
0.5
1
1.5
2
63.5‹14.9
54.4‹7.2
24.4‹5.8
22.1‹6.3
12.2‹4.7
<0.3d
29.4‹9.0
13.2‹2.4
3.4‹1.4
2.8‹0.6
1.0‹0.1c
<0.8d
62.6‹2.5
41.9‹8.3
23.2‹2.8
22.4‹7.0
6.0‹1.9
<0.3d
25.9‹5.7
12.8‹1.0
4.0‹1.1
3.4‹0.5
1.4‹0.7c
<0.8d
4
a
A single oral dose of the test compound (0.2 mmol/kg) was administered to four male ICR mice. Concentrations of penciclovir and 6-deoxy-
penciclovir in the blood at 0.25, 0.5, 1, 1.5, 2, and 4 h after dosing were determined by HPLC using a C18 reversed-phase column.
b
Mean‹SD of four animals.
Mean‹SD of three animals. In the fourth animal, the value was below the limit of reliable determination.
Below the limit of reliable determination in all animals (0.3 mM for penciclovir and 0.8 mM for 6-deoxypenciclovir).
c
d
the equivalent oral dose of famciclovir (Table 3).
Although the mean urinary recovery of 6-deoxy-
penciclovir over a 24-h period from both 4 and famci-
clovir was the same (each 11%), 4 (53%) achieved
slightly higher mean urinary recovery of penciclovir
compared with that from famciclovir (49%). Again, 4
and aforementioned, all the other postulated metabo-
lites were not detected at all in the urine. Although the
total urinary recovery of penciclovir and 6-deoxy-
penciclovir from 4 in mice (64% over a 24-h period) was
comparable to that in rats (63% over a 24-h period), the
amount of penciclovir in mice (53%) is much higher
than that in rats (36%), thus con®rming that more
extensive oxidation at the 6-position of the purine ring
occurred in mice. The in vivo antiviral ecacy of 4 on
HSV-1-induced mortality in BALB/c mice was eval-
uated and compared with those of famciclovir and
valaciclovir (Table 4). The mean survival time of HSV-
1-infected mice receiving no antiviral therapy was
6.5‹0.5 days (mean‹SEM), and no aminal survived
on the last day of evaluation (day 21). Following oral
administration of 4 (0.075 mmol/kg) twice daily for 5
consecutive days, it completely protected HSV-1-
induced mortality. Although both famciclovir and vala-
ciclovir caused increases in the survival rate (80 and
40%) and the mean survival time (13‹5.0 and 13‹1.6
days) as compared with those of untreated control, their
in vivo antiviral ecacy was lower than that of 4.
Treatment of DHBV-infected ducklings with 4 at the
dose of 30 mg/kg/day given orally in two equally divi-
ded doses for 4 weeks showed rapid decrease in serum
DHBV-DNA levels below detectable limits, reduction
of intrahepatic viral load and viral supercoiled DNA by
90 and 70%, respectively, and inhibition of Pre-S
expression by 30%.22 In contrast with SK 1875, after 4
weeks of drug-free follow-up, there was no di€erence in
the histological appearance in the liver between the
treated group and the control group, even though the
administered dose of 4 was twofold higher than that of
SK 1875.22 These results indicate that protection of
both hydroxyl groups in the acyclic moiety of the pro-
drug is necessary to minimize or eliminate the hepato-
toxicity so that the parent prodrug and its oxidized
metabolite can not be used as substrates for the host
cellular DNA polymerase in the liver.
As previously shown in the amino acid ester prodrugs of
acyclovir, the absolute stereochemistry of the prodrugs
might a€ect hydrolytic cleavage and absorption.28
However, it seems to be not the case for 4 since both
protecting groups of 4 were readily cleaved and each
enantiomer of 4 does not contain any distinguishable
Table 4. E€ects of 4, famciclovir, and valaciclovir on HSV-1-induced
mortality in BALB/c micea
Table 3. Urinary recovery of penciclovir and 6-deoxypenciclovir fol-
lowing oral administration of 2-amino-9-(3-acetoxymethyl-4-isopropoxy-
carbonyloxybut-1-yl)purine (4) and famciclovir to mice
Compd
% Survival
Mean survival time (days)b
Untreated control
4
Famciclovir
Valaciclovir
0
6.5‹0.5
>21
13‹5.0
13‹1.6f
Urinary recovery of penciclovir and
6-deoxypenciclovira (% dose)b
100c,d
80c
40e
Compd
Penciclovir
6-Deoxypenciclovir
Ten mice per group were infected i.p. with 3Â104 PFU of HSV-1
(KOS strain) and treated with oral dose of 0.075 mmol/kg of the test
compounds twice daily for 5 consecutive days postinfection.
a
4
Famciclovir
53
49
11
11
a
b
A single oral dose of the test compound (0.2 mmol/kg) was admi-
nistered to ®ve male ICR mice. The total amount of penciclovir and
6-deoxypenciclovir recovered in the urine over a 24-h period was
determined by HPLC using a C18 reversed-phase column.
Each value represents the mean‹SEM.
