The cyclohexene ring system as a furanose mimic: Synthesis and antiviral activity of both enantiomers of cyclohexenylguanine

Article abstract of DOI:10.1021/jm991171l

Both enantiomers of cyclohexenylguanine were synthesized in a stereospecific way starting from the same starting material: R-(-)-carvone. Both compounds showed potent and selective anti-herpesvirus activity (HSV-1, HSV-2, VZV, CMV). The binding of both cy

Full text of DOI:10.1021/jm991171l

736
J . Med. Chem. 2000, 43, 736-745
Th e Cycloh exen e Rin g System a s a F u r a n ose Mim ic: Syn th esis a n d An tivir a l
Activity of Both En a n tiom er s of Cycloh exen ylgu a n in e
J ing Wang,† Matheus Froeyen,† Chris Hendrix,† Graciela Andrei,‡ Robert Snoeck,‡ Erik De Clercq,‡ and
Piet Herdewijn*,†
Laboratories of Medicinal Chemistry and of Virology and Chemotherapy, Rega Institute for Medical Research,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received September 29, 1999
Both enantiomers of cyclohexenylguanine were synthesized in a stereospecific way starting
from the same starting material: R-(-)-carvone. Both compounds showed potent and selective
anti-herpesvirus activity (HSV-1, HSV-2, VZV, CMV). The binding of both cyclohexene
nucleosides in the active site of HSV-1 thymidine kinase was investigated, and a model for the
binding of both enantiomers is proposed. The amino acids involved in binding of the optical
antipodes are the same, but the interaction energy of both enantiomers is slightly different.
This may be attributed to the interaction of the secondary hydroxyl function of the nucleoside
analogues with Glu-225. Structural analysis has demonstrated the flexibility of the cyclohexenyl
system, and this may be considered as an important conformational characteristic explaining
the potent antiviral activity.
In tr od u ction
moiety is oriented axially with respect to the six-
membered hexitol ring (C1 conformation, N-type mimic).6
On the contrary, when the oligonucleotides consist of
modified six-membered nucleotides with an equatorially
oriented base moiety, no hybridization with DNA or
RNA is possible.7 It seems therefore possible that the
conformational requirements for a six-membered nu-
cleoside to function as a substrate for thymidine kinase
and DNA polymerase (as triphosphate) may be different.
This may lead us to infer that a certain degree of
conformational flexibility is important for the antiviral
activity of a nucleoside.
To prove this concept we synthesized the cyclohexene
nucleoside analogues 3-6 (Figure 1). Several cyclohex-
ane8-16 and cyclohexene14-19 nucleosides and unsatur-
ated hexitol nucleosides 20,21 have been described in the
literature. None of the cyclohexane nucleosides showed
antiviral activity. Also, no or low antiviral activity has
been described for the cyclohexene and unsaturated
hexitol congeners. The cyclohexene nucleosides and
unsaturated hexitol nucleosides described in the litera-
ture14-21 are analogues of the natural dideoxy nucleo-
sides, and the antiviral activity has mainly been deter-
mined against human immunodeficiency virus (HIV).
Poor anabolism (phosphorylation) inside the cells, or for
the phosphonate congeners poor penetration into the
cells,15,20 could account for this lack of activity. However,
the introduction of a secondary alcohol function in
cyclohexene nucleosides could increase binding to viral
(and cellular) kinases5 and facilitate phosphorylation of
the nucleoside analogue.
Anti-herpesvirus activity has been found for a whole
series of carbohydrate-modified nucleoside analogues,
the most commercial successful example being acyclo-
vir.1 Acyclovir (1) is a very flexible molecule, and it does
not provide us with much information about structural
requirements of a nucleoside analogue for antiviral
activity.2 During the last years, we and others3,4 have
synthesized conformationally restricted analogues of
natural nucleosides with the aim of establishing the
conformational preferences of the nucleoside for the
different enzymes (kinases, polymerases) involved in its
metabolic activation and action. These enzymes play a
crucial role in the antiviral activity of the nucleosides.
A conclusion of these studies is that nucleoside
analogues that mimic a natural furanose nucleoside in
its 3′-endo conformation (northern type nucleosides)
have the highest potential of showing anti-herpesvirus
activity. The reason for this has not been clearly
demonstrated. However, on the basis of further studies
an initial hypothesis can be formulated. A cocrystalli-
zation experiment3f of an antiviral anhydrohexitol nu-
cleoside (2) with herpes simplex virus (HSV) type 1
thymidine kinase showed a chair inversion when the
nucleoside analogue is bound in the active site (and thus
a conformation which is clearly different from the typical
northern type). This is due to the extra stabilization of
the anhydrohexitol moiety in the C1 conformation
(equatorial base) via hydrogen bonding of the secondary
hydroxyl group with Tyr-101 of the enzyme.5 When the
same nucleoside was used to construct an oligonucle-
otide, hexitol nucleic acids were obtained. These oligo-
nucleotides hybridize strongly with RNA and DNA.6 In
both the monomeric and oligomeric structures, the base
A second concept that has become increasingly im-
portant in the field of antiviral nucleosides is the
difference in biological behavior of enantiomers. It has
been recognized that D- and L-nucleosides might have
a different activity and toxicity spectrum.22,2 This is
most clearly demonstrated in the case of the anti-HIV
nucleoside 3TC (lamivudine). The L-(-)- and D-(+)-
enantiomers are equipotent, but 3TC, which corresponds
* To whom correspondence should be addressed. Tel: 016 33 73 87.
Fax: 016 33 73 40. E-mail: piet.herdewijn@rega.kuleuven.ac.be.
† Laboratory of Medicinal Chemistry.
‡ Laboratory of Virology and Chemotherapy.
10.1021/jm991171l CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/04/2000

