Synthesis and Biological Evaluation of 5-Substituted Derivatives of the Potent Antiherpes Agent (north)-Methanocarbathymine

Article abstract of DOI:10.1021/jm030241s

The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of

Full text of DOI:10.1021/jm030241s

J . Med. Chem. 2003, 46, 5045-5054
5045
Syn th esis a n d Biologica l Eva lu a tion of 5-Su bstitu ted Der iva tives of th e P oten t
An tih er p es Agen t (n or th )-Meth a n oca r ba th ym in e
,†
,‡,§
Pamela Russ, Pierre Schelling,
Leonardo Scapozza,‡ Gerd Folkers,‡ Erik De Clercq,| and
Victor E. Marquez*,†
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, 376 Boyles St.,
Frederick, Maryland 21702, Pharmaceutical Biochemistry and Chemistry, Swiss Federal Institute of Technology,
Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, and Rega Institute, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received May 20, 2003
The conformationally locked nucleoside, (north)-methanocarbathymine (1a ), is a potent and
selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2)
viruses. Hereby, we report on the synthesis and biological evaluation of a small set of
5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane
nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive
substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which
was significantly phosphorylated by the human cytosolic TK. The binding affinity constant
and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution
on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue
(3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a . Against
varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent
as its conformationally unlocked 2′-deoxyriboside equivalent BVDU. The three compounds were
also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell
lines, either deficient in TK (TK-), nontransduced, or stably transduced with HSV1 TK. The
5-iodo compound (2, CC50 25 ( 7 µM) was more efficient than ganciclovir (GCV, CC50 75 ( 35
µM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward
TK- cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively
similar dose-dependent manner.
In tr od u ction
the case of antiviral therapy against HSV1 and HSV2
especially, such nucleoside analogues have been used
successfully for years. However, in the case of other
herpesvirus infections and gene therapy trials, the very
same analogues used at higher concentrations demon-
strated various limitations in terms of specific activation
level by viral TKs, efficiency, and safety.6,17,18 For
example, the high level of GCV needed for tumor
regression during gene-therapy of cancer cells leads to
important side effects such as hematological toxicity
(neutropenia) and bone marrow depletion.6 In the case
of allo-BMT, the infusion of HSV1 TK gene-modified
T-cells was proposed as a way to improve the chances
of successful engraftment, thus allowing the control of
eventual GvHD reaction outcomes by the subsequent
administration of GCV.16,19 Unfortunately, CMV infec-
tions, which occur frequently after allo-BMT, are also
treated by GCV. Thus, a curative administration of GCV
would simultaneously lead to the depletion of the newly
infused T-cells. Thereby, antiviral therapy of repellent
infections as well as nascent HSV1 TK-based gene
therapy applications have increasingly shown urgent
needs for new and more potent molecules that would
overcome the limitations of the HSV1 TK/nucleoside
analogues paradigm. So far, several HSV1 TK mutants
with improved specificity toward a given substrate have
been engineered.20-25 Concomitantly, new prodrugs with
lower toxicity profiles, higher specificity for viral TK
activation, and DNA polymerase inhibition abilities
have been developed.26-29
On the basis of the broader substrate specificity of
viral-encoded thymidine kinase (TK), nucleoside-like
prodrugs have been successfully used for years in
antiviral therapy.1,2 After specific phosphorylation by
viral-encoded TKs and subsequent phosphorylation by
less specific cytosolic kinases, the triphosphate forms
of such nucleoside analogues act as competitive sub-
strates of the DNA polymerases. Once incorporated into
DNA, these alternate substrates lead to elongation
stops, reduced integrity, or instability of the newly
synthesized DNA.1,3-6 Classically, nucleoside analogues
such as 5-bromovinyl-2′-deoxyuridine (BVDU), 5-iodode-
oxyuridine (IdU), acyclovir (ACV), and ganciclovir (GCV)
have been used in the treatment of herpes simplex virus
type 1 (HSV1), type 2 (HSV2), cytomegalovirus (CMV),
and other herpesvirus infections.7-10 More recently,
gene transduction strategies based on the concomitant
use of HSV1 TK and nucleoside analogues have been
proposed against cancer cells11-13 and for controlling
graft versus host disease (GvHD) reactions after allo-
geneic bone-marrow transplantation (allo-BMT).14-16 In
* Corresponding author. Phone: (301) 846-5954(3). Fax: (301) 846-
6033. E-mail: marquezv@dc37a.nci.nih.gov.
These investigators contributed equally to this work.
† National Cancer Institute at Frederick.
‡ Swiss Federal Institute of Technology.
§ Current address: Developmental Immunology Laboratory, Mas-
sachusetts General Hospital and Harvard Medical School, 55 Fruit
St. GRJ 1402, Boston MA 02114.
| Katholieke Universiteit Leuven.
10.1021/jm030241s CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/14/2003

5046 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Russ et al.
Sch em e 1
F igu r e 1. Structures of the bicyclo[3.1.0]hexane nucleosides.
1a, 2-4: (north)-methanocarbathymine derivatives. 1b: (south)-
methanocarbathymine.
Sch em e 2
F igu r e 2. Fixed location of bicyclo[3.1.0]hexane nucleosides
(T ) thymine) in the pseudorotational cycle.
(north)-Methanocarbocyclic thymine [1a , (N)-MCT,
Figure 1] is a recently discovered, effective anti-herpes
virus agent that was shown to be 30 times more potent
than ACV against HSV1 and HSV2 in an in vitro plaque
reduction assay.26,30 Since the 5-substituent in pyrimi-
dine nucleosides is a modulator of antiherpes activity31
such as in the very effective antiviral compound BVDU,
we decided to explore a small set of substituents (Xd
Br, I, and CHdCH-Br) on this new class of locked
carbocyclic nucleosides built on a bicyclo[3.1.0]hexane
template.
viral plaque reduction (VPR) assays against HSV1,
HSV2, vaccinia, pox, and varicella-zoster virus. Finally,
cell growth inhibition assays on three osteosarcoma cell
lines, either stably transduced with HSV1 TK (143B-
TK+-HSV1-WT), TK deficient (143B-TK-), or nontrans-
duced (MG-63-hTK1), are presented.
The series was limited exclusively to the conforma-
tionally locked north analogues, since the structurally
related but conformationally opposite (south)-methano-
carbocyclic thymine [1b, (S)-MCT], locked in the 3E
envelope conformation of the southern hemisphere, was
found to be totally devoid of antiviral activity.26 We have
suggested that the difference between (N)-MCT and (S)-
MCT might be related to their antipodal pseudosugar
conformation (Figure 2) imposed by the rigid bicyclo-
[3.1.0]hexane system.26 Indeed, in the case of (N)-MCT,
the conformation of the pseudosugar mimics that of an
envelope (2E) 2′-deoxysugar locked in the northern
hemisphere of the pseudorotational cycle, whereas in
(S)-MCT the pseudosugar mimics a 2′-deoxysugar locked
in the (3E) envelope conformation of the southern
hemisphere.
Ch em istr y
Initially, a convergent approach starting from the
already known compound 532 was attempted by direct
coupling with 5-iodo-3-benzoyl-1,3-dihydropyrimidine-
2,4-dione under Mitsunobu conditions. To obtain this
modified nucleobase, the method reported for the ben-
zoylation of uridine and thymine was adapted for the
synthesis of 5-halo-N3-benzoylpyrimidines.33 Such a
convergent approach was plagued with low yields and
the concomitant formation of the O-alkylated product
(Scheme 1). However, despite this difficulty, we were
able to isolate the desired N-alkylated product 6 and
complete its conversion to the final target, 5-iodo
derivative (2), after two deprotection steps. In view of
the low yields obtained under Mitsunobu conditions, a
linear approach starting from the known carbocyclic
amine 834 was initiated (Scheme 2). Under similar
reaction conditions as those reported for the synthesis
of 1a ,34 in situ generated (2E)-ethoxy-1-oxoprop-2-
enisocyanate was reacted with amine 8 to give the
In this study, we report the synthesis of the new
compounds, their kinetic parameters as substrates for
cytosolic (hTK1) and herpes (HSV1 TK) thymidine
kinases. The antiviral activity of the same compounds
was measured by the cytopatogenic effect (CPE) and

5-Substituted Derivatives of Methanocarbathymine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5047
F igu r e 3. In vitro phosphorylation profiles of compounds (2-4) and thymidine performed by hTK1 (A) and HSV1 TK (B). Only
5-iodo (2) exhibited a significant phosphorylation level in the presence of hTK1, whereas all three compounds were significant
substrates of HSV1 TK. Color codes for the nucleobases are dashed for thymine, black for 5-bromovinyl (4), gray for 5-bromo (3),
and white for 5-iodo (2). Blank 1: mock experiment without TK. Blank 2: mock experiment without substrate.