Signi®cantly di€erent from untreated control group (p<0.001).
Signi®cantly di€erent from valaciclovir-treated group (p<0.01).
Signi®cantly di€erent from untreated control group (p<0.05).
Signi®cantly di€erent from untreated control group (p<0.01).
c
d
e
b
f
Mean values of ®ve animals.

D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
1721
protecting groups to show di€erent absorption rate in
the gastrointestinal tract. To prove this, it seemed
necessary to prepare each enantiomer of 4 and subject
each enantiomer for the biological evaluation. Initial
e€orts for HPLC separation of the racemic 4 using
chiral columns such as Chiralcel-OD (Daicel), Whelk-
OI (Regis) and Cyclobondi 2000-DMP (Astec), proved
unsuccessful under various chromatographic separation
conditions. We then decided to resort to enzymatic
reactions since there have been a lot of reports on the
asymmetric enzymatic acylations of 2-substituted-1,3-
propanediols or hydrosis of 1,3-diacetates of them.29 As
shown in Scheme 4, a few reactions were ®rst tried
with diols (6-deoxypenciclovir and 6-deoxy-6-chloro-
penciclovir) or diacetates (famciclovir and 22), using
three lipases from Pig Pancreas (PPL), Pseudomonas
¯uorescens (PFL), and Candida Antarctica (CAL), hop-
ing to get the corresponding mono-acetate 17 or 24 in
enantiomerically enriched form. It was disappointing to
®nd that only the racemic mixtures of mono-acetates 17
and 24 were obtained under various conditions. Achiwa
et al. have reported that the diol 23 can be converted to
the mono-acetate 25 in 81% enantiomeric purity using
PFL (supplied by Amano Pharmaceutical Co.).30 Thus,
we have repeated the same reaction using PFL (pur-
chased from Aldrich) under the identical condition, but
their result could not be reproduced by us. Disappoint-
ingly, only the 59:41 ratio of two enantiomers by HPLC
(Chiralcel OD) was the best we could get. We tentatively
attributed this discrepency to the di€erent sources of
PFL used. For now, it was concluded that either the
separation or synthetic preparation of each enantiomer
of 4 at this stage was too dicult. The study on the
stereochemical e€ect of the 2-amino-9-(3-acyloxy-
methyl-4-alkoxycarbonyloxybut-1-yl)purine prodrugs
will be undertaken when a new asymmetric synthesis of
each enantiomer is developed.
urinary recovery of penciclovir in mice and rats, and
concentrations of penciclovir in the blood in mice from
4 were slightly higher than those from famciclovir after
oral administration of the equivalent dose. The in vivo
antiviral ecacy of 4 in HSV-1-infected normal BALB/c
mice was higher than those of famciclovir and valaci-
clovir in terms of mortality and mean survival time.
Compound 4 demonstrated an e€ective anti-hepadna-
viral response against DHBV in vivo and did not cause
any signi®cant hepatotoxicity after 4 weeks of treat-
ment. On the basis of these results, the extensive pre-
clinical studies of 4 are presently under way in our
laboratory.
Experimental
Melting points were determined on a Mettler melting
point apparatus and are uncorrected. Infrared spectra
were recorded on
a Magna 750 FTIR spectro-
1
photometer. H NMR spectra were recorded on a Var-
ian Unity 300 spectrometer. The chemical shifts are
reported in parts per million (ppm) relative to internal
tetramethylsilane in CDCl3 or DMSO-d6. 1H noisy-
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 d 77.0 or 39.5, respectively. Fast-atom
bombardment mass spectra (FAB±MS) were obtained
on a VG Quattro mass spectrometer. Analytical thin-
layer chromatography (TLC) was performed on Merck
silica gel 60F-254 glass plates. Medium-pressure chro-
matography (MPLC) was performed using Merck silica
gel 60 (230±400 mesh) with a VSP-2200 ceramic pump
(Eyela). Elemental analyses were performed on a Carlo
Erba 1106 elemental analyzer.
2-Amino-6-chloro-9-(4-hydroxy-3-isopropoxycarbonyloxy-
methylbut-1-yl)purine (15) and 2-amino-9-(3-hydroxy-4-
hydroxymethylbut-1-yl)purin-6-ylpyridinium chloride (16).
To a cooled solution of 6-deoxy-6-chloropenciclovir
(537 g, 1.98 mol) in anhydrous pyridine (5.7 L) in an ice
bath under N2 was added dropwise isopropyl chloro-
formate (267 g, 2.18 mol) as a neat solution over 1.5 h,
In conclusion, 2-amino-9-(3-acetoxymethyl-4-isopropoxy-
carbonyloxybut-1-yl)purine (4) showed the highest oral
mean urinary recovery of penciclovir in rats in a series
of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxy-
but-1-yl)purines and 2-amino-9-(3-alkoxycarbonyloxy-
methyl-4-alkoxycarbonyloxy-but-1-yl)purines. The mean
Scheme 4. (a) Lipase (PPL, PFL or CAL), sodium phosphate bu€er (pH 7.0), rt; (b) lipase (PPL, PFL or CAL), vinyl acetate, acetone or toluene, rt.

