Inhibition of herpes simplex virus thymidine kinases by 2-phenylamino-6-oxopurines and related compounds: Structure-activity relationships and antiherpetic activity in vivo

Article abstract of DOI:10.1021/jm049059x

Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [ ³H] thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl) butyl]-6-oxopurine, was a competitive inhibitor, with K_i values of 0.03 and 0.005 μM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [³H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.

Full text of DOI:10.1021/jm049059x

J. Med. Chem. 2005, 48, 3919-3929
3919
Inhibition of Herpes Simplex Virus Thymidine Kinases by
2-Phenylamino-6-oxopurines and Related Compounds: Structure-Activity
Relationships and Antiherpetic Activity in Vivo
Andrzej Manikowski,† Annalisa Verri,‡ Andrea Lossani,‡ Bryan M. Gebhardt,§ Joseph Gambino,†
Federico Focher,‡ Silvio Spadari,‡ and George E. Wright*,†
GLSynthesis Inc., One Innovation Drive, Worcester, Massachusetts 01605, Istituto di Genetica Molecolare, Consiglio Nazionale
delle Ricerche, Pavia, Italy 27100, and LSU Eye Center, Louisiana State University Health Sciences Center,
New Orleans, Louisiana 70112
Received November 19, 2004
Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-
hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against
recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds
inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantita-
tively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in
inhibitory potency were generally consistent with the inhibitor/substrate binding site structure
based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl)
analogues with bulky tertiary amino substituents were among the most potent inhibitors.
Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-
decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and
0.005 µM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was
uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and
against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to
be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may
relate to its antiherpetic activity in vivo.
Introduction
there is a need for an antiviral drug that can enter the
CNS and effectively inhibit HSV infection.
Herpes simplex viruses (HSV-1 and HSV-2) establish
lifelong latency in neurons.1 Latency is interrupted
periodically by reactivation, leading to recurrent dis-
ease, but a significant proportion of infected individuals
may shed infectious virus without apparent signs of
disease. Thus, these individuals represent a real threat
for venereal spread. In addition, herpes simplex viruses
may cause life-threatening encephalitis, both in neo-
nates, as a result of virus exposure during birth, and
in immunocompromised adults. Herpetic encephalitis
results in high mortality, and survivors are often
severely handicapped. Herpetic encephalitis occurs in
about 3000 newborns each year in the United States. If
infection results in disseminated disease, central ner-
vous system (CNS) disease occurs in up to 50% of the
infants and can result in 75% mortality. For the
survivors of CNS involvement, the outcome is bleak,
with psychomotor retardation in 50-75% of the survi-
vors. Acyclovir (ACV) and foscarnet (PFA) have been
employed for HSV encephalitis and for therapy of
newborns with localized and disseminated HSV disease.
Although these treatments have reduced mortality from
encephalitis to 15%, only 50% of survivors develop
normally.2 Unfortunately, the overall mortality for
disseminated neonatal herpes remains very high (40-
65%), regardless of present antiviral therapy.3 Clearly
Competitive inhibitors of HSV thymidine kinases (TK)
prevent viral reactivation in vitro and in vivo, and
expression of viral DNA in the infected nerve ganglia
is reduced by treatment with TK inhibitors.4 These
results have demonstrated the validity of HSV TK as a
target for antiviral therapy and encouraged us to
proceed with the optimization and development of these
compounds for eventual clinical application. The first
task was to make available significant quantities of the
HSV enzymes, and this has been accomplished by
expression of recombinant TKs. Second, we have syn-
thesized several new candidate inhibitors to extend the
structure-activity relationships of 2-phenylamino-6-
oxopurines (“N2-phenylguanines”) and have found highly
potent inhibitors of both HSV-1 and HSV-2 TKs. Finally,
we have found that a lead compound, 2-phenylamino-
9-(4-hydroxybutyl)-6-oxopurine (HBPG, 1), effectively
* Corresponding author. Phone: (508) 754-6700. Fax: (508) 754-
7075. E-mail: george.wright@glsynthesis.com.
† GLSynthesis Inc.
‡ Consiglio Nazionale delle Ricerche.
§ Louisiana State University Health Sciences Center.
10.1021/jm049059x CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/11/2005

3920 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Manikowski et al.
Scheme 1
prevents lethal encephalitis in mice and is an efficient
alternate substrate for the viral TKs.
of each enzyme. Several concentrations of [3H]thymidine
(TdR) were incubated at 37 °C in the presence of a
limiting amount of enzyme, and the reaction velocity
(V) was determined at each substrate concentration (S).
By plotting V and S in double-reciprocal format, we
obtained the Lineweaver-Burke plots shown in Figure
1. These results indicate that recombinant HSV-1 and
HSV-2 TKs had Km values of 1 and 2 µM, respectively,
identical to those reported for the enzymes purified from
corresponding HSV-infected cells.5,6
Inhibition of HSV TKs by 9-Substituted-6-oxo-
purines. Screening of new compounds against the
recombinant TKs was done by measuring the effect of
each test compound on phosphorylation of [3H]TdR,
present at the Km value for each enzyme. In this way,
relative potencies, IC50s, have been obtained, and these
are summarized in Table 1. HBPG (1) inhibited the
recombinant HSV-1 and HSV-2 TKs with IC50 values
of 1.3 and 0.5 µM, respectively (Table 1). The activities
are of the same order of magnitude, but of opposite
Results
Synthesis of Inhibitors. 2-Bromohypoxanthine
served as common precursor of 2-phenylthio- and 2-phe-
noxy-6-oxopurine, as described previously for prepara-
tion of 2-phenylamino-6-oxopurine.5 These compounds
were converted into the corresponding 6-chloropurines
with phosphoryl chloride (Scheme 1). Alkylation of the
6-chloropurines with 4-acetoxybutyl bromide and sepa-
ration of the major 9-isomers, followed by alkaline
hydrolysis, gave 2-phenylthio (3) and 2-phenoxy (4)
analogues of 1 in high yields. A similar strategy with
2-phenylamino-6-chloropurine and other ω-substituted
alkyl halides gave several related 9-hydroxyalkyl, 9-meth-
oxybutyl, and 9-carboxypropyl compounds. Methylation
of the 6-chloro derivative of 1, followed by alkaline
hydrolysis, provided 2-(N-methyl-N-phenylamino)-9-(4-
hydroxybutyl)-6-oxopurine (5). Conversion of 1 to the
9-(4-iodobutyl) derivative (11), followed by reaction of
the latter intermediate with numerous secondary amines,
provided a series of 9-(4-aminobutyl)-2-phenylamino-6-
oxopurines, 12a-s (Scheme 1).
Cloning and Expression of HSV-1 and HSV-2
Thymidine Kinases. The complete coding sequences
of HSV-1 TK and HSV-2 TKs were expressed from the
pTrcHis vector as N-terminal fusion proteins. The fusion
proteins contain six tandem histidine residues that
allow one-step purification of the enzymes with a nickel-
chelating resin. Cloning and expression were performed
as described in the Experimental Section. One liter of
bacterial culture produced approximately 5 mg of tagged
TK, which was eluted from a Ni-NTA Superflow
column (1 mL) as a single sharp peak following step
elution with 250 mM imidazole.
Figure 1. Determination of Km values of recombinant HSV
TKs. Lineweaver-Burke (double reciprocal) plots of the activ-
ity of recombinant HSV TKs in the presence of increasing
concentrations of substrate, [3H]TdR, assayed as described in
the Experimental Section. HSV-1 TK (b), HSV-2 TK (O).
To determine the behavior of recombinant HSV TKs,
experiments were conducted in vitro to measure the Km

Herpes Simplex Virus Thymidine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3921
Table 1. Inhibition of HSV TKs by 9-Substituted-6-oxopurines

3922 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Table 1 (Continued)
Manikowski et al.
potencies, as those reported for the enzymes isolated
from virus-infected cells.7 The significance of the N2-
phenyl substituent in this family of inhibitors is em-
phasized by the weak inhibition afforded by 9-(4-
hydroxybutyl)guanine (2), a weak alternate substrate
of HSV TK and antiherpetic compound.8 The require-
ment of the 2-NH group of active compounds was probed
by the assay of 2-phenylthio (3) and 2-phenoxy (4)
analogues; both derivatives were potent inhibitors of
HSV-1 TK, but less potent against HSV-2 TK. In
contrast, the N-methyl analogue 5 was weakly inhibi-
tory to both TKs. Although no direct interactions of
2-NH were observed in the X-ray structure of 1 bound
to HSV-1 TK,9 the drastic reduction of affinity of 5 is
consistent with a significant steric effect of the N-Me
group.
The 9-(2-hydroxyethyl) (6) and 9-(3-hydroxypropyl) (7)
homologues of 1 inhibited both TKs within a few-fold
of 1, but the 9-(5-hydroxypentyl) compound (8) was 12-
fold and 108-fold weaker against HSV-1 and HSV-2
TKs, respectively. The congruence between the positions
of the hydroxybutyl side chain of 1 and the analogous
5′-hydroxymethyl and sugar ring atoms of TdR in the
TK binding site9 may explain the high affinity of 1 and
the weak affinity of 8 to the enzymes.
