Understanding how the herpes thymidine kinase orchestrates optimal sugar and nucleobase conformations to accommodate its substrate at the active site: A chemical approach

Article abstract of DOI:10.1021/ja053789s

The herpes virus thymidine kinase (HSV-tk) is a critical enzyme for the activation of anti-HSV nucleosides. However, a successful therapeutic outcome depends not only on the activity of this enzyme but also on the ability of the compound(s) to interact ef

Full text of DOI:10.1021/ja053789s

Published on Web 10/06/2005
Understanding How the Herpes Thymidine Kinase
Orchestrates Optimal Sugar and Nucleobase Conformations
To Accommodate Its Substrate at the Active Site: A Chemical
Approach
Victor E. Marquez,*,† Yongseok Choi,† Maria Julieta Comin,† Pamela Russ,†
Clifford George,‡ Mahmoud Huleihel,§ Tsipi Ben-Kasus,| and Riad Agbaria|
Contribution from the Laboratory of Medicinal Chemistry, Center for Cancer Research, National
Cancer Institute at Frederick, 376 Boyles Street, Frederick, Maryland 21702, Laboratory for the
Structure of Matter, NaVal Research Laboratory, Washington, D.C. 20375, and The National
Institute for Biotechnology and Department of Virology, and Department of Clinical
Pharmacology, Faculty of Health Sciences, Ben-Gurion UniVersity of the NegeV,
Beer-SheVa 84105, Israel
Received June 9, 2005; E-mail: marquezv@dc37a.nci.nih.gov
Abstract: The herpes virus thymidine kinase (HSV-tk) is a critical enzyme for the activation of anti-HSV
nucleosides. However, a successful therapeutic outcome depends not only on the activity of this enzyme
but also on the ability of the compound(s) to interact effectively with cellular kinases and with the target
viral or cellular DNA polymerases. Herein, we describe the synthesis and study of two nucleoside analogues
built on a conformationally locked bicyclo[3.1.0]hexane template designed to investigate the conformational
preferences of HSV-tk for the 2′-deoxyribose ring. Intimately associated with the conformation of the 2′-
deoxyribose ring is the value of the C-N torsion angle ø, which positions the nucleobase into two different
domains (syn or anti). The often-conflicting sugar and nucleobase conformational parameters were studied
using North and South methanocarbadeoxythymidine analogues (6 and 7), which forced HSV-tk to make
a clear choice in the conformation of the substrate. The results provide new insights into the mechanism
of action of this enzyme, which cannot be gleaned from a static X-ray crystal structure.
Introduction
infections are principally nucleoside analogues. Despite their
safety and efficacy, many nucleoside analogues have limited
Human herpes viruses HSV-1 and HSV-2 infections are
widespread and can be life-threatening to immunocompromised
patients.1 In the United States, HSV infections are a significant
public health problem affecting nearly 150 million Americans,
of which 40-60 million are infected with HSV-2 and an
additional 1 million infections occur each year.2 Genital herpes,
caused by either HSV-1 or HSV-2, has become the leading cause
of genital ulcers.3 Genital ulcers increase the risk of HIV
infection augmenting its morbidity and mortality.4 If the herpes
virus is not totally eradicated after the initial infection, it can
persist latently in host neurons, posing a risk of periodical
reactivation over a lifetime.5 The current treatments for HSV
oral bioavailability, must be administered early in the disease
process for maximal benefit, and can become ineffective due
to the development of drug resistance, particularly in immuno-
compromised patients.2,6 The need to overcome these problems
requires a commitment to develop novel drugs, including new
nucleoside analogues. The development of new nucleosides
analogues will require a better understanding of their mechanism
of action.
Successful therapy using nucleoside analogues depends on
the ability of the analogues to interact effectively with a triad
of anabolic kinases and with the target viral or cellular DNA
polymerases. In the specific case of HSV-infected cells, the viral
kinase (HSV-tk) accepts a broader range of substrates (nucleo-
side analogues) for the first phosphorylation step than the more
stringent cellular thymidine kinase (tk1), which phosphorylates
thymidine almost exclusively.7,8 Because only infected cells
contain HSV-tk, a favorable therapeutic index depends on the
differential specificity of HSV-tk and tk1 and the ability of
subsequent phosphorylation steps to produce a 5′-triphosphate
† National Cancer Institute at Frederick.
‡ Naval Research Laboratory.
§ The National Institute for Biotechnology and Department of Virology,
Ben-Gurion University of the Negev.
| Department of Clinical Pharmacology, Ben-Gurion University of the
Negev.
(1) Brady, R. C.; Bernstein, D. I. AntiViral Res. 2004, 61, 73-81.
(2) Firestine, S. M. Expert Opin. Ther. Pat. 2004, 14, 1139-1151.
(3) Celum, C.; Levine, R.; Weaver, M.; Wald, A. Bull. WHO 2004, 82, 447-
453.
(4) Sacks, S. L.; Griffiths, P. D.; Corey, L.; Cohen, C.; Cunningham, A.;
Dusheiko, G. M.; Self, S.; Spruance, S.; Stanberry, L. R.; Wald, A.; Whitley,
R. J. AntiViral Res., Suppl. 1 2004, 63, S3-S10.
(5) Schmid-Wendtner, M. H.; Korting, H. C. Skin Pharmacol. Appl. Skin
Physiol. 2004, 17, 214-218.
(6) Levin, M. J.; Bacon, T. H.; Leary, J. J. Clin. Infect. Dis. Suppl. 2004, 39,
S248-257.