1722
D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
1
and the mixture was stirred in an ice bath for 4 h after
the addition. The reaction mixture was evaporated to
dryness under the reduced pressure, and a mess of a
dark yellow solid was formed during the evaporation.
The resulting dark brown residue was suspended in
CHCl3 (2 L), and insoluble dark yellow solid was col-
lected by ®ltration. Filtrate was concentrated in vacuo,
and the gummy residue was partitioned between H2O
(1 L) and EtOAc (7Â500 mL). The combined organic
layers were dried over MgSO4, ®ltered, and evaporated
to dryness under the reduced pressure to give a dark
brown oil. The crude mixture was puri®ed by MPLC on
silica gel with CH3CN/EtOAc (1/9) as eluent to a€ord
67 g (9.5%) of 15 as an o€-white solid, which was crys-
tallized from EtOAc: mp 117ꢀC dec; IR (neat) 3347,
3209, 3104 (NH2 and OH), 1738 (CˆO); 1H NMR
(CDCl3) d 1.30 (d, J=6.0 Hz, 6H, CH(CH3)2), 1.85±
2.09 (m, 3H, CHCH2CH2), 2.38 (br s, 1H, OH), 3.71 (t,
J=4.2 Hz, 2H, CH2OH), 4.20±4.26 (m, 4H, NCH2 and
OCH2CH), 4.88 (septet, J=6.0 Hz, 1H, CH(CH3)2),
5.14 (br s, 2H, NH2), 7.80 (s, 1H, H-8); MS (FAB) m/z
358 (MH+). Anal. calcd for C14H20 ClN5O4: C, 47.00;
H, 5.63; N, 19.57. Found: C, 47.11; H, 5.67; N, 19.42.
The above obtained dark yellow solid was triturated
from MeOH/CHCl3 to give 360 g (52%) of 16: mp
152ꢀC dec (MeOH/CHCl3); IR (neat) 3345, 3292, 3257,
3182 (NH2 and OH), 1659, 1636, 1574, 1562 (CˆN and
cm 1; H NMR (DMSO-d6) d 0.99 (t, J=7.5 Hz, 3H,
CH2CH3), 1.60±1.70 (m, 1H, CH), 1.75±1.90 (m, 2H,
CHCH2CH2), 2.27 (q, J=7.5 Hz, 2H, CH2CH3), 3.42
(m, 2H, CHCH2OH), 3.95±4.06 (m, 2H, OCH2CH),
4.10 (t, J=7.2 Hz, 2H, NCH2), 4.64 (t, J=5.1 Hz, 1H,
OH), 6.47 (br s, 2H, NH2), 8.08 (s, 1H, H-8), 8.56 (s,
1H, H-6); 13C NMR (DMSO-d6) d 8.9, 26.7, 28.0, 37.6,
40.3, 60.5, 63.9, 126.9, 142.6, 148.9, 152.9, 160.4, 173.6;
MS (FAB) m/z 294 (MH+). Anal. calcd for C13H19
N5O3: C, 53.23; H, 6.53; N, 23.88. Found: C, 53.11; H,
6.43; N, 23.92.
General procedure for the preparation of 2-amino-9-(3-
acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purines 1±8.
A mixture of 17 or 18 (2.16 mmol), alkyl 4-nitrophenyl
carbonate (2.59 mmol), and a catalytic amount of
DMAP (26 mg, 0.21 mmol) in anhydrous pyridine
(7 mL) was heated at 80ꢀC under N2 for 20 h, and the
resulting reaction mixture was evaporated to dryness in
vacuo to a€ord a yellow residue. The crude product was
puri®ed by MPLC on silica gel (gradient elution: 2%
MeOH in CHCl3 followed by 3% MeOH in CHCl3) to
a€ord the titled compound as a white solid, which was
crystallized from a suitable solvent indicated.
2-Amino-9-(3-acetoxymethyl-4-methoxycarbonyloxybut-
1-yl)purine (1). Yield 86%; mp 71.0±72.5ꢀC (EtOAc/
1
1
CˆC) cm 1; H NMR (DMSO-d6) d 1.44±1.55 (m, 1H,
Et2O); IR (neat) 3338 (NH2), 1747, 1730 (CˆO) cm
;
CH), 1.76±1.88 (m, 2H, CHCH2CH2), 3.34±3.50 (m,
4H, 2 OCH2), 4.26 (t, J=7.4 Hz, 2H, NCH2), 4.58 (t,
J=5.0 Hz, 2H, 2 OH), 7.35 (br s, 2H, NH2), 8.46 (dd,
J=7.3 Hz, 6.2 Hz, 2H, H-30 and H-5), 8.59 (s, 1H, H-8),
8.93 (t, J=7.3 Hz, 1H, H-40), 10.08 (d, J=6.2 Hz, 2H,
H-20 and H-60); MS (FAB) m/z 315 (M Cl)+. Anal.
calcd for C15H19ClN6O2: C, 51.36; H, 5.46; N, 23.96.