Although the methyl ether (9) corresponding to 1 was
10-20-fold weaker than 1, the carboxy isostere (10) was
a potent inhibitor of both enzymes. In contrast, amino-
substituted derivatives displayed a wide range of activi-
ties. In general, 9-(4-aminobutyl) compounds were less
active against HSV-1 than HSV-2 TKs, but several
derivatives were more potent than 1 against both
enzymes. For example, the substituted piperidino com-
pounds 12h, 12i, 12k, and 12q had IC50 values that
were 2-9-fold lower and 6-38-fold lower than 1 against
HSV-1 and HSV-2 TKs, respectively. The bulky piperi-
dino groups of these compounds suggested better pack-
ing of the side chain at the 9 position compared with 1
or closely related analogues. Indeed, molecular modeling
studies with the X-ray coordinates of the HBPG:HSV-1
TK complex9 have revealed space in the binding site
that can accommodate such bulky substituents. (Details
of these studies will be presented elsewhere.)
Various 2-phenylamino-6-oxopurines and their 9-sub-
stituted derivatives such as 1 have been shown to be
competitive inhibitors of HSV TKs.5,7 To ensure that the
potent 9-(4-aminobutyl) derivatives were, in fact, bind-
ing at the active site of the viral TKs, the mechanism
of the most potent compound 12i was investigated.
When the inhibitory activity was measured at various
substrate concentrations and the results plotted in
double reciprocal (Lineweaver-Burke) format (see Sup-
porting Information), the results were consistent with
competitive kinetics. The Ki values of 12i obtained in
this way were 0.03 and 0.005 µM for HSV-1 and HSV-2
TKs, respectively.
Effect of TK Inhibitors on Experimental HSV
Infections in Mice. HBPG (1) was previously shown
to reduce the frequency of stress-induced reactivation
of latent HSV-1 in mice10 and HSV-1 ocular disease in
squirrel monkeys.11 To extend these observations to
latent disease caused by HSV-2 and to explore the
potential of TK inhibitors in HSV encephalitis, corre-
sponding experiments were conducted in mice with 1
and several new analogues.
Reactivation from Latency. Ocular infection of
mice with HSV establishes latency in the trigeminal
ganglia, and latent virus can be induced to reactivate
by heat stress. In previous studies, multiple intraperi-
toneal (ip) doses of 200 mg/kg of 1 to HSV-1-latent mice
reduced the presence of stress-induced virus in the eyes
to 15% compared with 47% for vehicle-treated animals.10

Herpes Simplex Virus Thymidine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3923
Table 3. Effect of Compounds on HSV Encephalitis in Micea
Table 2. Effect of Compounds on Ocular HSV-2 Reactivation
in Micea
HSV-1
HSV-2
dose, ip
(mg/kg)
HSV-2-positive
cultures/15 mice
mean
mean
compd
dose, ip survivors/ day of survivors/ day of
-
(corn oil)
200
11
4b
9
compd
(mg/kg)b
10c
deathc
10
death
1
corn oil
1 (HBPG)
-
100
200
400
100
100
200
200
25
0
3
9.0
9.5
11.8
11
0
4
14.1
15.7
18
7
200
8
200
8
6d
9d
1
9d
10d
0
3
200
10
11
11
10
10
7c
-
10
12f
12i
ACV
PFA
200
7
8
9.7
10.7
-
10.7
11.2
15.2
15.4
14.2
15.0
17.2
15.2
17.8
18.5
200
0
1
200
12f
12i
ACV
nte
nt
1
0
100
-
0
400
10
0
a Heat stress of latent HSV-2 infected mice, treatment with
compounds, and assay for infectious virus were done as described
in the Experimental Section. b p < 0.027. c p < 0.128.
50
3
11
2
100
100
200
400
7d
1
13
6d
0
PFA
9.2
10.4
12.3
5d
7d
4d
6d
We subsequently found that a single ip dose of 200 mg/
kg given 1 h before heat stress reduced the number of
eyes with infectious HSV-1 to 35% compared with 70%
for vehicle-treated animals.12 Consequently, we tested
1 and various analogues and control antiherpetic com-
pounds in latent HSV-2-infected mice under similar
conditions (see Experimental Section), and the results
are presented in Table 2. A single dose of 1 given 30
min prestress gave a significant reduction in infectious
virus in eyes 24 h later, i.e., 27% vs 73% for controls.
In contrast, none of the derivatives of 1 showed activity
at the same dose. Thus, neither the phenylthio isostere
3 nor the hydroxyalkyl homologues 7 or 8 reduced
reactivation of latent HSV-2. The carboxypropyl com-
pound 10 and two aminobutyl compounds, 12f and 12i,
were also devoid of activity at 200 mg/kg. Acyclovir
(ACV) at 100 mg/kg had no effect on HSV-2 reacti-
vation, but phosphonoformate (PFA), a pyrophosphate
analogue and moderately potent anti-HSV drug, had
statistically significant activity, but only at 400 mg/kg
(Table 2).
Herpetic Encephalitis. Ocular infection with HSV-1
(strain McKrae) or HSV-2 (strain G) with 106 plaque-
forming units per eye leads to lethal encephalitis rather
than self-resolving latency.13 Under these conditions
untreated mice die on average at 9 and 14 days
postinfection with HSV-1 and HSV-2, respectively (see
Table 3). These models were validated by testing the
antiherpes drugs ACV and PFA. Both drugs had dose-
dependent protective effects when given ip in corn oil
suspension twice daily for 5 days, a common regimen
in such studies.14 ACV had ED50 values for both infec-
tions between 50 and 100 mg/kg and modestly prolonged
the mean day of death in each case. PFA was effective
but weaker, having ED50s of 200 mg/kg and above, and
mean days of death were also prolonged.
a Infection and treatment with compounds were done as de-
scribed in the Experimental Section. b Twice daily for 5 days.
c Animals alive at 30 days postinfection were “survivors”, and the
day of death was recorded for the remainder. d p < 0.05. e nt, not
tested.
Table 4. Conversion of Nucleoside Analogues to
Monophosphates (MP) by HSV TKs
HPLC tR, mina
relative % MPb
compd
parent
MP
HSV-1 TK
HSV-2 TK
1 (HBPG)
ACV
TdR
38.5
25.2
20.7
31.8
14.6
17.5
100c
32
70
100c
20
100
a tR ) retention time; HPLC conditions in Experimental Section.
b Compounds were assayed at 200 µM with excess TK as described
in the Experimental Section. c 100% for HBPG corresponds to 25%
conversion to HBPG-MP for both enzymes under the assay
conditions.
models raised a question of the relevance of TK inhibi-
tion in this series to antiherpetic activity in vivo. The
results suggested that compound 1, a close relative of
the weak antiherpes compound HBG (2) and its “iso-
stere” ACV, may owe its in vivo activity to a different
form of the compound, i.e., a metabolite, and/or a
different enzymatic target.
HBPG Is Efficiently Phosphorylated by HSV-1
and HSV-2 TKs. To evaluate whether 1 is a nonsub-
strate inhibitor or an alternate substrate of the viral
enzymes, we incubated excess HSV-1 TK and HSV-2 TK
with 1, and, as control, with the substrate TdR, under
conditions that maximized formation of the 5′-mono-
phosphate of the natural substrate. For comparison, the
incubations were also performed with the antiherpes
drug ACV, a weak alternate substrate15 for the enzymes.
Reaction products were resolved and quantitated by
HPLC, as described in the Experimental Section. Table
4 shows the retention times of substrates and mono-
phosphates, clearly indicating that peaks in the mono-
phosphate region were observed in all cases. The
identity of the peak at 31.8 min resulting from incuba-
tion of HBPG with TKs was confirmed by comparison
with authentic monophosphate, HBPG-MP. The relative
rates and extents of phosphorylation are seen in Figure
2 for HSV-1 TK (panel A) and HSV-2 TK (panel B). It
is apparent that the rates of conversion of 1 to the
monophosphate are similar to those of the substrate
TdR for both enzymes and that both are significantly
better substrates than ACV. Thus, 1 is not a pure
HBPG (1) and representative TK inhibitors were
tested in this model, by ip dosing in corn oil twice daily
for 5 days. Compound 1 was effective against both
HSV-1 and HSV-2 infections, with ED50 values between
100 and 200 mg/kg for HSV-1 and about 100 mg/kg
against HSV-2. The highest dose of 1 was completely
curative for the HSV-2 infection. The close hydroxyalkyl
homologues, 7 and 8, were ineffective at 100 mg/kg, and
the potent aminoalkyl derivatives 12f and 12i were
inactive at 200 mg/kg.