Cyclohexene Ring System as Furanose Mimic
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 737
selectivity of the desilylation reaction has been observed
before.13c The C3 alcohol 13 was converted to the
corresponding mesylate 14, and the C1-OTBDMS group
was removed using TBAF to give the alcohol 15, which
was oxidized using PDC in CH2Cl2. This oxidation was
accompanied by simultaneous elimination of the C3-
OMs group to afford directly the desired enone 17 (68%).
The latter proved rather unstable on silica gel and was
used as such following flash chromatography. Stereo-
selective reduction of enone 17 using NaBH4 in the
presence of CeCl3‚7H2O in MeOH gave 11 (75%). The
stereochemistry at C1 of 11 was established in a similar
way as for 9.23
Mitsunobu reaction was then applied for introduction
of the base moiety (Scheme 4). Upon reaction of 11 with
adenine in the presence of DEAD and PPh3 in dioxane,
the desired adenine derivative 18a was isolated in a
moderate 40% yield, together with 15% of the N7-isomer
18b (Scheme 4). Finally, removal of the benzoyl protect-
ing groups using K2CO3 in MeOH gave the L-adenine
cyclohexene nucleoside 5 in 77% yield. The correspond-
ing L-guanine nucleoside 6 was synthesized in an
analogous way. The allylic alcohol 11 was treated with
2-amino-6-chloropurine under the same reaction condi-
tions as described above for 10, and the 6-chloropurine
19 thus obtained was converted to the guanine deriva-
tive 20 using TFA-H2O (3:1) (58% yield from 11). Final
deprotection was carried out by heating 20 in a satu-
rated solution of NH3 in MeOH in a sealed vessel for 2
days, and reversed-phase HPLC purification gave pure
L-guanine cyclohexene nucleoside 6 in 73% yield.
F igu r e 1. Structures of acyclovir (1), anhydrohexitol nucleo-
side (2), D-cyclohexenyl nucleosides (3, 4), and L-cyclohexenyl
nucleosides (5, 6).
to the L-(-)-enantiomer, is less toxic than the
D-enantiomer. These results stimulated us to synthesize
and investigate the properties of both enantiomeric
forms of the cyclohexene guanine and adenine nucleo-
sides (3, 4 and 5, 6), although the former compounds
have only two chiral centers while the cyclohexene
nucleosides have three chiral centers.
Syn th esis
Recently we have developed an enantioselective ap-
proach toward the synthesis of D-cyclohexene nucleoside
3 (Scheme 1) using R-(-)-carvone (7) as an inexpensive
starting material.23 Here the endocyclic double bond is
treated as analogous to the furanosyl oxygenation in
natural nucleosides, hence resulting in the D- and
L-designations for the cyclohexenyl compounds. A se-
quence of chemical transformations led to intermediate
8, possessing four chiral centers and, disregarding the
protecting groups R1-R4, a plane of symmetry. Depend-
ing on the nature of R1-R4, intermediate 8 allows for
the synthesis of both the D- and L-cyclohexene nucleo-
sides 3, 4 and 5, 6, respectively.
As described, the synthesis of D-adenine cyclohexene