Found: C, 51.53; H, 5.59; N, 24.07.
1H NMR (CDCl3) d 1.90±2.05 (m, 3H, CHCH2CH2),
2.06 (s, 3H, COCH3), 3.80 (s, 3H, OCH3), 4.13±4.29 (m,
6H, NCH2 and 2 OCH2CH), 5.09 (br s, 2H, NH2), 7.78
(s, 1H, H-8), 8.70 (s, 1H, H-6); 13C NMR (CDCl3) d
20.8, 28.7, 35.1, 40.7, 53.9, 63.4, 67.3, 128.3, 142.1,
149.9, 153.2, 155.6, 159.9, 170.8; MS (FAB) m/z 338
(MH+). Anal. calcd for C14H19N5O5: C, 49.85; H, 5.68;
N, 20.76. Found: C, 49.97; H, 5.62; N, 20.65.
General procedure for the preparation of 2-amino-9-(3-
acyloxymethyl-4-hydroxybut-1-yl)purines 17 and 18.
To a stirred solution of 6-deoxypenciclovir (6.00 g,
2-Amino-9-(3-acetoxymethyl-4-ethoxycarbonyloxybut-1-
yl)purine (2). Yield 87%; mp 92.0±93.5ꢀC (EtOAc/
1
Et2O); IR (neat) 3336 (NH2), 1746, 1735 (CˆO) cm
;
1H NMR (CDCl3) d 1.32 (t, J=7.2 Hz, 3H, CH2CH3),
1.90±2.05 (m, 3H, CHCH2CH2), 2.06 (s, 3H, COCH3),
4.14±4.26 (m, 8H, NCH2 and 3 OCH2), 5.10 (br s, 2H,
NH2), 7.78 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13C NMR
(CDCl3) d 14.2, 20.8, 28.7, 35.1, 40.8, 63.5, 65.8, 67.0,
128.3, 142.2, 149.9, 153.2, 155.0, 159.9, 170.8; MS
(FAB) m/z 352 (MH+). Anal. calcd for C15H21N5O5:C,
51.28; H, 6.02; N, 19.93. Found: C, 51.37; H, 6.16; N,
19.81.
.
25.3 mmol) and a catalytic amount of p-TsOH H2O
(0.48 g, 2.53 mmol) in anhydrous DMF (70 mL) was
added dropwise trimethyl orthoacetate or triethyl
orthopropionate (30.4 mmol) via a syringe at room
temperature, and the resulting reaction mixture was
stirred at room temperature for 2 h. H2O (3 mL) was
added, and the mixture was stirred for an additional 1 h.
The reaction mixture was neutralized with NaHCO3
(0.21 g, 2.53 mmol) in 3 mL of H2O and was evaporated
to dryness in vacuo to a€ord a yellowish residue. The
crude product was puri®ed by MPLC on silica gel with
MeOH/CHCl3 (1/9) as eluent to a€ord the titled com-
pound as a white solid, which was crystallized from a
suitable solvent.
2-Amino-9-(3-acetoxymethyl-4-propoxycarbonyloxybut-
1-yl)purine (3). Yield 86%; mp 72.5±73.5ꢀC (EtOAc/
Et2O/hexanes); IR (neat) 3337 (NH2), 1750, 1735
1
(CˆO) cm 1; H NMR (CDCl3) d 0.96 (t, J=7.4 Hz,
3H, CH2CH2CH3), 1.60±1.74 (m, 2H, CH2CH2CH3),
1.90±2.05 (m, 3H, CHCH2CH2), 2.06 (s, 3H, COCH3),
4.11 (t, J=6.8 Hz, 2H, OCH2CH2), 4.15±4.24 (m, 6H,
NCH2 and 2 OCH2), 5.08 (br s, 2H, NH2), 7.78 (s, 1H,
H-8), 8.70 (s, 1H, H-6); 13C NMR (CDCl3) d 10.1, 20.8,
22.0, 28.7, 35.1, 40.8, 63.5, 67.1, 69.9, 128.3, 142.2,
149.9, 153.2, 155.2, 159.9, 170.8; MS (FAB) m/z 366
(MH+). Anal. calcd for C16H23N5O5: C, 52.60; H, 6.34;
N, 19.17. Found: C, 52.32; H, 6.39; N, 19.31.
2-Amino-9-(3-acetoxymethyl-4-hydroxybut-1-yl)purine
(17). Yield 95%; mp 164.2±164.5ꢀC (MeOH) (lit.11
166±168ꢀC); spectroscopic data were identical to those
reported.
2-Amino-9-(4-hydroxy-3-propionyloxymethylbut-1-yl)-
purine (18). Yield 92%; mp 149.5±150.3ꢀC (EtOH); IR
(neat) 3367, 3326, 3191 (NH2 and OH), 1727 (CˆO)

D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
1723
2-Amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxy-
but-1-yl)purine (4). Yield 89%; mp 106.5±107.7ꢀC
(EtOAc/hexanes); IR (neat) 3337 (NH2), 1744, 1736
4.88 (septet, J=6.3 Hz, 1H, CH(CH3)2), 5.10 (br s, 2H,
NH2), 7.78 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13C NMR
(CDCl3) d 9.0, 21.7, 27.4, 28.7, 35.2, 40.8, 63.3, 66.9,
72.3, 128.2, 142.1, 149.8, 153.2, 154.5, 159.9, 174.2; MS
(FAB) m/z 380 (MH+). Anal. calcd for C17H25N5O5: C,
53.82; H, 6.64; N, 18.46. Found: C, 53.56; H, 6.67; N,
18.43.