Sch em e 3
Sch em e 4
N-bromosuccinimide (NBS) gave the conformationally
locked version of carbocyclic BVDU (4).
corresponding acyclic intermediate 9, which underwent
smooth acid-catalyzed cyclization to the uridine ana-
logue 10 (Scheme 2). Removal of the benzyl protecting
group with boron trichloride afforded the already known
conformationally locked (north)-methanocarba-2′-deox-
yuridine analogue 11.26
As shown in Scheme 3, both 10 and 11 are convenient
starting materials for the synthesis of the 5-halo com-
pounds. Therefore, starting with compound 10, direct
iodination provided intermediate 12, which after re-
moval of the benzyl-protecting group afforded the 5-iodo
analogue 2. This compound proved to be identical to the
sample obtained via the direct Mitsunobu coupling in
Scheme 1. In a similar fashion, compound 11 could be
effectively brominated, and following base-catalyzed
hydrolysis the diacetate intermediate (13) was converted
to the 5-bromo analogue 3.
To obtain the BVDU analogue 4, the procedure
reported by Herdewijn and co-workers35 for the synthe-
sis of plain, carbocyclic (()-BVDU was followed (Scheme
4). Related procedures for the synthesis of carbocyclic
(+)- and (-)-BVDU have also been reported.36-38 Thus,
reaction of 2 with methylacrylate in the presence of
palladium(II) acetate, triphenyl phosphine, and triethy-
lamine afforded the 5-(2-carbomethoxyvinyl) intermedi-
ate 14, which after hydrolysis and treatment with
Resu lts a n d Discu ssion
En zym a tic Activity. The ADP/ATP ratios reflecting
the phosphorylation levels of methanocarba analogues
(2-4) by HSV1 TK or hTK1 are summarized in Figure
3. Significant HSV1 TK phosphorylation levels were
observed for all methanocarba analogues. However,
none of these appeared to be significant substrates for
the ensuing thymidylate kinase activity that HSV1 TK
may present with certain substrates,39 since no trace
of diphosphorylated compounds was detected. In the
presence of hTK1, 5-iodo (2) was significantly phospho-
rylated, whereas 5-bromo (3) and 5-bromovinyl (4) were
not significant substrates for this enzyme.
Since Km is equal to Ki in the case of HSV1 TK, as
previously demonstrated,40 both values can be referred
to as the binding affinity constant. The inhibition
constant values (Ki ( SD) obtained for compounds (2-
4) were compared to the previously published values of
their corresponding deoxyribosyl and carbocyclic coun-
terparts (Table 1). Since the catalytic turnover values
(kcat) for the deoxyribosyl and carbocyclic counterparts
for HSV1 TK have not been reported, it is not possible
to evaluate the impact of the restricted carbocyclic ring
on catalysis. Nevertheless, the 5-bromovinyl (4) has a
kcat value of 0.07 s-1 that is lower than those for ACV

5048 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Russ et al.
Ta ble 1. Comparison of the Inhibition Constant and Catalytic Turnover of Methanocarba, Carbocyclic, and Deoxyribosyl Analogues
of Thymine for HSV1 TK
HSV 1 TK
Thya
methanocarba analogues
carbocyclic analoguesc
deoxyribosyl analoguesc
(N)-MCT (1a )d
BrV (4)b
Br (3)b
I (2)b
C-BVdUe
C-IdUe
BVdU
IdU
Ki (µM)
kcat (s-1
0.2 ( 0.05
0.35 ( 0.01
16.1 ( 7.6
0.16 ( 0.04
62.9 ( 6.1
69.8 ( 6.5
85.8 ( 13.5
0.22
ND
0.21
ND
0.1
ND
0.09
ND
)
0.07 ( 0.01 0.15 ( 0.04 0.14 ( 0.04
a
b
Published as Km value.47 This work (values are the mean of at least four separate experiments ( SD). c Published in ref 43 (for
clarity reasons, SD values were omitted). Published in ref 41 e The values of both racemate are reported (i.e. (()-C-BVdU and (()-
CIdU); ND, not determined.
d
Ta ble 2. Antiviral Activity against Herpes Viruses HSV-1 and HSV-2 in HFFa Cellsg
HSV-1 CPE
HSV-2 CPE
HSV-1 VPR
HSV-2 VPR
b
c
b
c
b
c
b
c
compd
EC50
CC50
SId
EC50
CC50
SI
EC50
CC50
SI
EC50
CC50
SI
1a e
2
0.12
>275
1.23
6.41
2.22
>396
>275
>315
>291
3300
0
>256
>45
0.35
9.06
2.20
>291
10.21
>396
>275
>315
>291
1131
30.3
>143
0
0.040
0.095
1.10
>79
>1975
0.470
0.378
1.68
>79
>168
3
4
>315
>3315
>315
833
ACVf
a
b
HFF ) human skin fibroblast. EC50 ) inhibitory concentration (µM) required to reduce virus-induced cytopathogenic effect (CPE)
or cause virus plaque reduction (VPR) by 50%. c CC50 ) cytotoxic concentration that produces 50% inhibition of cell growth. SI ) selectivity
d
index (CC50/EC50). e Adapted from ref 26. f ACV ) acyclovir. The presented values were assessed by the NIAID according to Kern et
g
al.44
Ta ble 3. Antiviral Activity against Vaccinia Virus (VV) and Cowpox Virus (CV) in HFFa Cellse
VV CPE
VV VPR
CV CPE
CV VPR
b
c
b
c
b
c
b
c
compd
EC50
2.01
CC50
SId
EC50
8.19
CC50
SI
EC50
36.26
CC50
SI
EC50
10.72
CC50
SI
3
>315
>156
>315
>38
>315
>8
>315
>29
a
b
HFF ) human skin fibroblast. EC50 ) inhibitory concentration (µM) required to reduce virus-induced cytopathogenic effect (CPE)
or cause virus plaque reduction (VPR) by 50%. c CC50 ) cytotoxic concentration that produces 50% inhibition of cell growth. SI ) selectivity
d
index (CC50/EC50). e The presented values were assessed by the NIAID according to Kern et al.44
and GCV (both 0.10 s-1),41 whereas the turnover con-
stant values for compounds 3 and 2 are higher (kcat 0.15
and 0.14 s-1, respectively). A different contribution of
the nucleobase in the dipole-charge interaction influenc-
ing the catalytic turnover may also be involved,42
although structural rearrangements of the active site
are probably induced upon binding of such ligands. In
the case of (N)-MCT, the restricted ring induces a
change in the inhibition constant value, in agreement
with an altered binding mode of the sugar moiety with
respect to the flexible substrate, thymidine (Thy).25 On
the other hand, the presence of a simple carbocyclic ring
does not modify the Ki values, as the values for C-BVdU
and C-IdU remained comparable to that of thymidine.43
In summary, we believe that the higher inhibition
constants determined for all 5-substituted methano-
carba analogues reflect the combined effect of a re-
stricted carbocyclic ring and a bulky substitution at
position 5, which hinders accessibility to the active site
based on steric grounds and most likely requires struc-
tural rearrangement within the active site to be accom-
modated.
Biologica l Activity. An tivir a l Activity. The anti-
viral activity of these compounds was evaluated in two
different laboratories against different strains of DNA
viruses using previously published methodology.26,44,45
All concentrations for EC50 and CC50 were converted
from µg/mL to µM to best compare the results from the
two laboratories. In the CPE assay, the 5-bromo ana-
logue (3) was only ca. 2-fold more potent than ACV
against HSV1 and HSV2, while relative to (N)-MCT (1a )
it was somewhat weaker (Table 2). However, in the
more meaningful VPR assay, the 5-bromo compound
appeared to be much less cytotoxic and almost as potent
as 1a (Table 2). The 5-iodo (2) and 5-bromovinyl (4)
analogues were not very effective against HSV1 and
HSV2, which is in sharp contrast to the good in vitro
antiviral activity reported for the cyclopentyl versions
of these compounds.37
The recent interest in screening compounds against
viruses that pose a threat from terrorist attacks prompted
the National Institutes of Allergy and Infections Dis-
eases (NIAID) to test these compounds against vaccinia
and pox viruses according to method of Kern at al.44
Against these viruses, only the 5-bromovinyl analogue
(3, Table 3) showed modest activity.
At the Rega Institute, the compounds were evaluated
against thymidine kinase positive (TK+) and negative
(TK-) strains of VZV (Table 4) according to standard
published methods.45 Consistent with the expected
required activation of these drugs by viral TK, the
strains that do not express adequate levels of thymidine
kinase were not sensitive to the drugs. On the other
hand, against the TK+ strains all the compounds were
more potent than ACV, particularly the 5-bromovinyl
analogue (4). It should be noted that the plain carbocy-
clic analogue of BVDU (C-BVDU) with a flexible cyclo-
pentane rings was reported to have good activity
against VZV (EC50 ) 0.02-0.04 µg/mL35 or 0.06-0.12
µM), which makes it ca. 10-fold less potent than BVDU
itself. From the results in Table 4, it seems that the
presence of the bicyclo[3.1.0]hexane appears to be able
to recover the loss in potency since the anti-VZV activity
of compound 4 was virtually indistinguishable from that
of BVDU.