nucleoside 3 was successfully accomplished by using a
Mitsunobu type reaction on the allylic alcohol 9 to
introduce the adenine base moiety on the cyclohexenyl
ring.23 The corresponding guanine derivative 4 has now
been synthesized in a similar way. The allylic alcohol 9
was reacted with 2-amino-6-chloropurine in the pres-
ence of DEAD and triphenylphosphine in dioxane to give
10 (Scheme 2), which was converted to 4 by treatment
with TFA-H2O (3:1). Under these reaction conditions
the two TBDMS protecting groups were simultaneously
removed. The overall yield starting from 9 was 46%.
Analytically pure 4 is obtained by reversed-phase HPLC
purification.
The synthesis of the L-cyclohexene nucleosides 5 and
6 was carried out by protection of the C4-CH2OH and
C5-OH groups of 8b, followed by conversion of the OR2
and OR4 groups into allylic alcohol 11. This compound
was then used for the introduction of the base moiety
according to the same strategy as used for the D-series.
Previously described intermediate 8b (R1 ) R3 ) H; R2
) R4 ) TBDMS) was converted into dibenzoate 12
(Scheme 3) using standard reaction conditions (Bz2O,
DMAP, CH2Cl2, 98%). The equatorial C3-OTBDMS
protecting group was selectively removed in the pres-
ence of the axial C1-OTBDMS to give 13 by using 1
equiv of TBAF in THF at room temperature (74%). The
Resu lts a n d Discu ssion
An tivir a l Activity. The anti-herpesvirus activity of
D-cyclohexenyl-G (4) and L-cyclohexenyl-G (6) and the
corresponding adenine analogues was determined in
human embryonic skin-muscle fibroblast (E6SM: HSV-
1, HSV-2) and human embryonic lung (HEL) cells
[varicella-zoster virus (VZV), cytomegalovirus (CMV)]
(Table 1). The source of the viruses and the methodology
used to monitor antiviral activity have been described
previously.24,25 The antiviral activity was compared with
the activity of known and approved antiviral drugs of
which two possessed purine bases (acyclovir, ganciclovir)
and two possesed pyrimidine bases (brivudine, cido-
fovir). The adenine nucleoside analogues only showed
very low antiviral activity and, therefore, will not be
further discussed.
D-Cyclohexenyl-G as well as L-cyclohexenyl-G did not
show toxicity in four different cell lines (HeLa, Vero,
E6SM, HEL) (Table 2), pointing to their selective
antiviral mode of action, as reflected by the high
selectivity index of the compounds (Table 1). A salient
feature is that the activity spectrum of both enantiomers
is remarkably similar. Both compounds displayed the
same activity against HSV-1 and HSV-2. Against VZV
and CMV the potency of L-cyclohexenyl-G was about
2-fold lower than that of D-cyclohexenyl-G. Against
HSV-1, the cyclohexenyl-G nucleosides were as active
as acyclovir and brivudin. Against HSV-2, their activity
was very similar to that of acyclovir. The cyclohexenyl-G
nucleosides retained activity against the TK- strains
of HSV-1 and VZV, although the activity was reduced
as compared to the activity against the wild type. The