1
(CˆO) cm 1; H NMR (CDCl3) d 1.30 (d, J=6.3 Hz,
6H, CH(CH3)2), 1.90±2.05 (m, 3H, CHCH2CH2), 2.06
(s, 3H, COCH3), 4.15±4.24 (m, 6H, NCH2 and 2
OCH2), 4.88 (septet, J=6.3 Hz, 1H, CH(CH3)2), 5.10
(br s, 2H, NH2), 7.78 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13
C
NMR (CDCl3) d 20.8, 21.7, 28.7, 35.1, 40.8, 63.5, 66.8,
72.3, 128.3, 142.1, 149.9, 153.2, 154.5, 159.9, 170.8; MS
(FAB) m/z 366 (MH+). Anal. calcd for C16H23N5O5: C,
52.60; H, 6.34; N, 19.17. Found: C, 52.45; H, 6.30; N,
19.19.
General procedure for the preparation of 2-amino-9-(3-
alkoxycarbonyloxymethyl-4-alkoxycarbonyloxybut-1-yl)-
purines 9±12. A stirred suspension of 6-deoxypenciclovir
(0.50 g, 2.11 mmol), alkyl 4-nitrophenyl carbonate
(4.43 mmol) and a catalytic amount of DMAP (26 mg,
0.21 mmol) in anhydrous pyridine (8 mL) was heated at
80ꢀC for 24 h, and the resulting homogeneous mixture
was evaporated to dryness in vacuo to a€ord a yellow
residue. The crude product was puri®ed by MPLC on
silica gel (gradient elution: 2% MeOH in CHCl3 fol-
lowed by 3% MeOH in CHCl3) to a€ord the titled
compound as a white solid, which was crystallized from
a suitable solvent indicated.
2-Amino-9-(4-methoxycarbonyloxy-3-propionyloxymethyl-
but-1-yl)purine (5). Yield 93%; mp 72.0±73.0ꢀC (EtOAc/
Et2O); IR (neat) 3383, 3335 (NH2), 1746, 1652 (CˆO)
1
cm
;
1H NMR (CDCl3) d 1.14 (t, J=7.8 Hz, 3H,
COCH2CH3), 1.95±2.08 (m, 3H, CHCH2CH2), 2.34 (q,
J=7.8 Hz, 2H, COCH2CH3), 3.80 (s, 3H, OCH3), 4.16-
4.24 (m, 6H, NCH2 and 2 OCH2), 5.14 (br s, 2H, NH2),
7.78 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13C NMR (CDCl3)
d 9.0, 27.4, 28.7, 35.1, 40.7, 54.9, 63.2, 67.3, 128.2, 142.1,
149.8, 153.2, 155.6, 159.9, 174.1; MS (FAB) m/z 352
(MH+). Anal. calcd for C15H21N5O5: C, 51.28; H, 6.02;
N, 19.93. Found: C, 51.35; H, 6.15; N, 20.08.
2-Amino-9-(3-methoxycarbonyloxymethyl-4-methoxycarb-
onyloxybut-1-yl)purine (9). Yield 83%; mp 111.0±
112.5ꢀC (EtOAc/hexanes); IR (neat) 3460, 3309 (NH2),
1742, 1732 (CˆO) cm 1; 1H NMR (CDCl3) d 1.97±2.11
(m, 3H, CHCH2CH2), 3.79 (s, 6H, 2 OCH3), 4.20±4.24
(m, 6H, NCH2 and 2 OCH2CH), 5.03 (br s, 2H, NH2),
7.78 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13C NMR (CDCl3)
d 28.6, 35.2, 40.8, 55.0, 67.0, 128.3, 142.2, 149.9, 153.2,
155.6, 159.8; MS (FAB) m/z 354 (MH+). Anal. calcd
for C14H19N5O6: C, 47.59; H, 5.42; N, 19.82. Found: C,
47.68; H, 5.52; N, 19.85.