(7) De Clercq, E.; Descamps, J.; Verheslt, G.; Walker, R. T.; Jones, A. S.;
Torrence, P. F.; Shugar, D. J. Infect. Dis. 1980, 141, 563-574.
(8) Snoek, R. Int. J. Antimicrob. Agents 2000, 16, 157-159.
9
10.1021/ja053789s CCC: $30.25 © 2005 American Chemical Society
J. AM. CHEM. SOC. 2005, 127, 15145-15150
15145

A R T I C L E S
Marquez et al.
and on host or viral polymerases incorporating the resulting
triphosphate. Although the second and third phosphorylation
steps are normally carried out by cellular kinases, HSV-tk can
also function as a thymidylate kinase, performing the second
phosphorylation step.9,10 In the end, it is the fine balance between
the ability of a nucleoside to function as substrate both for
kinases and for polymerases that determines its usefulness as a
drug.
We have demonstrated, in previous studies, that the confor-
mation of the sugar ring plays a critical role in determining the
affinity of kinases and polymerases for nucleosides.11 Using
nucleoside analogues built on a rigid template, the bicyclo[3.1.0]-
hexane scaffold, we produced two isomers of methanocarba
(MC) thymidine (T), one with the pseudosugar moiety locked
in the North (N) hemisphere of the pseudorotational cycle (1,
N-MCT) and the other with it locked in the antipodean South
(S) conformation (2, S-MCT). These two isomers display
opposite affinities for HSV-tk and DNA polymerases.11 We
found that for the second and third phosphorylation steps S-MCT
was the preferred substrate, but that despite the high levels of
S-MCT-5′-triphosphate formed, only negligible amounts were
incorporated into DNA. On the other hand, because N-MCT
was a poorer substrate for these kinases, lower levels of N-MCT-
5′-triphosphate were produced. Nevertheless, N-MCT-5′-triph-
osphate was efficiently incorporated into DNA by the host
polymerase; as a result, N-MCT (1) is a potent anti-HSV drug
(EC50 ) 0.02 µM) while S-MCT (2) was completely ineffec-
tive.10,12
Figure 1. Pseudorotational cycle of the furanose ring in nucleosides. The
populated ranges for N (2E f 3T2
f f 3E)
3E) and S (2E f 2T3
conformations are indicated.
nucleosides, because of their rigid pseudoboat conformations,
lock the positions of N-MCT and S -MCT at E (342°) and E
2
3
(198°), respectively (Figure 1).
The strong preference of S-MCT for the second and third
phosphorylation steps was demonstrated by the S/N ratios of
phosphorylated metabolites of 4.6 and 2.5, respectively.11
Surprisingly, the first phosphorylation step performed by HSV-
tk did not show the same penchant, as the observed S/N ratio
of 0.9 even appeared to suggest an opposite preference.11
A
proposal to explain this apparent deviation from the behavior
of the other kinases is that the nucleobase in S-MCT is in an
orientation not easily recognized by the enzyme. Both X-ray
structures of N-MCT15 and S-MCT,16 and computational analy-
sis,11 showed that the preferred conformation for the nucleobase
in N-MCT was anti, while the preferred conformation for the
nucleobase in S-MCT was syn. The anti or syn conformation
describes the orientation of the nucleobase relative to the sugar
ring (see structures 1 and 2), and it is determined by the value
of the torsion angle ø of the C-N bond that connects the base
to the sugar.14 The crystallographic data and the ab initio
calculations for N-MCT and S-MCT mentioned above are in
perfect agreement with the trend observed with conventional
nucleosides where ø tends to be anti when the sugar pucker is
N and syn when the sugar pucker is S.17 This propensity is much
stronger for the locked bicyclo[3.1.0]hexane nucleosides because
the energy barriers between anti and syn are much larger for
N-MCT and S-MCT than for conventional nucleosides.11
Remarkably, the crystal structures of HSV-tk in complexes with
N-MCT or S-MCT show that the nucleobase in both compounds
is in the anti conformation with the fused cyclopropane ring
neatly tucked under the cyclopentane ring and away from
neighboring amino acids.18,19 Unfortunately, X-ray structures
are static models of stable end-points that do not shed useful
The contrasting conformational preference of kinases and
polymerases demonstrates the importance of the inherent
flexibility of the ribose (or 2′-deoxyribose) ring, which allows
it to adapt to the conformational demands of the two types of
enzymes. Conventional nucleosides in solution normally un-
dergo rapid equilibration between the N and S puckering
domains, as described in the pseudorotational cycle (0° f 360°,
Figure 1).13,14 The N conformations encompass a range between
3
3
342° and 18° (2E f T2 f E), where E and T correspond to
envelope (E) and twisted (T) conformations of the tetrahydro-
furan ring, and the super- or subscripted numbers denote the
atoms on the five-member ring that project above or below the
plane of the sugar ring. At the other extreme, the antipodean S
2
conformations oscillate between 162° and 198° (2E f T3 f
3
2
3E). The ideal conformations of T2 and T3 correspond to 0°
and 180° in the pseudorotational cycle. The bicyclo[3.1.0]hexane
(9) Chen, M. S.; Walker, J.; Prusoff, W. H. J. Biol. Chem. 1979, 254, 10747-
10753.
(10) Zalah, L.; Huleihel, M.; Manor, E.; Jonson, A.; Ford, H., Jr.; Marquez, V.
E.; Johns, D. G.; Agbaria, R. AntiViral Res. 2002, 55, 63-75.
(11) Marquez, V. E.; Ben-Kasus, T.; Barchi, J. J., Jr.; Green, K. M.; Nicklaus,
M. C.; Agbaria, R. J. Am. Chem. Soc. 2004, 126, 543-549.
(12) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J.; Wagner,
R. W.; Matteucci, M. D. J. Med. Chem. 1996, 39, 3739-3747.
(13) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205-8212.