The findings that only 1 demonstrated significant
antireactivation and antiencephalitis activity in these

3924 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Manikowski et al.
affinity of compounds. Indeed, several 9-(4-aminobutyl)
derivatives with bulky tertiary amino substituents were
highly potent inhibitors of both HSV-1 and HSV-2 TKs
(Table 1).
Early-generation HSV TK inhibitors did not inhibit
replication of virus in cell cultures,18 consistent with the
lack of requirement of the virus-encoded enzyme for
virus growth in proliferating cells. Even 1, now found
to be a substrate for the enzyme, had weak activity
(ED50 > 100 µM) against HSV-1 and HSV-2 in human
foreskin fibroblasts and no activity in systemic infection
with HSV-2 (E. Kern, personal communication). How-
ever, HBPG (1) was previously shown to reduce the
frequency of latent HSV-1 reactivation in mice10 and
squirrel monkeys.11 In the present work, we extended
the studies of 1 to latent infection with HSV-2 and have
demonstrated that the compound is effective in reducing
the rate of reactivation of HSV-2 in mice (Table 2). We
also asked if 1 and related TK inhibitors affected the
course of experimental HSV-1 and HSV-2 encephalitis
in mice. The present results demonstrate that 1 is an
effective antiencephalitic compound in both cases, nearly
as potent as the standard drug ACV and more potent
than the antiherpetic drug PFA (Table 3).
HBPG (1) was originally selected from a series of TK
“inhibitors” because of its favorable balance of water
solubility and octanol:water partition coefficient.7 Our
observations that 1 exerts antiherpetic reactivation and
encephalitic activity in vivo and that it is an efficient
substrate for the enzymes now suggest that TK inhibi-
tion alone is not responsible for the activities. Closely
related 9-hydroxyalkyl homologues 7 and 8 and very
potent, nonsubstrate inhibitors, i.e., 12a-s, albeit more
polar compounds whose penetration into the nervous
system is uncertain, did not show activity against HSV
reactivation or encephalitis in mice (Tables 2 and 3).
Interestingly, the 9-hydroxypropyl analogue 7 was not
apparently phosphorylated by the TKs under the assay
conditions employed (data not shown). It is possible that
it is the monophosphate of 1 that is active in vivo or
that further virus-specific metabolism of 1, for example
to the triphosphate, HBPG-TP, is responsible for that
activity.
Figure 2. Kinetics of phosphorylation of nucleoside analogues
by HSV TKs. Panel A, HSV-1 TK. Panel B, HSV-2 TK. TdR
(O), 1 (b), and ACV (0). Two units of enzyme was used in the
HSV-1 TK assay, but 8 U was used in the experiments with
HSV-2 TK (to allow a detectable phosphorylation of ACV).
Each nucleoside (200 µM) was incubated for the indicated time,
and the products of the reaction were resolved by HPLC as
described in the Experimental Section. (100% corresponds to
5 nmol of substrate converted to monophosphate).
inhibitor of HSV TKs but an efficient alternate substrate
for the enzymes.
HSV-1 TK possesses thymidylate kinase (TMPK)
activity, as determined by HPLC analysis of enzyme
incubated with TMP, and we have shown that this
activity is inhibited by HBPG and related, nonsubstrate
inhibitors.16 When authentic HBPG-MP was incu-
bated with recombinant HSV-1 TK in the absence of
substrate, no product consistent with a diphosphate was
detected by HPLC, suggesting that the TMPK activity
does not phosphorylate this monophosphate (data not
shown).
Experimental Section
All new compounds were fully characterized by 1H NMR and
elemental analyses or high-resolution mass spectra (HR-MS).
Elemental analyses (C, H, N) were obtained from the Micro-
analysis Laboratory, University of MassachusettssAmherst
and agree to (0.4% of calculated values. HR-MS were ob-
tained from the Mass Spectrometry Facility, University of
MassachusettssAmherst, in EI mode. NMR spectra were
determined in Me2SO-d6 with a Bruker Avance 300 instru-
ment; chemical shifts are in ppm (δ) from internal Me4Si. All
chemicals were reagent grade. 2-Bromohypoxanthine, 2-phenyl-
amino-6-oxopurine, and 2-phenylamino-6-chloropurine were
prepared as described in ref 5, and 1 and 6 were prepared as
described in ref 7. HBG (2) was synthesized from 2-amino-6-
chloropurine as described for 1;7 the compound had proper-
ties identical to those reported by Yamazaki.18 Acyclovir (ACV)
and phosphonoformate (PFA) were purchased from Sigma-
Aldrich.
Discussion
A series of 2-phenylamino-6-oxopurine analogues
related to HBPG (1) was synthesized and studied to
investigate the structural basis of HSV TK inhibition,
and to search for more potent inhibitors suitable for
antiviral testing in vivo. Although we have identified
highly potent, competitive inhibitors of HSV-1 and
HSV-2 TKs, these “nonsubstrate” analogues did not
show activity in relevant mouse infection models. The
X-ray structure of the HBPG:HSV-1 TK complex9 re-
vealed that the inhibitor occupies the active, TdR-
binding site in a manner similar to the binding modes
of ACV and GCV.17 Interestingly, the presence of the
phenyl ring of 1 causes rotation of a tyrosine (Y132) by
ca. 90° from its position in the TdR and ACV complexes
and close to that found in the BVdU:TK complex.17 The
space available for packing of the 9-substituents sug-
gested that bulkier substituents might enhance the
Bacterial media components were from Difco, and Ni-NTA
Superflow resin was from QIAGEN. Restriction and modifi-
cation enzymes were purchased from Promega, Sigma, or
Boehringer. IPTG was from Sigma, and [3H-methyl]thymidine
(TdR), 20 Ci/mmol, was from New England Nuclear.

Herpes Simplex Virus Thymidine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3925
9-(4-Acetoxybutyl)-2-phenoxy-6-chloropurine, 4b. 4-Bro-
9-(4-Hydroxybutyl)-2-phenylthio-6-oxopurine,3.2-Phen-
ylthio-6-chloropurine, 3a. A suspension of 2-bromohypox-
anthine (3.0 g, 14 mmol) in 2-methoxyethanol (50 mL) was
treated with thiophenol (4.3 mL, 3 equiv) and anhydrous
K2CO3 (3.86 g, 2 equiv) and heated at 100 °C for 30 min. The
brownish solution was brought to room temperature and
neutralized with 10% aq HCl. The solution was extracted with
hexane until the sulfur smell was gone, and the solvent was
evaporated under vacuum to obtain 2-(phenylthio)hypoxan-
thine as an oil, used without further purification. Phosphoryl
chloride (5.2 mL, 4 equiv) was added to a well-stirred solution
of 2-(phenylthio)hypoxanthine, triethylamine (1.94 mL, 2
equiv), and triethylamine hydrochloride (0.96 g, 0.5 equiv) in
dry DMF (100 mL). The mixture was heated in an oil bath at
70 °C for 2 h. The brown solution was evaporated under
reduced pressure, and the residue was treated with water-
ice, and a saturated solution of NaHCO3 was added in small
portions to bring the pH to ca. 7. The solution was extracted
with CHCl3 (3 × 80 mL), and the organic layer was dried over
anhydrous Na2SO4. After evaporation of solvent under reduced
pressure, the residue was purified on a silica gel column in a
gradient of 0-4% MeOH in CHCl3 to give 3.2 g (87%) of
product as yellow solid foam. An analytical sample was
crystallized from Et2O. Mp: 177-179 °C. 1H NMR: δ 7.50 (m,
3H), 7.66 (m, 2H), 8.52 (s, 1H), 13.77 (s, 1H). Anal. (C11H7-
ClN4S): C, H, N.
mobutyl acetate (0.48 mL, 1.3 equiv) and anhydrous K2CO
3
(0.35 g, 1 equiv) were added to a solution of 2-phenoxy-6-
chloropurine (0.63 g, 2.55 mmol) in dry DMF (10 mL), and the
mixture was heated at 50 °C for 20 h. The solvent was removed
under reduced pressure to leave a yellow oil, which was mixed
with CHCl3:MeOH (1:1) and coevaporated with silica gel to
dryness. The material was purified on a silica gel column in a
gradient of 0-3% MeOH in CHCl3 to give 0.59 g (64%) of
product as a pale yellow solid. Mp: 75-77 °C (from Et2O). 1H
NMR: δ 1.51, 1.85 (2×quint, 2H), 1.98 (s, 3H), 3.98 (t, 2H),
4.16 (t, 2H), 7.28 (m, 3H), 7.46 (m, 2H), 8.57 (s, 1H). Anal.
(C17H17ClN4O3): C, H, N.
A suspension of 4b (0.4 g, 1.11 mmol) in 0.25 N aqueous
NaOH (50 mL) was heated under reflux for 20 h. The pale
yellow solution was brought to room temperature and treated
with concentrated acetic acid to bring the pH to ca. 5.