2-Amino-9-(4-ethoxycarbonyloxy-3-propionyloxymethyl-
but-1-yl)purine (6). Yield 93%; mp 65.0±66.2ꢀC
(EtOAc/Et2O); IR (neat) 3387, 3336 (NH2), 1746, 1652
1
(CˆO) cm 1; H NMR (CDCl3) d 1.14 (t, J=7.8 Hz,
3H, COCH2CH3), 1.31 (t, J=7.2 Hz, 3H, OCH2CH3),
1.95±2.07 (m, 3H, CHCH2CH2), 2.34 (q, J=7.8 Hz, 2H,
COCH2CH3), 4.17±4.24 (m, 8H, NCH2 and 3 OCH2),
5.14 (br s, 2H, NH2), 7.78 (s, 1H, H-8), 8.69 (s, 1H, H-
6); 13C NMR (CDCl3) d 9.0, 14.2, 27.4, 28.7, 35.1, 40.7,
63.3, 64.2, 67.0, 128.2, 142.1, 149.8, 153.2, 155.0, 159.9,
174.1; MS (FAB) m/z 366 (MH+). Anal. calcd for
C16H23N5O5: C, 52.60; H, 6.34; N, 19.17. Found: C,
52.83; H, 6.46; N, 19.06.
2-Amino-9-(3-ethoxycarbonyloxymethyl-4-ethoxycarbonyl-
oxybut-1-yl)purine (10). Yield 91%; mp 72.5±73.5ꢀC
(EtOAc/hexanes); IR (neat) 3383, 3334 (NH2), 1743
1
(CˆO) cm 1; H NMR (CDCl3) d 1.31 (t, J=7.2 Hz,
6H, 2 OCH2CH3), 1.95±2.15 (m, 3H, CHCH2CH2),
4.17±4.25 (m, 10H, NCH2 and 4 OCH2), 5.04 (br s, 2H,
NH2), 7.78 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13C NMR
(CDCl3) d 14.2, 28.5, 35.2, 40.7, 64.3, 66.7, 128.2, 142.2,
149.8, 153.2, 154.9, 159.8; MS (FAB) m/z 382 (MH+).
Anal. calcd for C16H23N5O6: C, 50.39; H, 6.08; N,
18.36. Found: C, 50.12; H, 6.01; N, 18.51.
2-Amino-9-(3-propionyloxymethyl-4-propoxycarbonyloxy-
but-1-yl)purine (7). Yield 94%; mp 63.0±64.5ꢀC (Et2O/
hexanes); IR (neat) 3387, 3336 (NH2), 1747, 1650
1
(CˆO) cm 1; H NMR (CDCl3) d 0.96 (t, J=7.5 Hz,
3H, OCH2CH2CH3), 1.14 (t, J=7.8 Hz, 3H, COCH2
CH3), 1.64±1.76 (m, 2H, OCH2CH2CH3), 1.96±2.07 (m,
3H, CHCH2CH2), 2.34 (q, J=7.8 Hz, 2H, COCH2
CH3), 4.13 (t, J=6.9 Hz, 2H, OCH2CH2CH3), 4.17±
4.24 (m, 6H, NCH2 and 2 OCH2), 5.09 (br s, 2H, NH2),
7.78 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13C NMR (CDCl3)
d 9.0, 10.1, 21.9, 27.4, 28.7, 35.2, 40.8, 63.3, 67.1, 69.9,
128.3, 142.2, 149.9, 153.2, 155.1, 159.9, 174.2; MS (FAB)
m/z 380 (MH+). Anal. calcd for C17H25N5O5: C, 53.82;
H, 6.64; N, 18.46. Found: C, 53.60; H, 6.65; N, 18.59.
2-Amino-9-(3-propoxycarbonyloxymethyl-4-propoxycarb-
onyloxybut-1-yl)purine (11). Yield 83%; mp 72.0±73.5ꢀC
(EtOAc/hexanes); IR (neat) 3401, 3332 (NH2), 1746
1
(CˆO) cm 1; H NMR (CDCl3) d 0.96 (t, J=7.5 Hz,
6H, 2 OCH2CH2CH3), 1.65±1.75 (m, 4H, 2 OCH2CH2
CH3), 1.95±2.17 (m, 3H, CHCH2CH2), 4.10 (t, J=
6.8 Hz, 4H, 2 OCH2CH2CH3), 4.20±4.25 (m, 6H, NCH2
and 2 OCH2), 5.04 (br s, 2H, NH2), 7.78 (s, 1H, H-8),
8.69 (s, 1H, H-6); 13C NMR (CDCl3) d 10.1, 22.0, 28.6,
35.3, 40.8, 66.8, 69.9, 128.3, 142.3, 149.8, 153.2, 155.1,
159.8; MS (FAB) m/z 410 (MH+). Anal. calcd for
C18H27N5O6: C, 52.80; H, 6.65; N, 17.10. Found: C,
52.47; H, 6.69; N, 17.15.