(14) Saenger, W. Principles in Nucleic Acid Structure; Springer-Verlag: New
York, Berlin, Heidelberg, 1984; pp 9-28.
(15) Altman, K. H.; Kesselring, R.; Francotte, E.; Rihs, G. Tetrahedron Lett.
1994, 35, 2331-2334.
(16) Altman, K. H.; Imwinkelried, R.; Kesselring, R.; Rihs, G. Tetrahedron Lett.
1994, 35, 7625-7628.
(17) Saenger, W. Principles in Nucleic Acid Structure; Springer-Verlag: New
York, Berlin, Heidelberg, 1984; pp 51-104.
(18) Prota, A.; Vogt, J.; Pilger, B.; Perozzo, R.; Wurth, C.; Marquez, V. E.;
Russ, P.; Schulz, G. E.; Folkers, G.; Scapozza, L. Biochemistry 2000, 39,
9597-9603.
9
15146 J. AM. CHEM. SOC. VOL. 127, NO. 43, 2005

Herpes Virus Thymidine Kinase
A R T I C L E S
Scheme 1
Scheme 2
Figure 2. Structures of the D- (7) and L-isomer (6) of N-MCdT. The optical
antipode (compound 6) has a bicylo[3.1.0]hexane ring that mimics the S
conformation. In both compounds, the disposition of the nucleobase is anti.
light on the energy required for the ligand to reach the bound
conformation. These X-ray structures also confirmed that
S-MCT was the better substrate mimic because the locked
cyclopentane ring of the bicyclo[3.1.0]hexane pseudosugar
perfectly followed the contour of the sugar ring of the natural
substrate, thymidine.11 Moreover, the critical 3′-OH group of
S-MCT is in an axial disposition and formed a well-defined
bifurcated H-bond with the OH of Tyr101 and the carboxylate
group of Glu225, identical to what was observed with thymi-
dine.19 In contrast, the equatorial 3′-OH in N-MCT was
displaced 3.7 Å away from Glu225 and 4.1 Å away from
Tyr101, showing that the N sugar pucker prevented the
formation of the H-bonds.18 The puzzling question was why
the first phosphorylation step of HSV-tk was not so efficient
when S-MCT appeared to be such a good mimic? Was the
higher energy barrier required to switch the nucleobase from
syn to anti the critical factor negatively affecting the first
phosphorylation step? If this is the problem, could we shift the
position of the nucleobase in S-MCT from syn to anti and
generate an analogue that would be efficiently phosphorylated?
Figure 3. Crystal structure of S-MCdT (6). Displacement ellipsoid plot
drawn at the 50% probability level. The numbering system used here
corresponds to the IUPAC name.
Scheme 3
Design of an anti-South Analogue
has the desired shape to be considered a South mimic, which,
according to the numbering shown (nucleoside numbering), is
Ab initio calculations suggested that the fused cyclopropane
ring adjacent to the C-N bond was the source of the high-
energy barrier between the syn and anti conformations of
S-MCT.11 The integrity of the bicyclo[3.1.0]hexane, which
maintains a rigid pseudoboat conformation, suggested that we
could transpose one bond of the fused cyclopropane ring in
S-MCT (2) to the opposite side of the molecule, generating the
isomeric bicyclo[3.1.0]hexane nucleoside 3 shown in Scheme
1. This rearrangement would maintain the desired S-like
conformation; however, it produced a structure that, based on
literature precedent, would be unstable and prone to ring-opening
to give 4, even at room temperature (Scheme 1).20 We then
considered removing the OH function, as in compound 5, which
after an identical bond transposition would produce a stable
dideoxy analogue, S-MCdT (6, Scheme 2). As shown is Scheme
2, the new compound 6 corresponds to the optical antipode of
the dideoxy analogue of N-MCdT (compound 7). These two
compounds, the L- and D-isomers of N-MCdT (Figure 2), are
the two compounds used to test our hypothesis. The L-isomer 6
equivalent to an E conformation identical to that of S-MCT
3
(2).21 As expected, the X-ray structure of 6 (Figure 3) confirmed
the anti disposition of the base. Because both compounds (6
and 7) now have the C-N bond in the anti conformation, the
enzyme’s choice between 6 and 7 would be governed exclu-
sively by its preference for a specific sugar conformation, N or
S.
Synthesis
Synthesis of the D-isomer (N-MCdT, 7) was performed by
catalytic hydrogenation of compound 922 (Scheme 3). Starting
with the same chiral cyclopentenol (8)23,24 used for the synthesis
(21) Because there are six carbons in the bicyclo[3.1.0]hexans system, but only
five are required to define the shape of the embedded five-member ring in
the pseudorotational cycle, one carbon has to be omitted. Normally, as in
compound 7, the omitted carbon is the one at the tip of the fused
cyclopropane ring. However, in the case of compound 6, the numbering
system corresponds to that of the progenitor compound 5; thus the carbon
formerly at the tip of the cyclopropane ring was omitted. The new five
member ring envisioned in 6 assumes a virtual bond between carbons 1
and 5.
(22) Choi, Y.; George, C.; Comin, M. J.; Barchi, J. J., Jr.; Kim, H. S.; Jacobson,
K. A.; Balzarini, J.; Mitsuya, H.; Boyer, P. L.; Hughes, S. H.; Marquez,
V. E. J. Med. Chem. 2003, 46, 3292-3299.
(23) Ezzitouni, A.; Russ, P.; Marquez, V. E. J. Org. Chem. 1997, 62, 4870-
4873.