Saturated aq NH4Cl was added, and the solution was extracted
with CHCl3 (8 × 30 mL). The combined organic extracts were
dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was purified on a silica gel column in a
gradient of 0-11% MeOH in CHCl3 to give 0.24 g (72%) of 4
as a pale yellow solid. Mp: 185-188 °C (from Me2CO). 1H
NMR: δ 1.23, 1.66 (2×quint, 2H), 3.29 (q, 2H), 3.85 (t, 2H),
4.35 (t, 1H), 7.30 (m, 3H), 7.46 (m, 2H), 7.91 (s, 1H), 12.57 (s,
1H). Anal. (C15H16N4O3): C, H, N.
9-(4-Hydroxybutyl)-2-(N-methyl-N-phenylamino)-6-oxo-
purine, 5. A solution of t-BuOK in THF (20 mL, 1.3 equiv)
was added to a solution of 9-(4-hydroxybutyl)-2-phenylamino-
6-chloropurine7 (0.5 g, 1.57 mmol) in Me2SO:THF (1:1, 10 mL)
at room temperature. After 0.5 h, MeI (1.3 equiv) was added
in one portion to the yellow solution, and the solution was
stirred at room temperature for 4 h. A small volume of
saturated aq NH4Cl was added, the solution was extracted
with EtOAc (3 × 20 mL), and the organic layer was washed
with saturated aq NH4Cl and dried over anhydrous Na2SO4.
Evaporation of the solvent under reduced pressure gave 0.35
g (67%) of 9-(4-hydroxybutyl)-2-(N-methyl-N-phenylamino)-6-
chloropurine as a pale yellow solid, used in the next step
without further purification. 1H NMR: δ 1.35, 1.83 (2×quint,
2H), 3.39 (q, 2H), 3.51 (s, 3H), 4.08 (t, 1H), 4.42 (t, 1H) 7.24
(m, 1H), 7.41 (m, 4H), 8.26 (s, 1H).
A suspension of the above intermediate (0.33 g, 1.01 mmol)
in 10% aqueous NaOH (10 mL) was heated under reflux for
44 h. After cooling, the pH was brought to neutrality with
concentrated acetic acid. The solvent was removed under
reduced pressure, and the residue was dissolved in CHCl3:
MeOH (1:1, 30 mL) and coevaporated with silica gel to dryness.
The residue was purified on a silica gel column in a gradient
of 0-11% MeOH in CHCl3 to give crude product. Crystalliza-
tion from Me2CO/Et2O provided 0.14 g (44%) of product as pale
yellow crystals. Mp: 163-165 °C. 1H NMR: δ 1.35, 1.77
(2×quint, 2H), 3.37 (q, 2H), 3.40 (s, 3H), 3.96 (t, 1H), 4.42 (br
s, 1H) 7.27 (m, 3H), 7.43 (m, 2H), 7.77 (s, 1H), 10.51 (br s,
1H). Anal. (C16H19N5O2‚0.25H2O): C, H, N.
9-(3-Hydroxypropyl)-2-phenylamino-6-oxopurine, 7.
9-(3-Acetoxypropyl)-2-phenylamino-6-chloropurine, 7a.
Potassium carbonate (3.4 g, 0.6 equiv) was added in one
portion to a solution of 2-phenylamino-6-chloropurine (10.0 g,
40.7 mmol, 1 equiv) in dry DMF (250 mL) at room tempera-
ture. 3-Chloropropyl acetate (7.8 g, 1.4 equiv) was added to
the mixture, followed by NaI (0.6 g, 0.1 equiv). The reaction
mixture was stirred at 55 °C for 40 h. After cooling to room
temperature, 20 mL of glacial acetic acid was added slowly to
the reaction mixture. The solvents were evaporated under
reduced pressure to leave a yellow slurry. Addition of water
and extraction of the product with CHCl3 (3 × 200 mL), drying
of the combined organic extracts over Na2SO4, and evaporation
gave a yellow solid. This material was stirred in 150 mL of
EtOH at room temperature for 2 h, and the suspension was
filtered to give a white solid containing both 7- and 9-isomers
(NMR). Purification of the mixture on a silica gel column in a
9-(4-Acetoxybutyl)-2-phenylthio-6-chloropurine, 3b.
4-Bromobutyl acetate (2.22 mL, 1.3 equiv) and anhydrous
K2CO3 (1.96 g, 1.2 equiv) were added to a solution of 2-phenyl-
thio-6-chloropurine (3.1 g, 11.80 mmol, 1 equiv) in dry DMF
(30 mL), and the mixture was stirred overnight at 50 °C.
Evaporation of solvent under reduced pressure gave a yellow
oil, which was mixed with CHCl3 and coevaporated with silica
gel to dryness. The product was purified on a silica gel column
in a gradient of 0-4% MeOH in CHCl3 to yield 3.3 g (74%) of
the 9-isomer as a yellow foam. Mp: 66-68 °C (from Et2O). 1H
NMR: δ 1.44, 1.78 (2×quint, 2H), 1.99 (s, 3H), 3.94 (t, 2H),
4.09 (t, 2H), 7.47 (m, 3H), 7.66 (m, 2H), 8.56 (s, 1H). Anal.
(C17H17ClN4O2S): C, H, N.
A suspension of 3b (3.25 g, 8.62 mmol) in 0.5 N aq NaOH
(100 mL) was heated under reflux for 30 h. The pale yellow
solution was brought to room temperature and treated with
concentrated acetic acid to bring the pH to ca. 5. Saturated
aq NH4Cl was added, and the solution was extracted with
CHCl3 (6 × 80 mL). The combined organic extracts were dried
over anhydrous Na2SO4 and evaporated under reduced pres-
sure. The residue was purified on a silica gel column in a
gradient of 0-11% MeOH in CHCl3 to give 2.55 g (93%) of 3
1
as white crystals. Mp: 184-186 °C (from MeOH). H NMR:
δ 1.19, 1.60 (2×quint, 2H), 3.28 (q, 2H), 3.82 (t, 2H), 4.37 (t,
1H), 7.49 (m, 3H), 7.63 (m, 2H), 7.94 (s, 1H), 12.66 (s, 1H).
Anal. (C15H16N4O2S): C, H, N.
9-(4-Hydroxybutyl)-2-phenoxy-6-oxopurine, 4. 2-Phe-
noxy-6-chloropurine, 4a. A mixture of 2-bromohypoxanthine
(1.0 g, 4.65 mmol) and K2CO3 (1.93 g, 3 equiv) in phenol (15
mL) was heated under Ar at 160 °C for 20 h. The pale yellow
solution was washed with Et2O, and the white residue of crude
2-phenoxyhypoxanthine was used without further purification.
Phosphoryl chloride (2.6 mL, 6 equiv) was added to a well-
stirred solution of 2-phenoxyhypoxanthine, triethylamine (1.3
mL, 2 equiv), and triethylamine hydrochloride (1.28 g, 0.5
equiv) in dry DMF (20 mL). The flask was heated in an oil
bath at 70 °C for 5 h. The solution was evaporated under
reduced pressure, and the residue was treated with water-
ice, and saturated aqueous NaHCO3 was added in small
portions to bring the pH to ca. 7. The solution was extracted
with CHCl3 (4 × 80 mL), and the combined organic extracts
were dried over anhydrous Na2SO4. After evaporation of
solvent under reduced pressure, the residue was purified on
a silica gel column in a gradient of 0-4% MeOH in CHCl3 to
give 0.66 g (57%) of product as a yellow solid. Mp: 192-195
°C (from MeOH). 1H NMR: δ 7.28 (m, 3H), 7.46 (m, 2H), 8.49
(s, 1H), 13.70 (s, 1H). Anal. (C11H7ClN4O): C, H, N.
gradient of 0-11% EtOH in CHCl3
gave 6.8 g (48%) of the
1
9-isomer 7a as a white solid. Mp: 181-183 °C. H NMR: δ

3926 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Manikowski et al.
1.91 (s, 3H), 2.21 (quint, 2H), 4.03 (t, 2H), 4.27 (t, 2H) 6.98 (t,
1H), 7.32 (t, 2H), 7.81 (d, 2H), 8.32 (s, 1H), 9.97 (s, 1H). Anal.
(C16H16ClN5O2): C, H, N.
4.19 (t, 1H), 6.98 (t, 1H), 7.31 (t, 2H), 7.81 (d, 2H), 8.32 (s,
1H), 9.97 (s, 1H). Anal. (C16H18ClN5O‚0.5H2O): C, H, N.
A suspension of 9a (4.3 g, 13.1 mmol) in 1 N NaOH (100
mL) was heated under reflux for 40 h. After cooling, the pH
was brought to neutrality with concentrated acetic acid to yield
a white precipitate. The solid was filtered and washed several
times with water. The wet solid was treated with EtOH:Me2CO
(8:1) and stirred for 1 h at room temperature. The solid was
filtered to give 4.0 g (58%) of 9 as a white solid. Mp: >300 °C
(from MeOH).1H NMR: δ 1.48, 1.85 (2×quint, 2H), 3.19 (s,
3H), 3.32 (t, 2H), 4.06 (t, 1H), 7.04 (t, 1H), 7.35 (t, 2H), 7.65
(d, 2H), 7.84 (s, 1H), 8.80 (s, 1H), 10.49 (s, 1H). Anal.