Cellu la r Gr ow th In h ibition . Cell growth inhibition
activity was measured on three different types of
osteosarcoma cells: either stably transfected with HSV1

5-Substituted Derivatives of Methanocarbathymine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5049
Ta ble 4. Antiviral Activity against Varicella-Zoster Virus
roborated the ones measured in HFF cells by VPR or
CPE assays (Table 2). In contrast, however, the CC50
values determined by the VPR or CPE assays were
significantly different from those obtained in HSV1 TK
stably transfected cells. The results from both CPE and
VPR are from virally infected cells, and thus cytoplasmic
production of the whole panel of viral enzymes, includ-
ing viral TK and viral DNA polymerase, are involved.
On the contrary, the 143B-TK+-HSV1-WT cells feature
only one viral enzyme, namely HSV1 TK. Thus, a
difference in CC50 values can be easily explained by the
presence or absence of viral DNA polymerase inhibition,
depending on the assay.
In summary, we have developed methods for the
synthesis of several 5-substituted uracil analogues built
on a rigid bicyclo[3.1.0]hexane template. Due to the poor
yield of the Mitsunobu coupling with uracil analogues,
the method of choice was the linear approach starting
from the corresponding carbocyclic amine 8. The uracil
analogue built from 8 was directly halogenated to
provide analogues 2 and 3, whereas the 5-bromovinyl
(4) derivative was constructed from the 5-iodo analogue
2 following the same methodology used for the synthesis
of BVDU. Moreover, these three nucleoside analogues,
which are restricted in the north conformation and
feature a bulky residue at position 5 of the nucleobase,
are significant substrates of HSV1 TK. The antiviral
activity of these compounds (2-4) reflects a different
modulating role for the 5-substituent on the uracil ring
when linked to the bicyclo[3.1.0]hexane template com-
pared to equivalent 2′-deoxyriboside analogues and
plain cyclopentyl nucleosides. Furthermore, modulation
of the specific phosphorylation by virus-encoded TKs
upon substitution in position 5 of the thymine ring was
observed.
(VZV)f
antiviral activity
(plaque reduction) EC50a(µM)
TK+ VZV
YS OKA
strain strain strain strain
TK- VZV
cytotoxicity (µM)
07/1 YS/R
MCCb
CC50
c
compd
2
3
4
0.12
0.20
0.007
2.4
0.16
0.20
0.005
2.4
>5
>20
>5
3
4
>5
19
>5
>50
>200
>200
>200
488
>5
ACVd
30
>200
>150
BVDUe 0.008
0.005
>150
>150
>400
a
EC50 ) inhibitory concentration (µM) required to reduce virus
b
plaques by 50%. MCC ) minimum cytotoxic concentration that
causes microscopically detectable alterations of cell morphology.
c CC50 ) cytotoxic concentration that produces 50% inhibition of
cell growth. ACV ) acyclovir. e BVDU ) (E)-5-(2-bromovinyl)-
d
2′-deoxyuridine. f The presented values were assessed by the Rega
Institute according to Andrei et al.45
TK (143B-TK+-HSV1-WT), or TK-negative (143B-TK-),
or nontransfected (MG-63-hTK1). Cell survival was
determined using the tetrazolium-based (XTT) colori-
metric assay performed after 4.5 days of incubation with
five different concentrations of each compound. Three
series of independent experiments were performed in
triplicate with good reproducibility (Figure 4). The
5-bromovinyl (4) did not lead to a typical TK-mediated
inhibition growth pattern and demonstrated increasing
inhibition only at high concentration (>50 µM) in all
three cell lines. Since growth inhibition appeared not
to be linked to TK activity, an intrinsic toxicity of the
compound at high concentration might be responsible
for the observed cytotoxicity. The 5-bromo (3) analogue
showed a dose-dependent reduction of cell growth in cell
lines possessing TK activity. Doses required for 50% cell
growth inhibition (CC50) were 150 ( 80 µM in 143B-
TK+-HSV1-WT and 330 ( 30 µM in MG-63-hTK1.
Interestingly, the inhibition profiles between transfected
and nontransfected cells for the 5-bromo (3) were
relatively close to each other. Although, the compound
is not a substrate for hTK1 (Figure 3), it is possible that
it interferes with thymidine monophosphate (TMP)
biosynthesis within the cell. Thus, the cytosolic amount
of thymidine monophosphate (TMP) would decrease,
resulting in a reduced cell growth rate in nontransfected
cells. The additional growth inhibition observed in
HSV1 TK-transfected cells would thus be enhanced by
the phosphorylated form of compound 3 that inhibits
enzymes beyond the initial phosphorylation step.
Finally, 5-iodo (2) showed no significant cell growth
inhibition in TK- cells within the experimental range
(CC50 > 500 µM). The CC50 values for 5-iodo (2) were
300 ( 30 µM in MG-63-hTK1 cells and 25 ( 7 µM in
HSV1 TK-transfected cells with an ensuing measurable
growth inhibition beyond 5 µM. By comparison, GCV
showed nonspecific growth inhibition starting at 5 µM
for TK- cells (CC50 ) 200 ( 100 µM) and MG-63-hTK1
cells (CC50 ) 300 ( 100 µM), while a specific effect was
observed against HSV1 TK-transfected cells (CC50 ) 75
( 35 µM). Thus, the 5-iodo analogue (2) appeared more
potent and more selective than GCV in the inhibition
of HSV1 TK-transfected cells. Moreover, the 5-iodo (2)
was less inhibitory than GCV toward TK- and non-
transfected dividing cells. Remarkably, the CC50 values
assessed in the nontransfected MG-63-hTK1 cells cor-
Exp er im en ta l Section
Ma ter ia ls. All chemical reagents were commercially avail-
able. Reported melting points were determined on a Fisher-
J ohnson melting point apparatus and are uncorrected. Column
chromatography was performed on silica gel 60, 230-240 mesh
(E. Merck), and analytical TLC was performed on Analtech
1
Uniplates silica gel GF. Routine IR and H, 13C, and 19F NMR
spectra were recordered using standard methods. Specific rota-
tions were measured in a Perking-Elmer model 241 polarim-
eter. Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA. Pyruvate kinase and L-lactate dehydroge-
nase purified from rabbit muscle and their respective sub-
strates phosphoenol-pyruvate and NADH were purchased from
Boehringer Mannheim; 1,4-dithio-DL-threitol (DTT), thymi-
dine, and ATP were from Fluka. ACV and GCV were pur-
chased from Glaxo-Wellcome and Roche, respectively. [methyl-
3H]-thymine was obtained from Amersham. Cell cultures
mediums and reagents were obtained from Life Technology.
5-Bromodeoxyuridine (5-BrdU), XTT (2,3-bis[methoxy-4-nitro-
5-sulfophenyl]-2H-tetrazolin-5-carboxyanilide), and Menadion
(vitamin K3) were obtained from Sigma. The following os-
teosarcoma cells were used for cell growth inhibition assays:
143B-TK- cells (ATCC No. CRL 8303), MG-63-hTK1 cells
(ATCC No. CRL-1427), and HSV1 TK stably transduced 143B-
TK+cells (ATCC No. CRL-8304) (143B-TK+-HSV1-WT).
Assessm en t of P h osp h or yla tion P r ofile. Both enzymes
HSV1 TK and hTK1 were expressed and purified as previously
published.25,46 Fully active HSV 1 TK and hTK1 featuring wild-
type kinetic properties were obtained and showed purity
higher than 95%, when stained after SDS-PAGE and mea-
sured by densitometry. Phosphorylation of thymidine and the
methanocarba analogues was monitored by reverse-phase ion-
pair high-performance liquid chromatography (HPLC) as

5050 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Russ et al.