2-Amino-9-(4-isopropoxycarbonyloxy-3-propionyloxy-
methylbut-1-yl)purine (8). Yield 94%; mp 72.5±73.5ꢀC
(Et2O/hexanes); IR (neat) 3386, 3336 (NH2), 1742, 1651
1
(CˆO) cm 1; H NMR (CDCl3) d 1.14 (t, J=7.5 Hz,
3H, COCH2CH3), 1.30 (d, J=6.3 Hz, 6H, CH(CH3)2),
1.94±2.10 (m, 3H, CHCH2CH2), 2.34 (q, J=7.5 Hz, 2H,
COCH2CH3), 4.17±4.24 (m, 6H, NCH2 and 2 OCH2),
2-Amino-9-(3-isopropoxycarbonyloxymethyl-4-isopropoxy-
carbonyloxybut-1-yl)purine (12). Yield 85%; mp 74.5±
75.0ꢀC (EtOAc/hexanes); IR (neat) 3397, 3335 (NH2),

1724
D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
1
1738 (CˆO) cm
;
1H NMR (CDCl3) d 1.30 (d,
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 ®l-
tered (0.45-mm), and the concentrations of penciclovir
and 6-deoxypenciclovir were analyzed by HPLC as fol-
lows. A C18 reversed-phase column equipped with a
compatible guard column was eluted at a ¯ow rate of
1 mL/min with the following three-step gradient: (step
1) a 10-min isocratic elution with 100% bu€er A (0.1%
phosphoric acid), (step 2) a 25-min linear gradient from
100% bu€er A to 55% bu€er A and 45% bu€er B (80%
MeCN in 0.1% phosphoric acid), and (step 3) a 4-min
isocratic elution with 55% bu€er A and 45% bu€er B.
The column was equilibrated with 100% bu€er A for
10 min before each sample injection. The UV absor-
bance of the column e‚uent was monitored at 248 nm.
J=6.3 Hz, 12H, 2 CH(CH3)2), 1.95±2.10 (m, 3H,
CHCH2CH2), 4.21±4.25 (m, 6H, NCH2 and 2 OCH2),
4.87 (septet, J=6.3 Hz, 2H, 2 CH(CH3)2), 5.12 (br s,
2H, NH2), 7.79 (s, 1H, H-8), 8.69 (s, 1H, H-6); 13C
NMR (CDCl3) d 21.7, 28.6, 35.2, 40.7, 66.6, 72.3, 128.2,
142.2, 149.8, 153.2, 154.5, 159.9; MS (FAB) m/z 410
(MH+). Anal. calcd for C18H27N5O6: C, 52.80; H, 6.65;
N, 17.10. Found: C, 52.72; H, 6.69; N, 17.17.
9-(3-Hydroxymethyl-4-isopropoxycarbonyloxybut-1-yl)-
guanine (19). A mixture of 15 (420 mg, 1.17 mmol) and
adenosine deaminase (42 mg; Sigma, Type VII from
Calf Intestinal Mucosa) in phosphate bu€er solution
(80 mL, pH 7.5) was vigorously stirred at room tem-
perature for 5 days. The reaction mixture was ®ltered
through a Celite pad, and the ®ltrate was evaporated to
dryness under the reduced pressure. The resulting resi-
due was puri®ed by MPLC on silica gel (gradient elu-
tion: 15% MeOH in CHCl3 followed by 20% MeOH in
CHCl3) to a€ord 370 mg (93%) of 19 as a white solid,
which was crystallized from absolute EtOH: mp 143ꢀC
Pharmacokinetics in mice. Four male ICR mice (25±
30 g) were each given a single dose of 4 and famciclovir
(0.2 mmol/kg/10 mL) with an intragastric needle. Blood
was collected at 0.25, 0.5, 1, 1.5, 2, and 4 h after dosing
by cardiac puncture using heparinized syringe. An ali-
quot (150 mL) of each sample was immediately mixed
with trichloroacetic acid (150 mL) (8% ®nal concentra-
tion) in a separate tube and centrifuged. The super-
natant (200 mL) was neutralized with 1/5 volume
(40 mL) of saturated aqueous NaHCO3. The concentra-
tions of 4, penciclovir, 6-deoxypenciclovir, and other
postulated metabolites of 4, SK 1875, 17, 19, 20, and 21,
were analyzed by HPLC as follows. A C18 reversed-
phase column equipped with a precolumn was eluted at
a ¯ow rate of 1 mL/min with the following four-step
gradient: (step 1) a 5-min isocratic elution with 99%
solution A (5 mM K2HPO4) and 1% solution B (80%
MeOH in 5 mM K2HPO4), (step 2) a 10-min linear gra-
dient from 99% solution A and 1% solution B to 85%
solution A and 15% solution B, (step 3) a 19-min linear
gradient from 85% solution A and 15% solution B to
5% solution A and 95% solution B, and (step 4) a 5-min
isocratic elution with 5% solution A and 95% solution
B. Before each sample injection, the column was re-
equilibrated to initial conditions. The UV absorbance of
the column e‚uent was monitored at both 256 and
300 nm with dual l absorbance detector.
dec; IR (neat) 3434, 3317, 3197 (NH2 and OH), 1741,
1
1635 (CˆO) cm
;
1H NMR (DMSO-d6) d 1.21 (d,
J=6.0 Hz, 6H, CH(CH3)2), 1.63±1.84 (m, 3H, CHCH2
CH2), 3.39 (t, J=5.1 Hz, 2H, CH2OH), 3.96±4.11 (m,
4H, NCH2 and OCH2), 4.66 (t, J=5.1 Hz, 1H,
CH2OH), 4.73 (septet, J=6.0 Hz, 1H, CH(CH3)2), 6.42
(br s, 2H, NH2), 7.69 (s, 1H, H-8),10.51 (br s, 1H, NH);
MS (FAB) m/z 340 (MH+). Anal. calcd for C14H21
N5O5: C, 49.55; H, 6.24; N, 20.64. Found: C, 49.67; H,
6.29; N, 20.52.