(C16H19N5O2): C, H, N.
A suspension of 7a (6.8 g, 19.7 mmol) in 10% aqueous NaOH
(100 mL) was heated at reflux for 48 h. The pale yellow
solution was brought to room temperature and treated with
concentrated acetic acid to bring the pH to ca. 5, producing a
yellowish precipitate. Collection of the solid by filtration and
washing with water and Me2CO gave crude product. Crystal-
lization from aqueous NH4OH/MeOH gave 3.7 g (72%) of 7 as
yellowish crystals. Mp: >300 °C. 1H NMR: δ 1.95 (quint, 2H),
3.41 (q, 2H), 4.1 (t, 2H), 4.63 (t, 1H), 7.03 (t, 1H), 7.34 (t, 2H),
7.67 (d, 2H), 7.81 (s, 1H), 8.81 (s, 1H), 10.50 (s, 1H). Anal.
(C14H15N5O2): C, H, N.
9-(3-Carboxypropyl)-2-phenylamino-6-oxopurine, 10.
Sodium hydride (60% dispersion in mineral oil, 1.2 equiv)) was
added in one portion to a solution of 2-phenylamino-6-chloro-
purine (3.0 g, 12.2 mmol) in dry DMF (100 mL) at 0 °C. After
0.5 h the reaction mixture was warmed to room temperature,
ethyl 4-bromobutyrate (3.3 g, 1.4 equiv) was added, and the
mixture was stirred at room temperature for 26 h. The solvents
were evaporated under reduced pressure, the residue was
treated with MeOH:CHCl3 (1:1, 50 mL), and the solution was
coevaporated with silica gel to dryness. The mixture was
purified on a silica gel column in a gradient of 0-15% Me2CO
in CHCl3 to give 1.92 g (44%) of the 9-isomer of the ethyl ester,
9-(5-Hydroxypentyl)-2-phenylamino-6-oxopurine, 8.
2-(N-Formyl-N-phenylamino)-6-chloropurine, 8a. Phos-
phoryl chloride (41 mL, 2 equiv) was added dropwise to a well-
stirred suspension of 2-phenylamino-6-chloropurine (50 g, 0.22
mol, 1 equiv), triethylamine (30.7 mL, 1 equiv), and triethyl-
amine hydrochloride (15.1 g, 0.5 equiv) in dry DMF (150 mL).
The solid disappeared almost immediately to give a dark
solution which was heated in an oil bath at 60 °C. After 4 h,
the suspension of yellow solid was brought to room tempera-
ture, and solvents were evaporated under reduced pressure
to ca. 100 mL of yellow slurry. This residue was treated with
water-ice, and a saturated solution of aqueous NaHCO3 was
added in small portions to bring the pH to ca. 7. After standing
overnight, the yellow precipitate was collected by filtration,
washed with water, and dried to give 49.7 g (83%) of product
1
used in the next step without further purification. H NMR:
δ 1.10 (t, 3H), 2.14 (quint, 2H), 2.36 (t, 2H), 3.96 (q, 2H), 4.22
(t, 2H), 6.98 (t, 1H), 7.31 (t, 2H), 7.81 (d, 2H), 8.30 (s, 1H),
9.95 (s, 1H).
1
as a yellow-orange solid. H NMR: δ 7.30 (t, 2H), 7.40-7.54
A solution of the above ester (1.72 g, 4.78 mmol) in a mixture
of 2 N aqueous NaOH (100 mL) and MeOH (15 mL) was heated
for 40 h under reflux. The pale yellow solution was brought to
room temperature and treated with concentrated acetic acid
to bring the pH to ca. 5, producing a white precipitate.
Collection of the solid by filtration and washing with water
and Me2CO gave 1.4 g (93%) of 10 as an amorphous white
(m, 3H), 8.53 (s, 1H), 9.72 (s, 1H), 13.81 (br s, 1H). HR-MS:
Calcd for C12H8ClN5O, 273.0417, found 273.0437.
9-(5-Acetoxypentyl)-2-(N-formyl-N-phenylamino)-6-chlo-
ropurine, 8b. Sodium hydride (1.60 g, 60% dispersion in
mineral oil) was added in one portion to a solution of 8a (10.0
g, 36.5 mmol, 1 equiv) in dry DMSO:THF (1:1, 200 mL) at 0
°C. The mixture was stirred for 0.5 h at 0 °C, and then brought
to room temperature. 5-Bromopentyl acetate (7.31 mL, 1.2
equiv) was added to the mixture, followed by NaI (0.55 g, 0.1
equiv). The reaction mixture was stirred at room temperature
for 24 h. THF was removed under reduced pressure, and 100
mL of water was added. The product was extracted with EtOAc
(3 × 200 mL), and the combined organic extracts were washed
with brine and dried over anhydrous Na2SO4. Purification on
a silica gel column in a gradient of 0-5% Me2CO in CHCl3
gave the 9-isomer as a yellow oil. Crystallization from Et2O-
petroleum ether gave, in two crops, 7.93 g (52%) of product as
1
solid. Mp: >300 °C. H NMR: δ 2.07 (quint, 2H), 2.26, 4.09
(2×t, 2H), 7.03 (t, 1H), 7.35 (t, 2H), 7.67 (d, 2H), 7.82 (s, 1H),
8.80 (s, 1H), 10.49 (s, 1H), 12.15 (s, 1H). Anal.(C14H15N5O2):
C, H, N.
9-(4-Iodobutyl)-2-phenylamino-6-oxopurine, 11 (IBPG).
Trimethylsilyl iodide (13.9 g, 4 equiv) was added to a stirred
suspension of 1 (5.21 g, 17.4 mmol, 1 equiv) in sulfolane (80
mL) at room temperature. The solid dissolved immediately,
and the resulting pale brown solution was heated in an oil
bath at 60 °C for 22 h. The reaction mixture was brought to
room temperature and quenched with a saturated solution of
Na2SO3. The resulting pale yellow solution was treated with
water (100 mL) and stirred for 1 h to give a white precipitate.
Filtration, washing with water and Me2CO, and drying of the
material gave 7.05 g (99%) of 11 as a white solid. Mp: >250
°C (dec). 1H NMR: δ 1.75, 1.90 (2×quint, 2H), 4.08 (t, 2H),
7.04 (t, 1H), 7.35 (t, 2H), 7.64 (d, 2H), 7.83 (s, 1H), 8.78 (s,
1H), 10.48 (s, 1H). Anal. (C15H16IN5O): C, H, N.
General Method for Synthesis of 9-(Substituted-butyl-
2-phenylamino-6-oxopurines, 12a-s. The appropriate amine
(4-10 equiv) was added in one portion to a suspension of 11
(1 equiv) in dry DMF. The reaction mixture was stirred at room
temperature until all starting material was consumed (TLC,
usually 3-72 h). After this time, the mixture was evaporated
to dryness under reduced pressure to yield a white or pale
yellow residue. The residue was treated with water-MeOH,
and the solid was filtered and washed with Me2CO. The solid
was dissolved in aq 2-4 N HCl to obtain a yellow solution.
Coevaporation several times with EtOH or Me2CO gave a
white precipitate, which was filtered and washed with a small
amount of cold EtOH or Me2CO. Products were crystallized
from EtOH or MeOH.
1
pale yellow crystals. Mp: 73-75 °C. H NMR: δ 1.20, 1.54,
1.80 (3×quint, 2H), 1.97 (s, 3H), 3.93 (t, 2H), 4.14 (t, 2H), 7.30
(m, 2H), 7.50 (m, 3H), 8.58 (s, 1H), 9.77 (s, 1H). Anal. (C19H20-
ClN5O3): C, H, N.
A suspension of 8b (7.29 g, 17.5 mmol) in 1 N aqueous
NaOH (400 mL) was heated under reflux for 41 h. The pale
yellow solution was brought to room temperature and treated
with concentrated acetic acid to bring the pH to ca. 5,
producing a pale pink precipitate. Collection of the solid by
filtration and washing with water and Me2CO gave the crude
title compound. Crystallization from DMF gave 4.42 g (80%)
of 8 as pale pink crystals. Mp: >300 °C. 1H NMR: δ 1.29,
1.46, 1.82 (3×quint, 2H), 3.37 (q, 2H), 4.04 (t, 2H), 4.36 (t, 1H),
7.03 (t, 1H), 7.34 (t, 2H), 7.66 (d, 2H), 7.82 (s, 1H), 8.92 (br s,
1H), 10.59 (br s, 1H). Anal. (C16H19N5O2): C, H, N.
9-(4-Methoxybutyl)-2-phenylamino-6-oxopurine, 9. 9-(4-
Methoxybutyl)-2-phenylamino-6-chloropurine, 9a. Potas-
sium carbonate (2.14 g, 0.7 equiv) was added to a solution of
2-phenylamino-6-chloropurine (5.43 g, 22.1 mmol, 1 equiv) and
4-methoxybutyl bromide (4.8 g, 1.3 equiv) in dry DMF (80 mL).