F igu r e 4. Cell growth profiles obtained after incubation with compounds (2-4), plus GCV as control. Plots are representative
of three independent series of assays performed in triplicate with concentration of compounds ranging from 0.5 to 500 µM. (Cell
lines are 143B-TK+-HSV1-WT, 0 and dotted line; 143B-TK-, O and full line; and MG-63-hTK1, 4 and dashed line).
described previously.47 Briefly, 2 mM of substrate was incu-
given 0.1 mg/mL 5-BrdU for TK- cell-selection. In the case of
MG-63-hTK1 osteosarcoma cells, MEM was enriched with 1
mM sodium pyruvate and 1× MEM supplements. The same
supplementation plus 1× HAT was used for 143B-TK+-HSV1-
WT osteosarcoma cells stably transduced with HSV1 TK wild
type. Cells were incubated at 37 °C with 5% CO2, subcultured
by trypsinisation, and resuspended in fresh medium every
third or fourth day. The respective cell growth inhibition rates
induced by the compounds (2-4) and GCV were determined
using the tetrazolium-based XTT colorimetric assay.51,52 Single-
cell suspensions obtained by trypsinization were seeded at 2
× 103 cells/well for both 143B-TK+-HSV1-WT and 143B-TK-
lines and 1.5 × 103 cells/well for MG-63-hTK1 line in 96-well
tissue culture plates. Various concentrations of nucleoside
analogues in the appropriate buffers were added up to a final
volume of 200 µL per well and incubated 4.5 days at 37 °C
with 5% CO2. Then, 50 µL of prewarmed (37 °C) XTT (1.0 mg/
mL) in RPMI 1640 medium supplemented with 100 µM
Menadion and 25 mM HEPES pH 7.5 was added to each well.
After 9 h incubation at 37 °C, the absorbance was read on a
microplate reader (Versamax, Molecular Devices) at 450 nm
with a reference at 750 nm. The percentage of cell growth was
compared to the control wells containing cells, medium, and
XTT and calculated according to the following equation: %
cell growth ) 100 × [OD drug-treated/OD control]. The
reported values are the results of three independent series of
measurements performed in triplicate.
bated 60 min at 37 °C with respectively 4 µg of HSV1 TK or 4
µg of hTK1 in the presence of 5 mM ATP and 5 mM of Mg2+
.
Blank reactions without enzyme or without substrate were run
concurrently to account for background ATP hydrolysis. The
detection limit for phosphorylated substrates was 20 nmol.47
In h ibition Con sta n t Assessm en t. Conversion of [methyl-
3H]-T to its monophosphate in the presence of various con-
centrations of methanocarba analogue was performed as
previously described.48,49 Reactions were carried out in a final
volume of 30 µL containing 50 mM Tris pH 7.5, 5 mM MgCl2,
5 mM ATP, and 2.5 mg/mL bovine serum albumin. Substrate
and enzyme concentrations were chosen in order to respect
Michaelis-Menten conditions for initial velocity measure-
ments. The concentration of methanocarba analogue was
varied, and measured slopes were drawn on a reciprocal plot
(Lineweaver-Burk). Inhibition constants (apparent Ki) arose
from data using three concentrations of test compounds and
were analyzed using the double reciprocal plot method (Lin-
eweaver-Burk’s plot) as previously described.50 The presented
values are the results of six independent assays.
Ca ta lytic Tu r n over Con sta n t Assessm en t. Reaction
mixtures with a final volume of 75 µL containing 50 mM Tris
pH 7.5, 1 mM DTT, 0.21 mM phosphoenol-pyruvate, 2.5 mM
MgCl2, 5 mM ATP, 0.18 mM NADH, 0.8 µg pyruvate kinase,
0.5 µg L-lactate dehydrogenase, and 1.0 mM of substrate were
incubated at 37 °C. Two minutes later, 1.5-4.6 µg of HSV-1
TK was added in order to initiate the reaction. The changes
in absorbance driven by TK-dependent reaction were moni-
tored during 10 min at 37 °C using a Cary 50 spectrophotom-
eter (Varian). As previously published, a direct relationship
exists between the decrease of absorbance over time of nucleo-
side analogues and their respective catalytic turnover con-
stants (kcat).41 The data presented are the result of five
independent series of measurements performed in triplicate.
Control experiments were performed concurrently to account
for spontaneous hydrolysis of ATP under the experimental
conditions.
Gen er a l Syn th esis of Mon oben zoyla ted P yr im id in es.
A stirred suspension of 24 mmol of 5-X-uracil (X ) Br, I) in
pyridine (24 mL) and acetonitrile (60 mL) maintained under
an argon atmosphere was treated dropwise with benzoyl
chloride (6 mL, 52 mmol) and stirring was continued for 4 days.
Volatiles were evaporated under vacuum and reconcentrated
three times from toluene (100 mL). The residue was treated
with 0.25 M K2CO3 (90 mL) and dioxane (90 mL) and stirred
at room temperature for 2 h. Dioxane was removed under
vacuum and the remaining suspension was diluted with water
(100 mL). The obtained solids were recrystallized from 95%
ethanol to give the desired N3-benzoyl analogues in yields
ranging from 77%-83%.
Cell Gr ow th In h ibition Assa ys. The three cell lines were
cultivated in Minimum essential medium (MEM) supple-
mented with 10% fetal calf serum and appropriate antibiotics.
The adherent 143B-TK- osteosarcoma cells were additionally
5-Br om o-3-b en zoyl-1,3-d ih yd r op yr im id in e-2,4-d ion e:
77% yield, mp 187-189 °C. Anal. (C11H7BrN2O3) C, H, N.

5-Substituted Derivatives of Methanocarbathymine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5051
5-Iod o -3-b en zoyl-1,3-d ih yd r op yr im id in e -2,4-d ion e:
83% yield, mp 203-205 °C. Anal. (C11H7IN2O3) C, H, N. Anal.
Calcd for C11H7IN2O3: C, 38.62; H, 2.06; N, 8.19. Found: C,
38.84; H, 2.07; N, 8.13.
stirred suspension of AgNCO (6.0 g, 36 mmol) that was
predried at 100 °C for 2 h. The resulting mixture was refluxed
under argon for 45 min and cooled to room temperature. A 40
mL aliquot of the organic supernatant was then added
dropwise to a solution of carbocyclic amine 834 (2.08 g, 9 mmol)
in DMF (50 mL), which was precooled to 0 °C (ice/salt) under
a blanket of argon. The reaction mixture was stirred overnight
and allowed to reach room temperature. Volatiles were re-
moved under vacuum to provide 9 (3.19 g, 95%) as a glassy
yellow solid. An analytically pure sample was obtained after
silica gel flash chromatography using 50% EtOAc/hexanes
followed by 100% EtOAc as eluants: 1H NMR (CDCl3) δ 9.64
(br s, 1 H), 8.85 (br d, 1 H, J ) 7.0 Hz), 7.69 (d, 1 H, J ) 12.2
Hz), 7.40 (m, 5 H), 5.41 (d, 1 H, J ) 12.4 Hz), 4.83 (t, 1 H, J
) 8.4 Hz), 4.64 (AB q, 2 H, J ) 11.9 Hz), 4.29 (t, 1 H, J ≈ 6.7
H z), 4.02 (q, 2 H, J ) 7.1 Hz), 3.92 (AB d, 1 H, J ) 9.7 Hz),
3.51 (AB d, 1 H, J ) 9.7 Hz), 2.36 (br s, 1 H), 2.11 (irregular
q, 1 H), 1.60 (m, 1 H), 1.43 (t, 3 H, J ) 7.1 Hz), 1,03 (irregular
t, 1 H), 0.96 (m, 1 H), 0.70 (dd, 1 H, J ≈ 7.9, 5.9 Hz). Anal.
(C20H26N2O5‚0.25H2O) C, H, N.
(1S,2S,4S,5R)-5-Iod o-3-(p h en ylca r bon yl)-1-{4-p h en yl-
m eth oxy)-5-[(p h en ylm eth oxy)m eth yl]-bicyclo[3.1.0]h ex-
2-yl}-1,3-d ih yd r op yr im id in e-2,4-d ion e (6). A solution of
diethyl azodicarboxylate (DEAD, 0.53 mL, 3.31 mmol) in THF
(20 mL) was added dropwise to a stirred solution of 5-iodo-3-
(benzoyl)-1,3-dihydropyrimidine-2,4-dione (1.34 g, 3.92 mmol),
532 (0.536 g, 1.65 mmol), and triphenylphosphine (0.866 g, 3.31
mmol) in THF (50 mL), all under an atmosphere of argon. After
overnight stirring at room temperature, volatiles were removed
under vacuum. A crude mixture of both N- (6) and O-alkylated
products was obtained after silica gel flash chromatography
using a step gradient (hexanes f 25% EtOAc/hexanes). The
O-alkylated product (0.074 g, 7%, TLC, Rf ) 0.78, 40% EtOAc/
hexanes) was obtained as a slightly contaminated solid after
a second flash column chromatography on silica gel using a
similar step gradient (hexanes f 20% EtOAc in hexanes). Pure
6 (0.117 g, 11%) was obtained as a white glass as the gradient
was increased from 25% f 30% EtOAc/hexanes: 1H NMR
(CDCl3) δ 9.00 (s, 1 H), 7.30-8.10 (m, 15 H), 5.07 (d, 1 H, J )
6.8 Hz), 4.62-4.84 (m, 3 H), 4.50 (AB q, 2 H, J ) 11.7 Hz),
4.20 (AB d, 1 H, J ) 9.7 Hz), 3.15 (AB d, 1 H, J ) 9.7 Hz),
2.05-2.23 (m, 1 H), 1.77-1.93 (m, 1 H), 1.40-1.50 (m, 1 H),
(1S,2S,4S,5R)-1-{4-Hydr oxy-5-[(ph en ylm eth oxy)m eth yl]-
b icyclo[3.1.0]h e x-2-yl}-1,3-d ih yd r op yr im id in e -2,4-d i-
on e (10). A solution of 9 (3.19 g, 85 mmol) in 95% EtOH (100
mL) was treated with 1 M H2SO4 (100 mL) and heated to reflux
for 1 h. Ethanol was removed under vacuum and the aqueous
solution was neutralized with 2 N NaOH to pH 7 followed by
extraction in chloroform (3 × 100 mL). The combined organic
extracts were washed with saturated NaHCO3, dried (MgSO4),
and concentrated under vacuum. Purification by silica gel flash
chromatography with EtOAc afforded 10 (2.02 g, 73%) as a
1.10 (irregular t, 1 H), 0.86 (irregular t, 1 H). Anal. (C32H29
IN2O5) C, H, N.