(19) Schelling, P.; Claus, M. T.; Johner, R.; Marquez, V. E.; Schulz, G. E.;
Scapozza, L. Biochemical and structural characterization of (South)-
methanocarbathymidine that specifically inhibits growth of herpes simplex
virus type 1 thymidine kinase-transduced osteosarcoma cells. J. Biol. Chem.
2004, 279, 32832-32838.
(20) Creary, X. J. Org. Chem. 1975, 40, 3326-3331.
9
J. AM. CHEM. SOC. VOL. 127, NO. 43, 2005 15147

A R T I C L E S
Scheme 4
Marquez et al.
Table 1. Levels of N-MCdT (7) and S-MCdT (6) Phosphates
(pmol/106 cells) in HSV-1-Infected Vero Cellsa
N-MCdT
S-MCdT
6 h
24 h
6 h
24 h
MP
DP
TP
60.7 ( 3.21
1.214 ( 0.21
0.721 ( 0.16
706 ( 21
45.6 ( 6.30
9.40 ( 1.40
137 ( 8.65
1.221 ( 0.16
0.215 ( 0.08
1609 ( 18
37.9 ( 2.68
5.06 ( 0.80
a MP ) monophosphate; DP ) diphosphate; TP ) triphosphate. Cells
were treated with 10 µM, 10 µCi/mL of drug. Data are mean ( SD (n )
3).
S/N ratio from 0.9 to 2.3 for the first phosphorylation step clearly
indicates that if the nucleobase is not constrained in the syn
conformation, HSV-tk has a clear preference for the substrates
in the S conformation.
The relatively low levels of di- and triphosphate metabolites
of N-MCdT and S-MCdT produced in the ensuing step, and
the surprising lack of discrimination between N and S beyond
the first step, contrasts with what was seen with N-MCT and
S-MCT, suggesting that the 3′-OH group plays a critical role in
substrate recognition by HSV-tk for the second phosphorylation
step. We know that HSV-tk is a multifunctional enzyme that
exhibits thymidylate kinase activity, but that, in this role, it has
a narrower range of substrates.9,25 Consistent with this more
stringent substrate recognition, the 3′-OH appears to play a
critical role for the second phosphorylation step. On the other
hand, for the first phosphorylation step HSV-tk is shown here
to be exquisitely sensitive to the conformation of the sugar and
the syn/anti disposition of the nucleobase as we predicted. The
low overall metabolism of N-MCdT and S-MCdT, which leads
to very low levels of 5′-triphosphates in HSV-infected Vero
cells, explains why these compounds poorly inhibited the growth
(IC50 > 1000 µM) of murine tumor cells (MC38) transfected
with HSV-tk (data not shown).
Evidently, modified nucleosides such as N-MCdT (7) and
S-MCdT (6) might partially behave as substrates and partially
as inhibitors of the enzyme. Although future kinetic experiments
will address this issue, preliminary IC50 values measuring the
ability of these compounds to inhibit thymidine phosphorylation
by HSV-tk (9.91 and 2.65 µM for 7 and 6, respectively)26 clearly
show that the better inhibitor with the lower IC50 value is also
the better substrate as demonstrated by direct phosphorylation
with the radiolabed substrates (Table 1).
Scheme 5
of 7, the intermediate 10 was generated according to our
published methodology.24 Following the hydroxyl-directed cy-
clopropanation of 10, the key bicyclo[3.1.0]hexane carbocyclic
precursor 11 was obtained (Scheme 4). From that point onward,
the synthesis mirrored the approach used for the synthesis of 9
to give the optical antipode 20 (Schemes 4 and 5). Catalytic
hydrogenation of 20 afforded the L-isomer (S-MCdT, 6).
Phosphorylation by HSV-tk and Discussion
The unfavorable ratio of S/N metabolites that was originally
determined for S-MCT (2, 72 ( 5.4 pmol/106 cells)11 and
N-MCT (1, 76.0 ( 0.9 pmol/106 cells)11 for the first phospho-
rylation step in HSV-infected Vero cells after 6 h was 0.9.11
The poor selectivity of the S conformation was proposed to be
the result of conflicting conformational preferences of HSV-tk
for the sugar pucker and the nucleobase disposition. Under the
same experimental conditions as those used for S-MCT and
N-MCT, there was only a slight drop in monophosphate
production (60.7 pmol/106 cells, Table 1) when N-MCdT, which
lacks the 3′-OH, was the substrate. Levels of all phosphate
metabolites in uninfected cells were very low and varied
between 0.1 and 0.4 pmol/106 cells.
Conclusions
The relocation of the cyclopropane ring from the fused
bicyclo[3.1.0]hexane system in compound 5 created S-MCdT
(6, Scheme 2). This bond transposition was used to free the
nucleobase from the preferred syn conformation. The resulting
conformational change allowed HSV-tk to recognize S-MCdT
as the preferred substrate, relative to the antipodal N-MCdT (7),
based on the shape of the pseudosugar ring. If one subtracts 5
kcal/mol for the intramolecular hydrogen bond energy between
the 5′-OH and the 2-oxopyrimidine carbonyl, which is favored
in calculations performed in a vacuum, from the total calculated
energy barrier of 15 kcal/mol needed to flip the pyrimidine ring
in S-MCT from syn into anti,11 10 kcal/mol would be required
In contrast to the low and invariant levels of monophosphate
metabolites generated from S-MCT, N-MCT, and N-MCdT, the
yield of the S-MCdT monophoshate increased to 137 pmol/106
cells in HSV-tk infected cells, which translated to a favorable
S/N ratio of 2.3 after 6 and 24 h (Table 1). The increase in the
(25) Chen, M. S.; Summers, W. P.; Walker, J.; Summers, W. C.; Prusoff, W.