9-(3-Acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)-
guanine (20). To a solution of 19 (80 mg, 0.24 mmol)
and a catalytic amount of DMAP (3 mg, 0.02 mmol) in
anhydrous DMF (1 mL) at room temperature under N2
was added acetic anhydride (48 mg, 45 mL, 0.47 mmol),
and the mixture was stirred at room temperature for 1 h.
The reaction was quenched with MeOH (1 mL), and the
mixture was evaporated to dryness under the reduced
pressure. The resulting residue was puri®ed by MPLC
on silica gel (gradient elution: 5% MeOH in CHCl3
followed by 10% MeOH in CHCl3) to a€ord an o€-
white solid, which was crystallized from absolute
MeOH/Et2O to give 75 mg (83%) of 20: mp 186ꢀC dec;
IR (neat) 3319, 3161 (NH2), 1741, 1691, 1651 (CˆO)
Urinary recovery in mice. Five male ICR mice (25±30 g)
were each given a single dose of 4 and famciclovir
(0.2 mmol/kg/10 mL) with an intragastric needle. Urine
was collected for 24 h in a metabolic cage after dosing.
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. An ali-
quot (100 mL) of each sample was diluted with 1 mM
Na2HPO4 (pH 7.0) (500 mL), was loaded onto the
cation-exchange Sep-Pak cartridge pretreated with
MeOH (1 mL) and H2O (1 mL), and was then eluted
with 100 mM Na2HPO4 (pH 11.0) (1 mL). The con-
centrations of 4, penciclovir, 6-deoxypenciclovir, and
other postulated metabolites of 4, SK 1875, 17, 19, 20,
and 21 in the urine samples were analyzed by reversed-
phase HPLC using a C18 column with a UV detector as
afore-mentioned conditions in the pharmacokinetic
study.
cm 1; H NMR (DMSO-d6) d 1.21 (d, J=6.0 Hz, 6H,
1
CH(CH3)2), 1.75±1.82 (m, 2H, CHCH2CH2), 1.90±1.99
(m, 1H, CHCH2CH2), 2.00 (s, 3H, COCH3), 3.99±4.08
(m, 6H, NCH2 and 2 OCH2), 4.73 (septet, J=6.0 Hz,
1H, CH(CH3)2), 6.41 (br s, 2H, NH2), 7.70 (s, 1H, H-
8),10.53 (br s, 1H, NH); MS (FAB) m/z 382 (MH+).
Anal. calcd for C16H23N5O6: C, 50.39; H, 6.08; N,
18.36. Found: C, 50.47; H, 6.13; N, 18.41.
Urinary recovery in rats. The bioavailability of the test
compound was estimated by determining the total
amount of penciclovir and 6-deoxypenciclovir 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 two male
Sprague±Dawley rats (200±250 g). A 5% solution of

D.-K. Kim et al. / Bioorg. Med. Chem. 7 (1999) 1715±1725
1725
In vivo antiviral ecacy. HSV-1 (KOS strain) suspen-
sion (3Â104 PFU/0.1 mL) was inoculated i.p. to 4.5-
week-old female BALB/c mice (17±21 g). Each group
consisted of 10 mice. Test compounds were dissolved in
H2O to give a dose of 0.075 mmol/kg of body weight per
mouse in 10 mL. The compounds were administered by
oral gavage twice daily at 8 a.m. and 6 p.m. for 5 days,
commencing at 18 h postinfection. Mortality was
observed for 21 days after virus inoculation. The two-
tailed Fisher's exact test was used for determination of
signi®cant di€erence in survival rate, and the Mann±
Whitney rank sum test was used for determination of
signi®cant di€erence in mean survival time. P Values of
<0.05 were considered statistically signi®cant.
11. Harnden, M. R.; Jarvest, R. L.; Boyd, M. R.; Sutton, D.;
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1994, 42, 306±310.
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Oldhafer, K., Boker, K. H. W., Pichlmayr, R., Manns, M. P.
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Acknowledgements
18. Kim, D.-K.; Lee, N.; Im, G.-J.; Kim, Y.-W.; Chang, K.;
Kim, H.-T.; Cho, Y.-B.; Choi, W.-S.; Jung, I.; Kim, K. H.
Bioorg. Med. Chem. Lett. 1996, 6, 1849±1854.
19. Kim, D.-K.; Lee, N.; Kim, Y.-W.; Chang, K.; Im, G.-J.;
Choi, W.-S.; Kim, K. H. Bioorg. Med. Chem. 1999, 7, 419±
424.
This work was supported in part by a grant HMP-97-D-
1-0002 from the Korean Ministry of Health and Welfare.
20. Kim, D.-K.; Lee, N.; Kim, H.-T.; Im, G.-J.; Kim, K. H.
Bioorg. Med. Chem. 1999, 7, 565±570.
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