-
( 1 S ,2 S ,4 S ,5 R ) -5 -I o d o -1 -{4 -p h e n y l m e t h o x y ) -5 -
[(p h en ylm et h oxy)m et h yl]b icyclo[3.1.0]h ex-2-yl}-1,3-d i-
h yd r op yr im id in e-2,4-d ion e (7). A solution of 6 (0.110 g, 0.17
mmol) in methanol (14 mL) was treated with concentrated
NH4OH (1 mL) and stirred for 1 h at room temperature.
Volatiles were removed under vacuum and the white powder
obtained was purified by silica gel flash chromatography using
a step gradient (25% EtOAc/hexanes f 50% EtOAc in hexanes)
to give 7 (0.080 g, 87%) as a white glassy solid: 1H NMR
(CDCl3) δ 8.81 (s, 1 H), 8.29 (br s, 1 H), 7.25-7.50 (m, 10 H),
5.06 (d, 1 H, J ) 6.6 Hz), 4.73 (AB q, 2 H, J ) 12.2 Hz), 4.62
(irregular t, 1 H), 4.48 (AB q, 2 H, J ) 11.9 Hz), 4.17 (AB d, 1
H, J ) 10.0 Hz), 3.12 (AB d, 1 H, J ) 10.0 Hz), 2.05-2.15 (m,
1 H), 1.77-1.93 (m, 1 H), 1.37 (m, 1 H), 1.10 (m, 1 H), 0.82
(irregular t, 1 H). Anal. (C25H25IN2O4) C, H, N.
colorless foam: [R]25 ) +59.0 (c 0.16, MeOH); 1H NMR
D
(CDCl3) δ 8.35 (br s, 1 H), 8.04 (d, 1H, J ) 8.0 Hz), 7.41 (m, 5
H), 5.46 (dd, 1 H, J ) 8.0, 1.9 Hz), 5.07 (d, 1H, J ) 7.1 Hz),
4.92 (t, 1 H, J ≈ 8.5 Hz), 4.63 (AB q, 2 H, J ) 10.9 Hz), 4.19
(AB d, 1 H, J ) 10.0 Hz), 3.37 (AB d, 1 H, J ) 10.0 Hz), 2.06
(m, 1 H), 1.81 (m, 1 H), 1.45 (irregular dd, 1 H), 1.05 (m, 1 H),
0.85 (m, 1 H). Anal. (C18H20N2O4‚0.25H2O) C, H, N.
(1S,2S,4S,5R)-1-{4-Hydr oxy-5-[(ph en ylm eth oxy)m eth yl]-
bicyclo[3.1.0]h ex-2-yl}-5-iod o-1,3-d ih yd r op yr im id in e-2,4-
d ion e (12). A stirred solution of 10 (0.164 g, 0.5 mmol), iodine
(0.254 g, 1 mmol), and 1 N HNO3 (0.5 mL) in dioxane (5 mL)
was heated to 100 °C for 1 h. Volatiles were removed under
vacuum and the residue was reconcentrated thrice with
ethanol (5 mL) and thrice with chloroform (5 mL). The solid
obtained was purified by silica gel flash chromatography using
a step gradient (50% EtOAc/hexanes f 100% EtOAc) to give
12 (0.191 g, 84%) as an off-white solid: mp 85-88 °C; 1H NMR
(CDCl3) δ 8.68 (s, 1 H), 8.41 (br s, 1 H), 5.04 (d, 1 H, J ) 6.8
Hz), 4.91 (t, 1 H, J ≈ 8.5 Hz), 4.77 (AB q, 2 H, J ) 12.5 Hz),
4.08 (AB d, 1 H, J ) 10.2 Hz), 3.22 (AB d, 1 H, J ) 10.2 Hz),
2.06 (m, 1 H), 1.79 (m, 1 H), 1.43 (irregular dd, 1 H), 1.04
(irregular dd, 1 H), 0.80 (m, 1 H). Anal. (C18H19IN2O4‚0.5H2O)
C, H, N.
(1S,2S,4S,5R)-1-[4-Hyd r oxy-5-(h yd r oxym eth yl)bicyclo-
[3.1.0]h ex-2-yl]-5-iodo-1,3-dih ydr opyr im idin e-2,4-dion e (2).
Meth od A. A solution of 7 (0.031 g, 0.057 mmol) in CH2Cl2
(10 mL) was stirred at -78 °C (acetone/dry ice) and treated
with BCl3 (1.2 mL, 1 M solution in CH2Cl2) for 1 h. Methanol
was added (3 mL) and the reaction mixture was concentrated
under vacuum and reconcentrated three times from MeOH (5
mL). The residue was purified by silica gel flash chromatog-
raphy using a step gradient (CHCl3 f 5% MeOH/CHCl3). The
solid obtained was recrystallized from MeOH/CHCl3 to give 2
(0.012 g, 57%) as a white solid: mp 226-227 °C; [R]25D ) -3.0
(1R,2S,4S,5S)-[2-Acet yloxy-4-(5-b r om o-2,4-d ioxo(1,3-
d ih yd r op yr im id in yl))bicyclo[3.1.0]h exyl]m eth yl Aceta te
(13). Under a blanket of argon, compound 1126 (0.073 g, 0.31
mmol) was dissolved in acetic anhydride (2 mL) with gentle
warming. After cooling to room temperature, bromine (0.02
mL, 0.33 mmol) was added slowly and the solution was stirred
at room temperature for 30 min. The solution was stored in
the refrigerator overnight (ca. 4 °C), and the volatiles were
removed under reduced pressure. The residue was reconcen-
trated three times from toluene (5 mL), and treatment of the
orange residue with water gave 0.179 g of crude product, which
was purified by silica gel flash chromatography using a step
gradient (hexanes f 50% EtOAc/hexanes f EtOAc) to give
13 (0.117 g, 94%) as a reasonably pure colorless foam; 1H NMR
(CDCl3) δ 8.72 (br s, 1 H), 8.03 (s, 1 H), 5.63 (t, 1 H, J ≈ 8.4
Hz), 5.15 (d, 1 H, J ) 7.6 Hz), 4.79 (AB d, 1 H, J ) 12.4 Hz),
3.75 (AB d, 1 H, J ) 12.4 Hz), 2.39 (m, 1 H), 2.33 (s, 3 H), 2.16
(s, 3 H), 1.86 (m, 1 H), 1.60 (m, 1 H), 1.12 (m, 1 H), 1.02 (m, 1
H). 0.63 (irregular dd, 1 H). This product was used im-
mediately in the following step.
1
(c 0.1, MeOH); H NMR (CDCl3 + D2O) δ 8.53 (s, 1 H), 4.72
(d, 1 H, J ) 6.8 Hz), 4.55 (irregular t, 1 H), 4.08 (AB d, 1 H, J
) 11.5 Hz), 3.07 (AB d, 1 H, J ) 11.5 Hz), 1.82 (dd, 1 H, J ≈
14.7, 8.0 Hz), 1.50-1.55 (m, 1 H), 1.32 (irregular dd, 1 H), 0.82
(irregular t, 1 H), 0.63 (irregular dd, 1 H); 13C NMR (CDCl3) δ
153.13, 143.21, 138.93, 61.39, 61.05, 53.91, 48.55, 30.33, 29.22,
16.61, 2.34; FAB MS m/z (relative intensity) 365 (MH+, 100),
239 (b + 2H, 45). Anal. (C11H13IN2O4‚H2O) C, H, N.
Meth od B. A solution of 12 (0.072 g, 0.16 mmol) in CH2Cl2
(10 mL) was stirred at -78 °C (acetone/dry ice) and treated
with BCl3 (1.6 mL, 1 M solution in CH2Cl2) for 1 h. After a
similar workup and purification procedure, 2 (0.028 g, 50%)
was obtained as a white solid, mp 229-230 °C, which
was spectroscopically identical to the solid obtained under
method A.