H. J. Virol. 1979, 30, 942-945.
(26) Balzarini, J. Katholieke Universiteit Leuven, Rega Institute, Leuven,
Belgium. Personal communication.
(24) Marquez, V. E.; Russ, P.; Alonso, R.; Siddiqui, M. A.; Hernandez, S.;
George, C.; Nicklaus, M. C.; Dai, F.; Ford, H., Jr. HelV. Chim. Acta 1999,
82, 2119-2129.
9
15148 J. AM. CHEM. SOC. VOL. 127, NO. 43, 2005

Herpes Virus Thymidine Kinase
A R T I C L E S
beled compounds were quantitated on the basis of the known specific
activity of the parent tritiated compounds.
to flip the base. Removing this energy barrier allowed HSV-tk
to select its substrate based exclusively on sugar pucker; the
difference in the levels of monophoshates formed from two of
the substrates with opposite locked pseudosugar conformations
clearly supports our hypothesis that the S conformation is
preferred by HSV-tk.
Synthetic Methods. (1′S,2′S,5′R)-1-[5-(Hydroxymethyl)bicyclo-
[3.1.0]hex-2-yl]-5-methyl-1,3-dihydropyrimidine-2,4-dione (7). A
stirred solution of 922 (38.5 mg, 0.164 mmol) in methanol (5 mL) was
reduced under a hydrogen-filled balloon atomosphere in the presence
of 5% Pd/C for 1 h. The reaction mixture was filtered through a Celite
pad, and the filtrate was concentrated under vacuum. The residue was
purified by silica gel flash chromatography (CHCl3:MeOH, 25:1) to
give 7 (38 mg, 98%) as a white solid, mp 173-175 °C; [R]22D +66.6°
These results, in conjunction with data from our previously
published work on N-MCT and S-MCT,11 provide insights into
the multifaceted role of the sugar ring in nucleosides and
demonstrates the ability of kinases and polymerases to dis-
criminate among their substrates on the basis of sugar and
nucleobase conformations. Furthermore, our study highlights
how the substrate preferences for each step can be affected,
positively or negatively, by minor structural modifications. On
the basis of the results presented here, we can conclude that for
the first step HSV-tk activity is quite insensitive to the presence
or absence of a 3′-OH, but very responsive to both sugar and
nucleobase conformation preferring the sugar in the S conforma-
tion and the base in the anti disposition. For the second step,
however, HSV-tk becomes extremely sensitive to the 3′-OH and
still maintains its preference for substrates with the S conforma-
tion. The sensitivity of both kinases and polymerases to subtle
structural changes may help explain why ostensibly similar
nucleoside analogues often produce widely different biological
effects ranging from inactive to extremely toxic. It is hoped
that the results presented here will contribute to the design of
more specific and selective antiviral and antitumor agents such
as N-MCT. This compound is a very effective and selective
anti-herpes agent, both in vitro and in vivo, which, despite its
lower affinity for HSV-tk and cellular kinases, has excellent
properties as a substrate for DNA polymerases.10 The potential
use of N-MCT as a clinical agent is being actively pursued.
1
(c 0.35, MeOH); H NMR (DMSO-d6) δ 11.12 (br s, 1H, NH), 7.83
(dd, 1H, J ) 1.2 Hz, H-6), 4.98 (br s, 1H, OH), 4.68 (d, 1H, J ) 6.6
Hz, H-2′), 3.84 (d, 1H, J ) 11.1 Hz, CHHOH), 3.21 (d, 1H, J ) 11.1
Hz, CHHOH), 1.90-2.00 (irregular q, 1H, H-4′a), 1.60-1.78 (overlap-
ping s and m, 4H, CH3 and H-4′b), 1.40-1.58 (m, 2H, H-3), 1.16 (dd,
1H, J ≈ 8.8, 3.9 Hz, H-1′), 0.55 (dd, 1H, J ≈ 8.5, 5.2 Hz, H-6′a), 0.46
(irregular t, 1H, H-6′b); 13C NMR (DMSO-d6) δ 165.3, 151.7, 139.0,
109.7, 64.8, 57.7, 32.9, 29.9, 25.9, 25.3, 11.3, 11.2; FAB MS (m/z,
relative intensity) 237 (MH+, 100). Anal. Calcd for C12H16N2O3‚
0.30H2O: C, 59.58; H, 6.86; N, 11.59. Found: C, 59.32; H, 6.86; N,
11.56.
(1R,2S,4S,5S)-4-(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)-5-
[(phenylmethoxy)methyl]bicyclo[3.1.0]hexan-2-ol (11). Under an
argon atmosphere, a 1.0 M solution of diethyl zinc in hexanes (16 mL)
was added dropwise to a solution of 1023,24 (6.40 g, 14 mmol) in
anhydrous CH2Cl2 (100 mL) at 0 °C. After 15 min of stirring, a solution
of diiodomethane (1.3 mL, 16 mmol) in anhydrous methylene chloride
(7 mL) was rapidly added via syringe. Second equal portions of diethyl
zinc (16 mL) and diiodomethane (7 mL) solutions were added
sequentially after 15 min, and after an additional 15 min the reaction
mixture was allowed to reach room temperature. The reaction was
quenched after 4 h by the addition of a saturated solution of NH4Cl.