The mixture was heated in an oil bath at 50 °C for 20 h. The
solvent was removed under reduced pressure, CHCl3:MeOH
(1:1, 50 mL) was added, and the solution was coevaporated
with silica gel to dryness. The mixture was purified on a silica
gel column in a gradient of 0-4% MeOH in CHCl3 to give 4.35
g (59%) of 9a as pale yellow crystals. Mp: 191-193 °C. 1H
NMR: δ 1.50, 1.91 (2×quint, 2H), 3.20 (s, 3H), 3.34 (t, 2H),
2-Phenylamino-9-[4-(N,N-dimethylamino)butyl]-6-oxo-
purine Dihydrochloride, 12a. The general method with 2
N dimethylamine in MeOH gave 94% of product as white
crystals. Mp: 260-262 °C (from MeOH). 1H NMR: δ 1.70, 1.92
(2×quint, 2H), 2.66 (s, 6H), 3.05 (m, 2H), 4.22 (t, 2H), 7.08 (t,

Herpes Simplex Virus Thymidine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3927
1
1H), 7.38 (dd, 2H), 7.68 (d, 2H), 8.86 (s, 1H), 10.28 (s, 1H),
10.36 (s, 1H), 11.69 (s, 1H). Anal. (C17H22N6O‚2HCl): C, H,
N.
2-Phenylamino-9-[4-(N,N-diethylamino)butyl]-6-oxo-
purine Dihydrochloride, 12b. The general method with
diethylamine gave 75% of product. Mp: 249-255 °C (from
MeOH). 1H NMR: δ 1.14 (t, 6H), 1.68, 1.93 (2×quint, 2H), 3.00
(m, 6H), 4.23 (t, 2H), 7.08 (t, 1H), 7.39 (t, 2H), 7.68 (d, 2H),
8.86 (s, 1H), 10.18 (s, 1H), 10.29 (s, 1H), 11.70 (s, 1H). Anal.
(C19H26N6O‚2HCl): C, H, N.
2-Phenylamino-9-[4-(N-ethyl-N-methylamino)butyl]-6-
oxopurine Dihydrochloride, 12c. The general method with
ethylmethylamine gave 79% of product as white crystals. Mp:
260-263 °C (from MeOH-water). 1H NMR: δ 1.17 (t, 3H), 1.68,
1.92 (2×quint, 2H), 2.61 (s, 3H), 2.90-3.15 (m, 4H), 4.22 (t,
2H), 7.08 (t, 1H), 7.39 (t, 2H), 7.68 (d, 2H), 8.87 (s, 1H), 10.31
(s, 2H), 11.71 (s, 1H). An analytical sample was recrystallized
from MeOH/H2O. Mp: 260-263 °C. Anal. C18H24N6O‚2HCl‚
0.25H2O): C, H, N.
the product in 98% yield. Mp: 257-262 °C (from MeOH). H
NMR: δ 1.84, 1.97 (2×m, 2H), 2.97 (m, 1H), 3.21 (m, 4H), 3.59
(m, 1H), 4.21 (m, 3H), 4.44 (m, 1H), 7.10 (m, 2H), 7.22 (m,
3H), 7.36 (t, 2H), 7.68 (d, 2H), 8.91 (s, 1H), 10.34 (s, 1H), 10.92
(s, 1H), 11.74 (s, 1H). Anal. (C24H26N6O‚HCl): C, H, N.
2-Phenylamino-9-{4-[2-(1-piperidylmethyl)pyrrolidyl]-
butyl}-6-oxopurine Trihydrochloride, 12l. The general
method with 2-(1-piperidylmethyl)pyrrolidine and NaHCO3
gave the product in 74% yield. Mp: 178-188 °C (from MeOH).
1H NMR: δ 1.35 (m, 1H), 1.68-1.94 (m, 12H), 2.41 (m, 1H),
2.89, 3.03 (2×m, 4H), 3.45 (m, 5H), 3.73 (m, 1H), 3.82 (m, 1H),
4.25 (t, 2H), 7.08 (t, 1H), 7.41 (t, 2H), 7.70 (d, 2H), 8.99 (s,
1H), 10.38 (s, 1H), 11.04 (br s, 1H), 11.34 (br s, 1H), 11.76 (s,
1H). Anal. (C25H35N7O‚3HCl‚0.25H2O): C, H, N.
2-Phenylamino-9-{4-[1-(4-hydroxy)piperidyl)]butyl}-6-
oxopurine Dihydrochloride, 12m. The general method with
4-hydroxypiperidine gave 84% of product. Mp: 294-297 °C
1
(from aq EtOH). H NMR (two conformers): δ 1.70 (m, 4H),
1.89 (m, 4H), 2.77 (m, 1H), 3.02 (m, 3H), 3.16 (m, 1H), 3.32
(m, 1H), 3.52+3.90 (2×m, 1H), 4.22 (t, 2H), 7.09 (quint, 1H),
7.39 (t, 2H), 7.67 (d, 2H), 8.85 (d, 1H), 10.24 (d, 2H), 11.67 (s,
1H). Anal. (C20H26N6O2‚2HCl‚H2O): C, N; H, calcd 6.38, found
5.94.
2-Phenylamino-9-{4-[N,N-bis(2-hydroxyethyl)amino]-
butyl}-6-oxopurine Dihydrochloride, 12d. The general
method with bis(2-hydroxyethyl)amine gave 85% of product.
Mp: 240-246 °C (from EtOH). 1H NMR: δ 1.73, 1.91 (2×quint,
2H), 3.13-3.24 (m, 6H), 3.72 (t, 4H), 4.21 (t, 2H), 7.08 (t, 1H),
7.38 (t, 2H), 7.68 (d, 2H), 8.79 (s, 1H), 9.64 (s, 1H), 10.23 (s,
1H), 11.64 (s, 1H). Anal. (C19H26N6O3‚2HCl): C, H, N.
2-Phenylamino-9-[4-(N,N-diallylamino)butyl]-6-oxo-
purine Dihydrochloride, 12e. The general method with
diallylamine gave the product in 62% yield. Mp: 237-235 °C
2-Phenylamino-9-{4-[1-(3-hydroxy)piperidyl)]butyl}-6-
oxopurine Dihydrochloride, 12n. The general method with
3-hydroxypiperidine gave 90% of product. Mp: 275-279 °C
(from MeOH). 1H NMR (two conformers): δ 1.20+1.68 (2×m,
5H), 1.91 (m, 3H), 2.41-2.93 (4×m, 2H), 3.01 (m, 2H), 3.22
(m, 2H), 3.81+3.99 (2×m, 1H), 4.19 (t, 2H), 7.08 (t, 1H), 7.38
(h, 2H), 7.67 (d, 2H), 8.60+8.71 (2×s, 1H), 9.18+10.46 (2×br
s, 1H), 10.07+10.12 (2×s, 1H), 11.52 (br s, 1H). Anal.
(C20H26N6O2‚2HCl): H, N; C, calcd 52.75, found 53.10.
2-Phenylamino-9-{4-[1-(4-methoxy)piperidyl)]butyl}-
6-oxopurine Dihydrochloride, 12o. The general method
with 4-methoxypiperidine hydrochloride and equivalent an-
hydrous K2CO3 gave 73% of product. Mp: 241-251 °C (dec)
(from EtOH). 1H NMR (two conformers): δ 1.72 (m, 3H), 1.92
(m, 5H), 2.85 (m, 2H), 3.05 (m, 2H), 3.16-3.52 (m, 6H), 4.23
(t, 2H), 7.09 (t, 1H), 7.40 (t, 2H), 7.68 (d, 2H), 8.98 (d, 1H),
10.43 (s, 1H), 10.60 (br s, 1H), 11.82 (s, 1H). Anal. (C21H28N6O2‚
2HCl‚0.5H2O): C, H, N.
1
(from MeOH). H NMR: δ 1.73, 1.93 (2×quint, 2H), 2.99 (m,
2H), 3.62 (m, 4H), 4.22 (t, 2H), 5.44, 5.50 (2×d, 4H), 5.88, 6.03
(2×q, 2H), 7.09 (t, 1H), 7.37 (t, 2H), 7.67 (d, 2H), 8.90 (s, 1H),
10.34 (s, 1H), 11.01 (s, 1H), 11.74 (s, 1H). Anal. (C21H26N6O‚
2HCl‚0.25H2O): C, H, N.
2-Phenylamino-9-[4-(1-piperidyl)butyl]-6-oxopurine Di-
hydrochloride, 12f. The general method with piperidine gave
1
97% of product. Mp: 280-284 °C (from MeOH). H NMR: δ
1.31 (m, 1H), 1.70 (m, 7H), 1.91, 2.70 (2×quint, 2H), 3.00 (m,
2H), 3.30 (d, 2H), 4.22 (t, 2H), 7.09 (t, 1H), 7.39 (t, 2H), 7.67
(d, 2H), 8.84 (s, 1H), 10.18 (s, 1H), 10.27 (s, 1H), 11.68 (s, 1H).