(1S,2S,4S,5R)-N-((2E)-Eth oxypr op-2-en oyl)({4-h ydr oxy-
5-[(ph en ylm eth oxy)m eth yl]bicyclo[3.1.0]h ex-2-yl}am in o)-
ca r boxa m id e (9). 3-Ethoxypropenoyl chloride (2.14 g, 18
mmol) in benzene (50 mL) was added dropwise to a vigorously

5052 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Russ et al.
(1S,2S,4S,5R)-5-Br om o-1-[4-h ydr oxy-5-(h ydr oxym eth yl)-
bicyclo[3.1.0]h ex-2-yl]-1,3-d ih yd r op yr im id in e-2,4-d ion e
(3). A solution of 13 (0.111 g, 0.27 mmol) in saturated
methanolic ammonia (3 mL) was kept at room temperature
overnight and then heated at 50 °C for 24 h. Volatiles were
removed under vacuum, and the solid obtained (TLC, Rf )
0.32, 15% MeOH/CHCl3) was found by 1H NMR to still contain
one acetate group. The solid was then dissolved in THF (2.5
mL) and treated with 1 N NaOH (0.2 mL), and the solution
was stirred at room temperature for 4 h. Acetic acid (20 µL)
was added, and volatiles were removed under vacuum. The
crude solid was purified by reverse phase C-18 column
chromatography using a step gradient (H2O f 5% MeOH/H2O
f 10% MeOH/H2O), and following a second chromatography
on silica gel with a step gradient (CHCl3 f 10% MeOH/
CHCl3), 3 (0.026 g, 30%) was obtained as a white solid: mp
removed by filtration and the filtrate was concentrated under
vacuum. The crude product was purified by silica gel flash
column chromatography using a step gradient (CHCl3 f 5%
MeOH/CHCl3 f 10% MeOH/CHCl3). A second chromato-
graphic purification on silica gel (CH2Cl2 f 5% i-propanol/
CH2Cl2 f 10% i-propanol/CH2Cl2) and a final chromatography
on a C-18 reverse phase column using a step gradient (H2O f
20% MeOH/H2O) were required to obtain 4 (0.035 g, 25% yield)
as an off-white solid: mp 120-122 °C; [R]25 ) -23.0 (c 0.13,
D
1
MeOH); H NMR (CDCl3 + D2O) δ 8.29 (s, 1 H), 7.24 (d, 1 H,
J ) 13.6 Hz), 6.76 (d, 1 H, J ) 13.6 Hz), 4.76 (d, 1 H, J ) 6.8
Hz), 4.61 (t, 1 H, J ≈ 8.4 Hz), 4.14 (AB d, 1 H, J ) 11.5 Hz),
3.11 (AB d, 1 H, J ) 11.5 Hz), 1.85 (dd, 1 H J ≈ 14.7, 8.2 Hz),
1.58 (m, 1 H), 1.35 (irregular dd, 1 H), 0.84 (irregular t, 1 H),
0.65 (irregular dd, 1 H); FAB MS m/z (relative intensity) 343/
345 (MH+, 100/95), 217 (b + 2H, 35). Anal. (C13H15BrN2O4‚
0.5H2O) C, H, N.
218-219 °C; [R]25 ) 18.2 (c 0.11, MeOH); 1H NMR (CDCl3 +
D
D2O) δ 8.55 (s, 1 H), 4.74 (d, 1 H, J ) 6.6 Hz), 4.57 (t, 1 H, J
≈ 8.4 Hz), 4.11 (AB d, 1 H, J ) 11.2 Hz), 3.07 (AB d, 1 H, J )
11.2 Hz), 1.86 (dd, 1 H, J ) 14.4, 7.8 Hz), 1.58 (m, 1 H), 1.35
(m, 1 H), 0.83 (irregular t, 1 H), 0.65 (irregular dd, 1 H); 13C
NMR (CDCl3) δ 154.70, 151.75, 142.70, 87.64, 61.27, 53.85,
48.78, 30.07, 29.06, 16.52, 2.36; FAB MS m/z (relative inten-
Ack n ow led gm en t. Pierre Schelling was supported
by the Stipendienfonds der Basler Chemischen Indus-
trie. The authors wish to thank Dr. Christopher K.-H.
Tseng, NIAID, NIH, for arranging the antiviral tests
through his contractors and Dr. J ames A. Kelley of the
LMC for mass spectral analyses.
sity) 317/319 (MH+, 100/87), 191 (b + 2H, 30). Anal. (C11H13
-
BrN2O4) C, H, N.
(1S,2S,4S,5R)-Ethyl(2E)-3-{1-[4-Hydroxy-5-(hydroxymethyl)-
bicyclo[3.1.0]h ex-2-yl]-2,4-d ioxo(1,3-d ih yd r op yr im id in e-
5-yl)}p r op -2-en oa te (14). In a vial fitted with a Teflon-lined
cap, a solution of palladium(II) acetate (5.5 mg, 25 mmol) and
triphenylphosphine (0.013 g, 50 mmol) in dioxane (1 mL) was
allowed to stand for 10 min during which time the solution
turned red. Immediately after, triethylamine (0.055 mL, 0.4
mmol) and methyl acrylate (0.110 mL, 1.24 mmol), followed
by 2 (0.089 g, 0.25 mmol), were added. After adding additional
dioxane (2 mL), the vial was sealed and heated at 78 °C for 4
h. A similar reaction was set up separately, and the two
combined reactions were concentrated under reduced pressure.
Initial purification by silica gel flash chromatography using a
step gradient (CHCl3 f 5% MeOH/CHCl3 f 10% MeOH/
List of Abbr evia tion s
acyclovir, ACV; allogeneic bone-marrow transplanta-
tion, allo-BMT; cytomegalovirus, CMV; cytopatogenic
effect, CPE; ganciclovir, GCV; graft versus host disease,
GvHD; hypoxanthine, aminopterin, thymidine medium,
HAT; herpes simplex type 1, HSV1; herpes simplex type
2, HSV2; human cytosolic thymidine kinase, hTK1;
Minimum essential medium, MEM; National Institutes
of Allergy and Infectious Diseases, NIAID; (north)-
methanocarbocyclic thymine, (N)-MCT; (south)-metha-
nocarbocyclic thymine, (S)-MCT; thymidine kinase, TK;
thymidine monophosphate, TMP; thymidine, Thy; va-
ricella-zoster virus, VZV; viral plaque reduction, VPR;
2,3-bis[methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolin-5-
carboxanilide, XTT; (E)-5-(2-bromovinyl)-2′-deoxyuri-
dine, BVDU; 5-iodo-deoxyuridine, IdU
CHCl3) gave unreacted
2 (0.054 g) along with product-
containing fractions that were evaporated and triturated with
EtOAc to give 14 (0.076 g, 48%) as white crystals: mp 240-
241 °C; TLC, Rf ) 0.54, 15% MeOH/CHCl3; [R]25 ) -2.4 (c
D
1
2.3, MeOH); H NMR (CDCl3 + D2O) δ 8.57 (s, 1 H), 7.33 (d,
1 H, J ) 16.0 Hz), 6.86 (d, 1 H, J ) 16.0 Hz), 4.78 (d, 1 H, J
) 6.8 Hz), 4.61 (t, 1 H, J ≈ 8.4 Hz), 4.14 (AB d, 1 H, J ) 11.5
Hz), 3.70 (s, 3 H), 3.10 (AB d, 1 H, J ) 11.5 Hz), 1.89 (dd, 1 H,
J ) 14.3, 8.05), 1.58 (m, 1 H), 1.38 (irregular dd, 1 H), 0.58
(irregular t, 1 H), 0.63 (irregular dd, 1 H). Anal. (C15H18IN2O6‚
0.25H2O) C, H, N.
Refer en ces
(1) Griffiths, P. D. Progress in the clinical management of herpes-
virus infections. Antiviral Chem. Chemother. 1995, 6, 191-209.
(2) Shugar, D. Viral and host-cell protein kinases: Enticing antiviral
targets and relevance of nucleoside, and viral thymidine, ki-
nases. Pharmacol. Ther. 1999, 82, 315-335.
(3) Balzarini, J .; Bernaerts, R.; Verbruggen, A.; De Clercq, E. Role
of the incorporation of (E)-5-(2-iodovinyl)-2′-deoxyuridine and its
carbocyclic analogue into DNA of herpes simplex virus type
1-infected cells in the antiviral effects of these compounds. Mol.
Pharmacol. 1990, 37, 402-407.
(4) Reardon, J . E.; Spector, T. Herpes simplex virus type 1 DNA
polymerase. Mechanism of inhibition by acyclovir triphosphate.
J . Biol. Chem. 1989, 264, 7405-7411.