The aqueous layer was extracted with CH2Cl2 three times, and the
organic phase was dried (MgSO4) and concentrated under vacuum to
give a yellowish oil, which was used in the next step without further
purification. An analytical sample for characterization was purified by
silica gel flash chromatography (hexanes:EtOAc, 8:2) to afford
Experimental Methods
compound 10 as an oil; [R]22 -42.1° (c 1.52, CHCl3); 1H NMR
Preparation of Cell Extract for Metabolite Analysis. Vero cell
cultures (4 × 106/25 mL flask) were infected with 1 PFU/cell of HSV-
1. After a 2 h incubation period, the cells were incubated with
radioactive [Me-3H]-N-MCdT or [Me-3H]-S-MCdT, 10 µM, 5 µCi/mL.
These compounds were purchased from Moravek Biochemicals, Brea,
CA [specific activities of 1100 and 1250 dpm/pmol for [Me-3H]-N-
MCdT and [Me-3H]-S-MCdT, respectively]. As a control, uninfected
cells were treated with the radioactive compounds. At the end of 6
and 24 h incubation periods, the cells were washed three times with
PBS, trypsinized, and recovered by centrifugation. The dry pellets were
suspended in 250 µL of 60% methanol (HPLC grade) and heated at 95
°C for 3 min. After centrifugation at 12 000g for 10 min, the clear
supernatant fractions were evaporated under nitrogen and redissolved
in 250 µL of water. Aliquots of this solution were analyzed by anion-
exchanged (SAX-10) HPLC.
HPLC Separation of Metabolites. The separation of [3H]-N-MCdT,
[3H]-S-MCdT, and their phosphorylated metabolites was carried out
using a Hewlett-Packard 1100 HPLC with a diode-array UV absorption
detector. A Partisil-10 SAX column (250 × 4.6 mm) was used with
the following elution program: 0-5 min, 100% buffer A (0.01 M
ammonium phosphate, native pH); 5-20 min, linear gradient to 25%
buffer B (0.7 M ammonium phosphate with 10% methanol); 20-30
min, linear gradient to 100% buffer B; 30-40 min 100% buffer B;
40-55 min, linear gradient to 100% buffer A and equilibration. The
flow rate was 2 mL/min. One-minute fractions were collected, and
radioactivity was measured by scintillation spectrometry. The retention
times were as follows: N- and S-MCdT (4 min), N- and S-MCdT-
monophosphate (11 min), N- and S-MCdT-diphosphate (22 min), and
N- and S-MCdT-triphosphate (31 min). Fractions containing radiola-
D
(CDCl3) δ 7.56-7.63 (m, 4H, Ph), 7.18-7.36 (m, 11H, Ph), 4.71
(irregular sextet, 1H, H-2), 4.30-4.8 (overlapping doublets, 2H, J )
12.0 Hz and J ) 4.7 Hz, OCHHPh and H-4), 4.31 (d, 1H, J ) 12.0
Hz, OCHHPh), 4.23 (dd, 1H, J ) 9.5, 1.3 Hz, CHHOBn), 2.94 (d, 1H,
J ) 9.5 Hz, CHHOBn), 1.62 (dd, 1H, J ) 14.4, 7.6 Hz, H-3R), 1.52
(irregular quintet, 1H, H-1), 1.28 (br s, 1H, OH), 1.01 (s, 9H, C(CH3)3),
0.95 (m, 1H, H-3â), 0.65 (irregular t, 1H, H-6a), 0.58 (irregular t, 1H,
H-6b); 13C NMR (CDCl3) δ 138.7, 136.2, 136.0, 134.9, 133.9, 129.8,
129.7, 128.5, 127.9, 127.8, 127.7, 75.0, 73.0, 72.3, 70.6, 39.7, 34.5,
27.4, 27.2, 19.6, 10.4; FAB MS (m/z, relative intensity) 473 (MH+, 2),
455 (MH+ - H2O, 12). Anal. Calcd for C30H26O3Si‚0.25H2O: C, 75.49;
H, 7.60. Found: C, 75.49; H, 7.73.
(1R,2S,4S,5S)-4-(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)-5-
[(phenylmethoxy)methyl]bicyclo[3.1.0]hex-2-yl Benzoate (12). A
stirred solution of 11 (19.6 g, 41.4 mmol) in CH2Cl2 (250 mL) was
cooled to 0 °C and treated with pyridine (10 mL, 124 mmol) and
benzoyl chloride (5.8 mL, 49.7 mmol). The mixture was allowed to
reach room temperature and stirred for 3 h. After the addition of a
saturated solution of NaHCO3, the aqueous layer was extracted with
CH2Cl2. The combined organic extract was washed with NaHCO3, water
and brine, dried (MgSO4), and concentrated under vacuum. The residue
was purified by silica gel flash chromatography (hexanes:EtOAc, 60:
1) to give 12 (22 g, 92%) as a colorless syrup; [R]22 -52.1° (c 0.58,
D
1
CHCl3); H NMR (CDCl3) δ 7.88-7.92 (m, 2H, Ph), 7.60-7.66 (m,
4H, Ph), 7.41-7.46 (m, 1H, Ph), 7.18-7.36 (m, 13H, Ph), 5.69
(irregular sextet, 1H, H-2), 4.52 (d, 1H, J ) 4.8 Hz, H-4), 4.39 (AB q,
2H, J ) 11.9 Hz, OCH2Ph), 4.28 (dd, 1H, J ) 9.7, 1.3 Hz, CHHOBn),
2.98 (d, 1H, J ) 9.7 Hz, CHHOBn), 1.78-1.88 (m, 2H, H-1, H-3R),
9
J. AM. CHEM. SOC. VOL. 127, NO. 43, 2005 15149

A R T I C L E S
Marquez et al.