Anal. (C20H26N6O‚2HCl): C, H, N.
2-Phenylamino-9-{4-[4-(1-piperidyl)piperidyl]butyl}-6-
oxopurine Trihydrochloride, 12g. The general method with
4-(N-piperidyl)piperidine gave 77% of product. Mp: 295-298
°C (from MeOH). 1H NMR: δ 1.42 (m, 1H), 1.69-1.94 (m, 9H),
2.06-2.18 (m, 2H), 2.27 (m, 2H), 2.79-2.94 (m, 4H), 3.04 (m,
2H), 3.14-3.45 (m, 3H), 3.52 (m, 2H), 4.20 (t, 2H), 7.08 (t, 1H),
7.40 (t, 2H), 7.67 (d, 2H), 8.68 (s, 1H), 10.18 (s, 1H), 10.77 (s,
2H), 11.56 (s, 1H). Anal. (C25H35N7O‚3HCl‚H2O): C, H, N.
2-Phenylamino-9-{4-[1-(2-hydroxyethyl)piperidyl)]-
butyl}-6-oxopurine Dihydrochloride, 12h. The general
method with 2-(2-hydroxyethyl)piperidine gave 62% of product.
Mp: 225-232 °C (from MeOH). 1H NMR: δ 1.34-2.02 (m,
12H), 2.84-3.54 (m, 7H), 4.20 (m, 2H), 7.08 (t, 1H), 7.38 (t,
2H), 7.67 (d, 2H), 8.64 (s, 1H), 9.75 and 9.93 (2×s, 1H), 10.00
(s, 1H), 11.45 (s, 1H). Anal. (C22H30N6O2‚2HCl): C, H, N.
2-Phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxo-
purine Dihydrochloride, 12i. The general method with
cis,trans-decahydroquinoline gave 96% of product. Mp: 198-
206 °C (from MeOH). 1H NMR: δ 0.86-2.09 (m, 17), 2.54-
3.20 (4×m, 4H), 3.30 (d, 1H), 4.22 (t, 2H), 7.09 (t, 1H), 7.39 (t,
2H), 7.69 (d, 2H), 8.80 (s, 1H), 10.28 (s, 1H), 10.33 (s, 1H),
11.67 (s, 1H). Anal. (C24H32N6O‚2HCl): C, H, N.
2-Phenylamino-9-{4-[1-(3-methoxy)piperidyl)]butyl}-
6-oxopurine Dihydrochloride, 12p. The general method
with 3-methoxypiperidine hydrochloride and equivalent K2CO3
1
gave 86% of product. Mp: 239-246 °C (from aq MeOH). H
NMR (two conformers): δ 1.18-2.08 (4×m, 8H), 2.55-3.62 (m,
10H), 4.24 (t, 2H), 7.09 (t, 1H), 7.39 (h, 2H), 7.69 (d, 2H),
8.95+9.04 (2×s, 1H), 9.22+10.98 (2×br s, 1H), 10.52+10.58
(2×s, 1H), 11.87 (br s, 1H). Anal. (C21H28N6O2‚2HCl‚0.75H2O):
C, H, N.
2-Phenylamino-9-{4-[1-(2-phenyl)piperidyl]butyl}-6-
oxo-purine Dihydrochloride, 12q. The general method with
2-phenylpiperidine and anhydrous K2CO3 gave 53% of product.
1
Mp: 190-196 °C (from aq EtOH). H NMR: δ 1.60-2.08 (m,
8H), 2.12 (m, 2H), 2.71 (m, 2H), 2.96 (m, 1H), 3.55 (m, 1H),
4.13 (m, 3H), 7.10 (t, 1H), 7.38 (m, 5H), 7.63 (m, 4H), 8.82 (s,
1H), 10.31 (s, 1H), 11.02 (s, 1H), 11.76 (s, 1H). Anal. (C26H30N6O‚
2HCl‚H2O): C, H, N.
2-Phenylamino-9-{4-[1-(4-cyano-4-phenyl)piperidyl)]-
butyl}-6-oxopurine Dihydrochloride, 12r. The general
method with 4-cyano-4-phenylpiperidine and equivalent
NaHCO3 gave 69% of product. Mp: 265-270 °C (from EtOH).
1H NMR: δ 1.80 (m, 2H), 1.96 (m, 2H), 2.46 (t, 2H), 2.61 (t,
2H), 3.10-3.24 (2×m, 4H), 3.66 (m, 2H), 4.23 (t, 2H), 7.08 (t,
1H), 7.38-7.56 (m, 7H), 7.67 (d, 2H), 8.80 (s, 1H), 10.03 (s,
1H), 11.24 (s, 1H), 11.55 (s, 1H). Anal. (C27H29N7O‚2HCl): H,
N; C, calc 60.00, found 60.45.
2-Phenylamino-9-{4-[4-hydroxy-4-(4-chloro-3-trifluoro-
methylphenyl)piperidyl]butyl}-6-oxopurine Dihydro-
chloride, 12s. The general method with 4-hydroxy-4-[(4-
chloro-3-trifluoromethyl)]phenylpiperidine and equivalent K2CO3
gave 87% of product. Mp: 246-250 °C (from MeOH). 1H
NMR: δ 1.78 (m, 4H), 1.96 (m, 2H), 2.45 (m, 2H), 3.14+3.37
(2×m, 6H), 4.23 (t, 2H), 7.08 (t, 1H), 7.40 (t, 2H), 7.67 (d, 2H),
2-Phenylamino-9-[4-(2,6-dimethylpiperazinyl)butyl}-
6-oxopurine Trihydrochloride, 12j. The general method
with 2,6-dimethylpiperazine and NaHCO3 gave the product
in 76% yield. Mp: 257-263 °C (from EtOH). 1H NMR: δ 1.27,
1.29 (2×s, 6H), 1.78, 1.94 (2×m, 2H), 3.00-3.16 (m, 4H), 3.60-
3.75 (m, 4H), 4.22 (t, 2H), 7.08 (t, 1H), 7.40 (t, 2H), 7.68 (d,
2H), 8.80 (s, 1H), 10.05 and 10.11 (2×s, 2H), 11.59 (s, 1H),
11.98 (s, 1H). Anal. (C21H29N7O‚3HCl): C, H, N.
2-Phenylamino-9-{4-[2-(1,2,3,4-tetrahydroisoquinolyl)]-
butyl}-6-oxopurine Hydrochloride, 12k. The general method
with 1,2,3,4-tetrahydroisoquinoline and anhydrous K2CO3 gave

3928 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Manikowski et al.
7.78 (d, 2H), 7.92 (s, 1H), 8.80 (s, 1H), 10.07 (s, 1H), 10.75 (s,
was loaded on a Ni-NTA Superflow column (1 mL) at a flow
rate of 0.25 mL/min. The column was first washed with lysis
buffer and then with 50 mM sodium phosphate buffer (pH 8.0)
containing 300 mM NaCl and 20 mM imidazole. The protein
was then step-eluted with 250 mM imidazole in 50 mM sodium
phosphate buffer (pH 8.0) containing 300 mM NaCl at a flow
rate of 0.5 mL/min. Fractions were collected for enzymatic
activity analysis. The enzyme, collected from peak fractions,
was dialyzed against 50 mM Tris-HCl (pH 7.5) containing 20%
glycerol and 1 mM dithiothreitol (DTT) and then frozen in
liquid nitrogen until used.
Thymidine Kinase Assays. Recombinant HSV TKs were
assayed at 37 °C for 15 min in 25 µL of a mixture containing
30 mM HEPES K+, pH 7.5, 6 mM MgCl2, 6 mM ATP, 0.5 mM
DTT, and 1 or 2 µM (ca. Km concentration) of [3H-methyl]TdR
(2200 cpm/pmol). The reaction was terminated by spotting 20
µL of the incubation mixture on a 23 mm DEAE paper disk
(DE-81, Whatman). The disk was washed twice in an excess
of 1 mM ammonium formate, pH 5.6, to remove unconverted
nucleoside and finally in ethanol. Filters were dried, and
radioactivity was counted in 1 mL of Betamax (ICN) scintil-
lation fluid. One unit (U) is the amount of enzyme that
phosphorylates 1 nmol of TdR to TMP in 1 h at 37°C.
Inhibitor Assay. Stock solutions of inhibitors in DMSO
were serially diluted into enzyme assay mixtures. Enzymes
were assayed with at least five inhibitor concentrations in
duplicate assays to yield IC50, the inhibitor concentration
reducing activity by 50% in the above assay. When inhibitor
Kis were measured, the assay was carried out at several
concentrations of [3H]TdR.