(5) Ilsley, D. D.; Lee, S. H.; Miller, W. H.; Kuchta, R. D. Acyclic
guanosine analogues inhibit DNA polymerases alpha, delta, and
epsilon with very different potencies and have unique mecha-
nisms of action. Biochemistry 1995, 34, 2504-2510.
(6) Thust, R.; Tomicic, M.; Klocking, R.; Voutilainen, N.; Wutzler,
P.; Kaina, B. Comparison of the genotoxic and apoptosis-inducing
properties of ganciclovir and penciclovir in Chinese hamster
ovary cells transfected with the thymidine kinase gene of herpes
simplex virus-1: Implications for gene therapeutic approaches.
Cancer Gene Ther. 2000, 7, 107-117.
(7) Spadari, S.; Ciarrocchi, G.; Focher, F.; Verri, A.; Maga, G.;
Arcamone, F.; Iafrate, E.; Manzini, S.; Garbesi, A.; Tondelli, L.
5-Iodo-2′-deoxy-L-uridine and (E)-5-(2-bromovinyl)-2′-deoxy-L-
uridine: Selective phosphorylation by herpes simplex virus type
1 thymidine kinase, antiherpetic activity, and cytotoxicity stud-
ies. Mol. Pharmacol. 1995, 47, 1231-1238.
(8) Haynes, P.; Lambert, T. R.; Mitchell, I. D. Comparative in-vivo
genotoxicity of antiviral nucleoside analogues; penciclovir, acy-
clovir, ganciclovir and the xanthine analogue, caffeine, in the
mouse bone marrow micronucleus assay. Mutat. Res. 1996, 369,
65-74.
(1S,2S,4S,5R)-(2E)-3-{1-[4-Hyd r oxy-5-(h yd r oxym eth yl)-
bicyclo[3.1.0]h ex-2-yl]-2,4-d ioxo(1,3-d ih yd r op yr im id in e-
5-yl)}p r op -2-en oic Acid (15). A solution of 14 (0.165 g, 0.51
mmol) in 1.8 N KOH (2 mL) was stirred overnight at room
temperature. The solution was then acidified with concen-
trated HCl to pH 2 and the precipitate formed was collected
by filtration. The filtrate was reduced to dryness and the solid
residue was triturated with MeOH. The combined solids were
dissolved in hot MeOH, the solution was filtered, and the
filtrate was allowed to stand at room temperature. Since no
crystals formed, the methanolic solution was concentrated to
give 15 (0.137 g, 87%) as a white solid: mp 240 °C dec; 1H
NMR (CDCl3 + D2O) δ 8.53 (s, 1 H), 7.26 (d, 1 H, J ) 15.8
Hz), 6.79 (d, 1 H, J ) 15.8 Hz), 4.78 (d, 1 H, J ) 6.6 Hz), 4.61
(irregular t, 1 H), 4.14 (AB d, 1 H, J ) 11.5 Hz), 3.15 (AB d, 1
H, J ) 11.5 Hz), 1.87 (irregular dd, 1 H), 1.59 (m, 1 H), 1.37
(irregular dd, 1 H), 0.85 (irregular t, 1 H), 0.63 (irregular dd,
1 H). This product was used directly in the next step.
(1S,2S,4S,5R)-5-((1E)-2-Br om ovin yl)-1-[4-h yd r oxy-5-
(h yd r oxym eth yl)bicyclo[3.1.0]h ex-2-yl]-1,3-d ih yd r op yr i-
m id in e-2,4-d ion e (4). To a mixture of 15 (0.126 g. 0.41 mmol)
in DMF (2.5 mL) was added KHCO3 (0.118 g, 1.18 mmol)
followed by slow addition of NBS (0.072 g, 0.41 mmol) dissolved
in DMF (1 mL). After 2.5 h of stirring, insoluble materials were

5-Substituted Derivatives of Methanocarbathymine
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5053
(9) Fyfe, J . A.; Keller, P. M.; Furman, P. A.; Miller, R. L.; Elion, G.
B. Thymidine kinase from herpes simplex virus phosphorylates
the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine.
J . Biol. Chem. 1978, 253, 8721-8727.
(10) De Clercq, E. Antiviral agents: Characteristic activity spectrum
depending on the molecular target with which they interact. Adv.
Virus Res. 1993, 42, 1-55.
(11) Caruso, M. Gene therapy against cancer and HIV infection using
the gene encoding herpes simplex virus thymidine kinase. Mol.
Med. Today 1996, 2, 212-217.
(12) Culver, K. W.; Ram, Z.; Wallbridge, S.; Ishii, H.; Oldfield, E. H.;
Blaese, R. M. In vivo gene transfer with retroviral vector-
producer cells for treatment of experimental brain tumors.
Science 1992, 256, 1550-1552.
(28) Ostrowski, T.; Wroblowski, B.; Busson, R.; Rozenski, J .; De
Clercq, E.; Bennett, M. S.; Champness, J . N.; Summers, W. C.;
Sanderson, M. R.; Herdewijn, P. 5-Substituted pyrimidines with
a 1,5-anhydro-2,3-dideoxy-D-arabino-hexitol moiety at N-1: Syn-
thesis, antiviral activity, conformational analysis, and interac-
tion with viral thymidine kinase. J . Med. Chem. 1998, 41, 4343-
4353.
(29) Villarreal, E. C. Current and potential therapies for the treat-
ment of herpesvirus infections. Prog. Drug Res. 2001, 56, 77-
120.
(30) Zalah, L.; Huleihel, M.; Manor, E.; Konson, A.; Ford, H., J r.;
Marquez, V. E.; J ohns, D. G.; Agbaria, R. Metabolic pathways
of N-methanocarbathymidine, a novel antiviral agent, in native
and herpes simplex virus type 1 infected Vero cells. Antiviral
Res. 2002, 55, 63-75.
(31) Herdewijn, P. 5-Substituted-2′-deoxyuridines as anti-HSV-1
agents: Synthesis and structure activity relationship. Antiviral.
Chem. Chemother. 1994, 5, 131-146.
(13) Uckert, W.; Kammertons, T.; Haack, K.; Qin, Z.; Gebert, J .;
Schendel, D. J .; Blankenstein, T. Double suicide gene (cytosine
deaminase and herpes simplex virus thymidine kinase) but not
single gene transfer allows reliable elimination of tumor cells
in vivo. Hum. Gene Ther. 1998, 9, 855-865.
(14) Tiberghien, P.; Reynolds, C. W.; Keller, J .; Spence, S.; Des-
chaseaux, M.; Certoux, J . M.; Contassot, E.; Murphy, W. J .;
Lyons, R.; Chiang, Y. et al. Ganciclovir treatment of herpes
simplex thymidine kinase-transduced primary T lymphocytes:
An approach for specific in vivo donor T-cell depletion after bone
marrow transplantation? Blood 1994, 84, 1333-1341.
(15) Garin, M. I.; Garrett, E.; Tiberghien, P.; Apperley, J . F.;
Chalmers, D.; Melo, J . V.; Ferrand, C. Molecular mechanism for
ganciclovir resistance in human T lymphocytes transduced with
retroviral vectors carrying the herpes simplex virus thymidine
kinase gene. Blood 2001, 97, 122-129.
(32) Marquez, V. E.; Russ, P.; Alonso, R.; Siddiqui, M. A.; Hernandez,
S.; George, C.; Nicklaus, M. C.; Dai, F.; Ford, H., J r. Synthesis
of conformationally restricted carbocyclic nucleosides: The role
of the O(4′)-atom in the key hydration step of adenosine
deaminase. Helv. Chim. Acta 1999, 82, 2119-2129.
(33) Cruickshank, K. A.; J iricny, J .; Reese, Colin B. The benzoylation
of uracil and thymine. Tetrahedron Lett. 1984, 25, 681-684.
(34) Ezzitouni, A.; Russ, P.; Marquez, V. E. (1S,2R)-[(Benzyloxy)-
methyl]cyclopent-3-enol. A versatile synthon for the preparation
of 4′,1′a-methano- and 1′,1′a-methanocarbocyclic nucleosides. J .
Org. Chem. 1997, 62, 4870-4873.
(35) Herdewijn, P.; De Clercq, E.; Balzarini, J .; Vanderhaeghe, H.
Synthesis and antiviral activity of the carbocyclic analogues of
(E)-5-(2-halovinyl)-2′-deoxyuridines and (E)-5-(2-halovinyl)-2′-
deoxycytidines. J . Med. Chem. 1985, 28, 550-555.
(36) Balzarini, J .; Baumgartner, H.; Bodenteich, M.; De Clercq, E.;
Griengl, H. Synthesis and biological properties of (+)- and (-)-
(E)-5-(2-bromovinyl)-2′-deoxy-1′a-carbauridine. Nucleosides Nu-
cleotides 1989, 8, 855-858.