1.25-1.34 (m, 1H, H-3â), 1.03 (s, 9H, C(CH3)3), 0.72 (irregular t, 1H,
H-6a), 0.67 (irregular t, 1H, H-6b); 13C NMR (CDCl3) δ 166.9, 138.7,
136.2, 136.1, 134.7, 133.8, 133.0, 130.8, 129.9, 129.8, 129.7, 128.5,
127.9, 127.9, 127.8, 127.7, 76.1, 74.3, 73.1, 70.5, 36.7, 34.9, 27.3, 24.9,
J ) 11.4 Hz, CHHOSi), 3.48 (d, 1H, J ) 11.4 Hz, CHHOSi), 2.15
(dd, 1H, J ) 14.3, 8.1 Hz, H-3R), 1.74 (irregular quintet, 1H, H-1),
1.53 (m, 1H, H-3â), 1.00 (s, 9H, C(CH3)3), 0.68 (dd, 1H, J ) 5.6, 3.8
Hz, H-6a), 0.31 (dd, 1H, J ) 7.8, 5.8 Hz, H-6b); 13C NMR δ 166.8,
135.8, 133.0, 132.9, 132.8, 130.8, 130.3, 130.2, 129.8, 128.5, 128.2,
128.1, 76.5, 74.9, 67.2, 37.2, 34.6, 27.0, 25.2, 19.3, 11.0; FAB MS
(m/z, relative intensity) 487 (MH+, 24.5), 469 (MH+ - H2O, 86). Anal.
Calcd for C30H34O4Si: C, 74.04; H, 7.04. Found: C, 74.01; H, 6.93.
19.7, 11.3; FAB MS (m/z, relative intensity) 577 (MH+, 2), 455 (MH+
-
- PhCOOH, 15), 321 (MH+
TBPSiOH, 26). Anal. Calcd for
C37H40O3Si‚0.8H2O: C, 75.17; H, 6.96. Found: C, 75.20; H, 7.82.
(1R,2S,4S,5S)-4-Hydroxy-5-[(phenylmethoxy)methyl]bicyclo[3.1.0]-
hex-2-yl Benzoate (13). A stirred solution of 12 (16.7 g, 29 mmol) in
THF (150 mL) at room temperature was treated dropwise with a 1 M
solution of tetra-n-butylammonium fluoride (TBAF, 71.6 mL) in THF.
The reaction mixture was stirred overnight and concentrated under
vacuum. The residue was purified by silica gel flash chromatography
(1R,2S,4R,5S)-5-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)methyl]-
4-iodobicyclo[3.1.0]hex-2-yl Benzoate (16). Starting from 15 (1.51
g, 3.11 mmol), this compound was prepared in the same manner as its
23
optical antipode22 to give 16 (1.62 g, 88%) as a colorless syrup: [R]D
-27.2° (c 0.60 CHCl3). Anal. Calcd for C30H33IO3Si: C, 60.40; H,
5.58. Found: C, 60.73; H, 5.63.
(hexanes:EtOAc, 4:1) to give 12.05 g (89%) of 13 as a colorless syrup;
1
[R]22 -70.5° (c 0.42, CHCl3); H NMR (CDCl3) δ 7.94-7.98 (m,
D
(1R,2S,5S)-5-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)methyl]-
bicyclo[3.1.0]hex-3-en-2-yl Benzoate (17). Starting from 16 (1.57 g,
2.63 mmol), this compound was prepared in the same manner as its
2H, Ph), 7.46-7.50 (m, 1H, Ph), 7.22-7.38 (m, 7H, Ph), 5.78 (irregular
sextet, 1H, H-2), 4.50 (AB q, 2H, J ) 12.0 Hz, OCH2Ph), 4.38 (d, 1H,
J ) 5.1 Hz, H-4), 3.79 (d, 1H, J ) 10.4 Hz, CHHOBn), 3.46 (d, 1H,
J ) 10.4 Hz, CHHOBn), 2.14 (dd, 1H, J ) 14.2, 7.9 Hz, H-3R), 1.92
(irregular quintet, 1H, H-1), 1.56 (m, 1H, H-3â), 0.78 (dd, 1H, J )
5.7, 4.0 Hz, H-6a), 0.51 (dd, 1H, J ) 7.8, 5.7 Hz, H-6b); 13C NMR
(CDCl3) δ 166.8, 137.8, 133.0, 133.7, 129.8, 128.8, 128.5, 128.1, 128.0,
76.4, 74.4, 73.4, 72.8, 37.1, 33.3, 25.0, 11.4; FAB MS (m/z, relative
intensity) 339.2 (MH+, 3), 321 (MH+ - H2O, 34), 231 (MH+ - PhCH2-
OH, 22). Anal. Calcd for C21H22O4‚0.4H2O: C, 73.03; H, 6.65.
Found: C, 72.78; H, 6.75.
optical antipode22 to give 17 (640 mg, 52%) as a colorless syrup: [R]D
23
+42.1° (c 0.43 CHCl3). Anal. Calcd for C30H32O3Si: C, 76.88; H, 6.88.
Found: C, 76.55; H, 7.05.
(1R,2S,5S)-5-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)methyl]-
bicyclo[3.1.0]hex-3-en-2-ol (18). Starting from 17 (619 mg, 1.32
mmol), this compound was prepared in the same manner as its optical
antipode22 to give 18 (475 mg, 99%) as a colorless syrup: [R]D23 +11.2°
(c 0.43 CHCl3). Anal. Calcd for C23H28O2Si: C, 75.78; H, 7.74.
Found: C, 75.53; H, 7.79.