Substrate Assay. When nucleoside analogues were tested
as possible substrates of HSV TKs, each compound (200 µM)
was incubated at 37 °C for 30 min in 25 µL of a mixture
containing 30 mM HEPES-K+, pH 7.5, 6 mM MgCl2, 6 mM
ATP, 0.5 mM DTT, and approximately 2 U of HSV-1 TK or 8
U of HSV-2 TK. Samples were then heated at 100°C for 5 min
and centrifuged for 15 min at 10 000 rpm in a microcentrifuge.
Supernatants were transferred to a new tube for subsequent
HPLC analysis.
HPLC Separation of Nucleosides and Nucleotides.
Reverse phase chromatography employing a SHIMADZU
HPLC system was used to separate nucleosides from nucleo-
tides. A 4.6 mm × 25 cm ALLTIMA C18-NUC 100A 5U
(Alltech) column was used at room temperature under the
following conditions: injection volume, 20 µL; detection, UV
260 nm; eluents, buffer A (20 mM KH2PO4, pH 7.5), buffer B
(20 mM KH2PO4, pH 5.2, 60% MeOH); linear gradient, 30 min
from 0% to 100% buffer B; 20 min 100% buffer B; flow rate,
0.5 mL/min.
1H), 11.57 (s, 1H). Anal. (C27H28ClF3N6O2‚2HCl): C, H, N.
2-Phenylamino-9-(4-phosphoryloxybutyl)-6-oxopu-
rine, 13 (HBPG-MP). Phosphoryl chloride (553 mg, 3.61
mmol) was added to a cold slurry of 1 (400 mg, 1.34 mmol) in
trimethyl phosphate (5 mL), and the mixture was kept at 0
°C for 4.5 h. Cold water (15 mL) was poured into the reaction
mixture, and it was neutralized with triethylamine (1.09 g,
10.8 mmol). After stirring for 1 h, the white precipitate was
collected and washed with water to give 487 mg (1.28 mmol,
96%) of 13 as the free acid. HR-MS: calcd 378.0968, found
378.1034. A portion of the acid (20 mg) was neutralized with
triethylamine, dissolved in water (5 mL), and purified by ion
exchange chromatography (DEAE-Sephadex, 20 × 2.2 cm).
Elution was carried out in a linear gradient of triethylammo-
nium bicarbonate, 0.02-1.0 M (pH 7.9), during 8 h at a flow
rate of 4 mL/min with detection at 260 nm. The fractions
containing the desired product were combined and evaporated
under vacuum at 30 °C. The residue was dissolved in water
and passed through 15 mL of Dowex 50Wx8 (Na+ form), and
the aqueous solution was lyophilized to give 15 mg of the title
compound as the sodium salt. 1H NMR (D2O): δ 1.59-1.72
(m, 2H), 1.86-2.00 (m, 2H), 3.92 (q, 2H), 4.06 (t, 2H), 7.08 (m,
2H), 7.32 (m, 2H), 7.49 (m, 2H), 7.83 (s, 1H). 31P NMR (121
MHz, D2O): δ 2.22 (s).
Cloning and Expression of HSV Thymidine Kinases.
Construction of Recombinant Bacterial Expression Vec-
tor for HSV-1 TK. Eukaryotic expression vector pMC1 (kindly
provided by Dr. L. Magrassi, IGM-CNR, Pavia, Italy), which
contains the complete 1131 bp coding sequence of HSV-1 TK,
was amplified by using the following primers: 5′-GCTATG-
GCTAGCTACCCCGGCCATCAGCACGCGTCTGCG-3′ (primer
1, sense) and 5′-CGTGAATTCTCAGTTAGCCTCCCCCAT-
CTCCC-3′ (primer 2, antisense), containing the Nhe I and EcoR
I restriction sites, respectively. The amplified region (1143 bp
long) containing the complete TK coding sequence was inserted
into the multiple cloning site of pTrcHisA (Invitrogen), re-
stricted with Nhe I and EcoR I, to give the recombinant
bacterial expression vector pHisHSV-1-TK containing the
sequence that encodes for the complete 376 amino acid enzyme
with a 6-His tag at its NH2-terminus.
Construction of Recombinant Bacterial Expression
Vector for HSV-2 TK. Plasmid pYCA2 (kindly provided by
Dr. John Bishop, Center for Genome Research, University of
Edinburgh), which contains the complete 1128 bp coding
sequence of HSV-2 TK, was digested with Sac I and Hind III.
The 1916 bp fragment containing the HSV-2 TK coding
sequence was amplified by using the following primers: 5′-
GGTATGGCTAGCCACGCCGGCCAACAGC-3′ (primer 1, sense)
and 5′-CGTGAATTCTAAACTCCCCCCACCTCGC-3′ (primer
2, antisense), containing the Nhe I and EcoR I restriction sites,
respectively. The amplified region (1139 bp long) containing
the complete TK coding sequence was inserted into the
multiple cloning site of pTrcHisA, restricted with Nhe I and
EcoR I, to give the recombinant bacterial expression vector
pHisHSV-2-TK containing the sequence that encodes for the
complete 386 amino acid enzyme with a 6-His tag at its NH2-
terminus.
In Vivo Models. BALB/c female mice at 6 weeks of age
were used in these experiments. The animals were provided
food and water ad libitum and were maintained in an AAALAC
accredited animal care facility. The McKrae strain of HSV-1
was propagated in Vero cells, titered on the same cells, and
stored frozen as a stock until used to infect animals. The G
strain of HSV-2 was obtained from the American Type Culture
Collection, Manassas, VA, propagated and titered on Vero
cells, and stored frozen until use.
Reactivation. The establishment of latency and hyper-
thermic reactivation in mice were performed as described.10
Groups of 15 animals were treated intraperitoneally with
drugs suspended in corn oil or corn oil alone, subjected 30 min
later to hyperthermic stress consisting of immersion in 43 °C
water for 10 min, dried, and returned to their cages. After 24
h, the eyes of all animals were swabbed for the determination
of infectious virus at the ocular surface.
Encephalitis. Groups of 10 mice each were anesthetized
with a mixture of ketamine and xylazine, their corneas lightly
scratched with a 27 gauge needle, and 106 plaque forming units
of infectious virus in tissue culture medium were placed on
each cornea. The animals were returned to their cages for
recovery from anesthesia. Following the infection and recovery,
groups of 10 animals were given 0.1 mL intraperitoneal
injections of test compounds as suspensions in corn oil or corn
Expression and Purification of Recombinant HSV
TKs from Bacterial Cells. Expression and purification of the
HSV-1 and HSV-2 TK were carried out as described by the
manufacturer of the Ni-NTA Superflow resin (Qiagen). Briefly,
a fresh overnight saturated culture of E. coli (DH5R strain)
transformed with pHis-HSV-1 TK or pHis-HSV-2 TK was
diluted 1:100 in 2 L of 2× YT (yeast tryptone) broth containing
ampicillin (60 µg/mL) and incubated at 37 °C with shaking.
At 0.6OD600, isopropylthio-â-D-galactopyranoside (IPTG, Sigma)
was added to a final concentration of 1 mM, and the culture
was incubated for a further 4 h at 37 °C. The bacterial cell
pellet was resuspended in four volumes of lysis buffer (50 mM
sodium phosphate (pH 8.0), 300 mM NaCl, 10 mM imidazole,
1 mM PMSF and 1 mg/mL lysozyme) and incubated on ice for
30 min. Cells were then sonicated on ice, and the lysate was
centrifuged at 10 000g for 30 min at 4 °C. The supernatant

Herpes Simplex Virus Thymidine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3929
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simplex virus type-1 thymidine kinase of a novel, nonsubstrate
inhibitor: X-ray crystallographic comparison with binding of
aciclovir. FEBS Lett. 1999, 443, 121-125.
oil alone. Treatments were continued twice daily for 5 days.
Animals were observed daily for 30 days, and the day of death
of each animal was recorded. Statistical analysis of results was
done with Student’s t test comparing analysis of variance
between groups.
(10) Gebhardt, B. M.; Wright, G. E.; Xu, H.; Focher, F.; Spadari, S.;
Kaufman, H. E. 9-(4-Hydroxybutyl)-N2-phenylguanine, HBPG,
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guanine (HBPG), a thymidine kinase inhibitor, on clinical
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Acknowledgment. This work was supported by
small business grant AI43170 from the National Insti-
tutes of Health (to G.W.), NIH Core grant P30EY002377
and an unrestricted departmental grant from Research
to Prevent Blindness (both to LSU Eye Center), and by
FIRB grant no. RBAU01LSR4_001 (to F.F.). The au-
thors thank Dr. Andrew Maioli for synthesis of HBPG-
MP.
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Animal Models of Ocular Herpes Simplex Virus Infections
(Rabbits, Primates, Mice). In Handbook of Animal Models of
Infection; Academic Press: New York, 1999; Chapter 110; pp
919-926.
Supporting Information Available: Lineweaver-Burke
plots for inhibition of HSV-1 and HSV-2 TK by 12i and
elemental analysis results. This material is available free of
charge via the Internet at http://pubs.acs.org.
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