(37) Balzarini, J .; Baumgartner, H.; Bodenteich, M.; De Clercq, E.;
Griengl, H. Synthesis and antiviral activity of the enantiomeric
forms of carba-5-iodo-2′-deoxyuridine and carba-(E)-5-(2-bro-
movinyl)-2′-deoxyuridine. J . Med. Chem. 1989, 32, 1861-1865.
(38) Wyatt, P. G.; Anslow, A. S.; Coomber, B. A.; Cousins, R. P. C.;
Evans, D. N.; Gilbert, V. S.; Humber, D. C.; Pasternoster, I. L.;
Sollis, S. L.; Tapolczay, D. J .; Weingarten, G. G. A short high
yielding synthesis of the potent anti-VZV carbocyclic nucleoside
analogue carba-BVDU. Nucleosides Nucleotides 1995, 14, 2039-
2049.
(39) Degre`ve, B.; Esnouf, R.; De Clercq, E.; Balzarini, J . Mutation of
Gln125 to Asn selectively abolishes the thymidylate kinase
activity of herpes simplex virus type 1 thymidine kinase. Mol.
Pharmacol. 2001, 59, 285-293.
(40) Kussmann-Gerber, S.; Wurth, C.; Scapozza, L.; Pilger, B. D.;
Pliska, V.; Folkers, G. Interaction of the recombinant herpes
simplex virus type 1 thymidine kinase with thymidine and
aciclovir: A kinetic study. Nucleosides Nucleotides 1999, 18,
311-330.
(41) Schelling, P.; Folkers, G.; Scapozza, L. A Spectrophotometric
Assay for Quantitative Determination of kcat of Herpes Simplex
Virus Type 1 Thymidine Kinase Substrates. Anal. Biochem.
2001, 295, 82-87.
(42) Sulpizi, M.; Schelling, P.; Folkers, G.; Carloni, P.; Scapozza, L.
The rationale of catalytic activity of herpes simplex virus
thymidine kinase. A combined biochemical and quantum chemi-
cal study. J . Biol. Chem. 2001, 276, 21692-21697.
(43) Balzarini, J .; De Clercq, E.; Baumgartner, H.; Bodenteich, M.;
Griengl, H. Carbocyclic 5-iodo-2′-deoxyuridine (C-IDU) and
carbocyclic (E)-5-(2-bromovinyl)-2′-deoxyuridine (C-BVDU) as
unique examples of chiral molecules where the two enantiomeric
forms are biologically active: Interaction of the (+)- and (-)-
enantiomers of C-IDU and C-BVDU with the thymidine kinase
of herpes simplex virus type 1. Mol. Pharmacol. 1990, 37, 395-
401.
(44) Kern, E. R.; Hartline, C.; Harden, E.; Keith, K.; Rodriguez, N.;
Beadle, J . R.; Hostetler, K. Y. Enhanced inhibition of orthopox-
virus replication in vitro by alkoxyalkyl esters of cidofovir and
cyclic cidofovir. Antimicrob. Agents Chemother. 2002, 46, 991-
995.
(16) Tiberghien, P. “Suicide” gene for the control of graft-versus-host
disease. Curr. Opin. Hematol. 1998, 5, 478-482.
(17) Thust, R.; Tomicic, M.; Klocking, R.; Wutzler, P.; Kaina, B.
Cytogenetic genotoxicity of anti-herpes purine nucleoside ana-
logues in CHO cells expressing the thymidine kinase gene of
herpes simplex virus type 1: Comparison of ganciclovir, penci-
clovir and aciclovir. Mutagenesis 2000, 15, 177-184.
(18) Thust, R.; Schacke, M.; Wutzler, P. Cytogenetic genotoxicity of
antiherpes virostatics in Chinese hamster V79-E cells. I. Purine
nucleoside analogues. Antiviral Res. 1996, 31, 105-113.
(19) Tiberghien, P.; Ferrand, C.; Lioure, B.; Milpied, N.; Angonin,
R.; Deconinck, E.; Certoux, J . M.; Robinet, E.; Saas, P.; Petracca,
B.; J uttner, C.; Reynolds, C. W.; Longo, D. L.; Herve, P.; Cahn,
J . Y. Administration of herpes simplex-thymidine kinase-
expressing donor T cells with a T-cell-depleted allogeneic marrow
graft. Blood 2001, 97, 63-72.
(20) Hinds, T. A.; Compadre, C.; Hurlburt, B. K.; Drake, R. R.
Conservative mutations of glutamine-125 in herpes simplex
virus type 1 thymidine kinase result in a ganciclovir kinase with
minimal deoxypyrimidine kinase activities. Biochemistry 2000,
39, 4105-4111.
(21) Drake, R. R.; Wilbert, T. N.; Hinds, T. A.; Gilbert, K. M.
Differential ganciclovir-mediated cell killing by glutamine 125
mutants of herpes simplex virus type 1 thymidine kinase. J . Biol.
Chem. 1999, 274, 37186-37192.
(22) Black, M. E.; Newcomb, T. G.; Wilson, H. M.; Loeb, L. A. Creation
of drug-specific herpes simplex virus type 1 thymidine kinase
mutants for gene therapy. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 3525-3529.
(23) Christians, F. C.; Scapozza, L.; Crameri, A.; Folkers, G.; Stem-
mer, W. P. Directed evolution of thymidine kinase for AZT
phosphorylation using DNA family shuffling. Nat. Biotechnol.
1999, 17, 259-264.
(24) Kokoris, M. S.; Sabo, P.; Black, M. E. In vitro evaluation of
mutant HSV-1 thymidine kinases for suicide gene therapy.
Anticancer Res. 2000, 20, 959-963.
(25) Prota, A.; Vogt, J .; Pilger, B.; Perozzo, R.; Wurth, C.; Marquez,
V. E.; Russ, P.; Schulz, G. E.; Folkers, G.; Scapozza, L. Kinetics
and Crystal Structure of the Wild-Type and the Engineered
Y101F Mutant of Herpes simplex Virus Type 1 Thymidine
Kinase Interacting with (North)-methanocarba-thymidine. Bio-
chemistry 2000, 39, 9597-9603.
(45) Andrei, G.; Snoeck, R.; Reymen, D.; Liesnard, C.; Goubau, P.;
Desmyter, J .; De Clercq, E. Comparative activity of selected
antiviral compounds against clinical isolates of varicella-zoster
virus. Eu.r J . Clin. Microbiol. Infect. Dis. 1995, 14, 318-328.
(46) Prota, A. Structure elucidation of viral and human thymidine
kinases and enolase from Alternaria alternata. Thesis 13456,
Swiss Federal Institute of technology, Zurich, 1999; pp 61-93.
(47) Pilger, B. D.; Perozzo, R.; Alber, F.; Wurth, C.; Folkers, G.;
Scapozza, L. Substrate diversity of herpes simplex virus thymi-
dine kinase. Impact of the kinematics of the enzyme. J . Biol.
Chem. 1999, 274, 31967-31973.
(26) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang,
J .; Wagner, R. W.; Matteucci, M. D. Nucleosides with a twist.
Can fixed forms of sugar ring pucker influence biological activity
in nucleosides and oligonucleotides? J . Med. Chem. 1996, 39,
3739-3747.
(27) Hasegawa, Y.; Nishiyama, Y.; Imaizumi, K.; Ono, N.; Kinoshita,
T.; Hatano, S.; Saito, H.; Shimokata, K. Avoidance of bone
marrow suppression using A-5021 as a nucleoside analogue for
retrovirus-mediated herpes simplex virus type
I thymidine
kinase gene therapy. Cancer Gene. Ther. 2000, 7, 557-562.

5054 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Russ et al.
(48) Furlong, N. B. A rapid assay for nucleotide kinases using C14
-
(51) Roehm, N. W.; Rodgers, G. H.; Hatfield, S. M.; Glasebrook, A.
L. An improved colorimetric assay for cell proliferation and
viability utilizing the tetrazolium salt XTT. J . Immunol. Methods
1991, 142, 257-265.
(52) J ost, L. M.; Kirkwood, J . M.; Whiteside, T. L. Improved short-
and long-term XTT-based colorimetric cellular cytotoxicity assay
for melanoma and other tumor cells. J . Immunol. Methods 1992,
147, 153-165.
or H3-labeled nucleotides. Anal. Biochem. 1963, 5, 512-522.
(49) Gerber, S.; Folkers, G. A new method for quantitative determi-
nation of tritium-labeled nucleoside kinase products adsorbed
on DEAE-cellulose. Biochem. Biophys. Res. Commun. 1996, 225,
263-267.
(50) Ashida, N.; Watanabe, Y.; Miura, S.; Kano, F.; Sakata, S.;
Yamaguchi, T.; Suzutani, T.; Machida, H. Structure-activity
relationship of the affinity of 5-substituted uracil nucleoside
analogues for varicella-zoster virus thymidine kinase and their
activity against varicella-zoster virus. Antiviral. Res. 1997, 35,
167-175.
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