(1R,2S,4S,5S)-4-Hydroxy-5-(hydroxymethyl)bicyclo[3.1.0]hex-2-
yl Benzoate (14). A solution of 13 (10.18 g, 30.1 mmol) in ethanol
(200 mL) was stirred in the presence of 20% Pd(OH)2 on charcoal
(2.54 g) and cyclohexene (100 mL). As the reaction mixture was heated,
hydrogen evolution started at ca. 80 °C, and further heating at reflux
continued for 12 h. The reaction mixture was filtered through a pad of
Celite and the filtrate was concentrated under vacuum to give 7.4 g
(99%) of diol 14 as white solid, which was used directly in the next
step. An analytical sample was obtained after silica gel flash chroma-
tography (hexanes:EtOAc, 1:1), mp 81-82 °C; [R]22D -88.4° (c 0.63,
(1′R,2′R,5′S)-1-{5-[(2,2-Dimethyl-1,1-silapropoxy)methyl]bicyclo-
[3.1.0]hex-3-en-2-yl}-5-methyl-3-(phenylcarbonyl)-1,3-dihydropy-
rimidine-2,4-dione (19). Starting from 18 (480 mg, 3.41 mmol), this
compound was prepared in the same manner as its optical antipode22
to give 19 (340 mg, 45%) as a colorless syrup: [R]D23 -49.6° (c 0.16,
CHCl3). Anal. Calcd for C35H36N2O4Si‚0.7H2O: C, 71.33; H, 6.39; N,
4.75. Found: C, 71.30; H, 6.28; N, 4.64.
(1′R,2′R,5′S)-1-[5-(Hydroxymethyl)bicyclo[3.1.0]hex-3-en-2-yl]-
5-methyl-1,3-dihydropyrimidine-2,4-dione (20). Starting from 19 (310
mg, 0.53 mmol), this compound was prepared in the same manner as
its optical antipode22 to give 20 (90 mg, 75%) as a white solid: mp
182 °C; [R]D23 -166.5° (c 0.37, CH3OH). Anal. Calcd for C12H14N2O3‚
0.25H2O: C, 60.37; H, 6.12; N, 11.73. Found: C, 0.70; H, 6.37; N,
11.35.
1
CHCl3); H NMR (CDCl3) δ 7.93-7.98 (m, 2H, Ph), 7.44-7.50 (m,
1H, Ph), 7.33-7.39 (m, 2H, Ph), 5.79 (irregular sextet, 1H, H-2), 4.45
(d, 1H, J ) 5.0 Hz, H-4), 3.99 (br d, 1H, J ) 12.0 Hz, CHHOH), 3.56
(br d, J ) 12.0 Hz, CHHOH), 3.20-3.40 (broad overlapping singlets,
2H, OH), 2.15 (dd, 1H, J ) 14.5, 7.9 Hz, H-3R), 1.96 (irregular quintet,
1H, H-1), 1.61 (m, 1H, H-3â), 0.78 (dd, 1H, J ) 5.6, 3.8 Hz, H-6a),
0.51 (dd, 1H, J ) 7.5, 6.0 Hz, H-6b); 13C NMR (CDCl3) δ 167.1,
133.15, 130.50, 129.8, 128.5, 76.5, 74.9, 65.5, 37.6, 35.3, 24.6, 11.6;
(1′R,2′R,5′S)-1-[5-(Hydroxymethyl)bicyclo[3.1.0]hex-2-yl]-5-meth-
yl-1,3-dihydropyrimidine-2,4-dione (6). Starting from 20 (38.5 mg,
0.164 mmol), this compound was prepared in the same manner as its
optical antipode (compound 7) to give 6 (38 mg, 98%) as a white
-
FAB MS (m/z, relative intensity) 249 (MH+, 6.7), 231 (MH+ H2O,
23
solid: mp 173-175 °C; [R]D -67.0° (c 0.17, CH3OH).
63). Anal. Calcd for C14H16O4: C, 67.73; H, 6.50. Found: C, 67.63;
H, 6.49.
Acknowledgment. This paper is dedicated to the memory of
Muttaiya Sundaralingam.
(1R,2S,4S,5S)-5-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)methyl]-
4-hydroxybicyclo[3.1.0]hex-2-yl Benzoate (15). Imidazole (4.91 g,
72.2 mmol) was added to a stirred solution of diol 14 (8.15 g, 32.80
mmol) in CH2Cl2 (200 mL) at 0 °C. Immediately after, tert-butylchlo-
rodiphenyl silane (9.0 mL, 34.44 mmol) dissolved in CH2Cl2 (30 mL)
was added dropwise. The mixture was stirred at 0 °C for 20 min and
quenched with brine (20 mL). The organic phase was washed with
brine, dried (MgSO4), and concentrated under vacuum. The residue
was purified by silica gel flash column chromatography (hexanes:
EtOAc, 9:1) to give 12.34 g (77%) of 15 as a syrup: [R]22D -81.2° (c
0.49, CHCl3); 1H NMR (CDCl3) δ 7.93-7.98 (m, 2H, Ph), 7.58-7.64
(m, 4H, Ph), 7.44-7.49 (m, 1H, Ph), 7.31-7.41 (m, 8H, Ph), 5.80
(irregular sextet, 1H, H-2), 4.43 (d, 1H, J ) 4.8 Hz, H-4), 4.08 (d, 1H,
Supporting Information Available: Complete crystallo-
graphic data on compound 6, including crystal data and structure
refinement, atomic coordinates, bond lengths and angles,
anisotropic displacement parameters, hydrogen coordinates and
isotropic displacement parameters, torsion angles, and hydrogen
bonds (seven tables). X-ray crystallographic data, in CIF format.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA053789S
9
15150 J. AM. CHEM. SOC. VOL. 127, NO. 43, 2005