738 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Wang et al.
Sch em e 1
Sch em e 2
2′-endo mimic of a furanose nucleoside. The energy of
3
2
interconversion between H2 and H3 of cyclohexene (via
the boat conformation) is about 23.4 kJ /mol.27 The
energy barrier for interconversion of the most stable
conformers of a cyclohexene nucleoside (Figure 3) is not
known. The energy difference between both low-energy
conformations of cyclohexenyl-G (1.6 kJ /mol)23 is similar
to the energy difference between the 3′-endo and 2′-endo
forms of a deoxyribose nucleoside (2.1 kJ /mol).28 The
proposed cyclohexene nucleosides are flexible mol-
ecules,23 and structurally, they may be considered as
the best six-membered mimics of natural furanoses. The
reason for this is that the π-σ* interaction in cyclohex-
enyl nucleosides takes over the role of the anomeric
effect in furanose nucleosides.23,29 The anomeric effect
in furanose nucleosides drives the S T N equilibrium
in the northern direction. Unfavorable 1,3-steric pseudo-
diaxial interactions (the base moiety is generally more
sterically demanding than the CH2OH group) together
with the gauche effect (3′-OH and ring oxygen atom)
favor the southern type conformation. In cyclohexene
activity of D-cyclohexenyl-G against TK+ and TK- VZV
strains was higher than the respective activities of
acyclovir and brivudin against these viruses. Finally,
D-cyclohexenyl-G showed the same potency against
CMV as ganciclovir. In conclusion, the activity spectrum
of the newly synthesized cyclohexenyl nucleosides is
very similar to that of the known antiviral compounds
possessing a guanine base moiety (acyclovir, ganciclo-
vir). Both the D- and L-enantiomers of cyclohexenyl-G
are antivirally active. The high selectivity indexes
observed for D- and L-cyclohexenyl-G warrant further
in vivo studies with these compounds against herpes-
virus infections.
3
nucleosides the π-σ* interaction favors the H2 confor-
2
mation while steric effects favor the H3 conformation
23,29
(Figure 3).
Molecu la r Mod elin g. D- And L-cyclohexenyl-G are
the first examples of two enantiomeric nucleosides
showing similar activity against a whole range of
herpesviruses (HSV-1, HSV-2, VZV, CMV). When evalu-
ated against TK- strains of HSV-1, both nucleoside
analogues showed reduced antiviral activity. It is there-
fore reasonable to hypothesize that intracellular phos-
phorylation in the virus-infected cells is an important
step in the enzymatic activation of cyclohexenyl-G
nucleosides. In an effort to understand how enantio-
meric nucleosides can be phosphorylated by the same
kinases (we assume that the viral kinase is responsible
for the first phosphorylation step because the activity
of both enantiomeric nucleosides was reduced against
TK- strains of HSV-1), we carried out a molecular
modeling study using D- and L-cyclohexenyl-G and
HSV-1 thymidine kinase as players.
Con for m a tion a l An a lysis of Cycloh exen e Nu cle-
osid es. The conformational globe of a puckered cyclo-
hexene is more complex than the pseudo-rotation cycle
of five-membered rings (Figure 2).26 The energetically
most favorable puckered ring forms are located on the
surface of the globe. The chair and inverted chair forms
of the cyclohexene ring are located at the two poles of
the globe. The half-chair forms are located at θ ) 52.7°,
φ2 ) 210° and at θ ) 127.3°, φ2 ) 30°. The half-chair-
half-chair interconversion occurs via the boat forms.
Cyclohexene rings are often locked in energy valleys
along the ellipsis formed by the different boat and half-
chair forms (Figure 3). For simplicity and to make
comparison with furanose nucleosides possible, we
designated the 2H3 conformation of the cyclohexene
When D-cyclohexenyl-G was analyzed as a single
molecule, it was observed that the conformation with
the pseudo-axial base (Figure 3, N′-conformation) and
an anti-glycoside torsion angle was (calculations were
carried out in a vacuum) more stable than the confor-
mation with a pseudo-equatorial base moiety (Figure
3, S′-conformation). These data are in agreement with
3
nucleoside as S′-type and the H2 conformation as N′-
type (Figure 3). The 3H2 conformation may be considered
as a mimic of a furanose nucleoside in its 3′-endo form,
while the 2H3 conformation might be considered as a

Cyclohexene Ring System as Furanose Mimic
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 739
Sch em e 3a
a
a. Bz2O, DMAP, CH2Cl2, 0 °C, 98%; b. TBAF (1 equiv), THF, rt, 74%; c. MsCl, Et3N, CH2Cl2, 0 °C, 98%; d. TBAF, THF, rt, 86%; e.
PDC, CH2Cl2, rt, 68%; f. NaBH4, CeCl3‚7H2O, MeOH, rt, 75%.
Sch em e 4a
of D-cyclohexenyl-G (4) is bound via four hydrogen
bounds (covering the Watson-Crick base-pairing func-
tionality of guanine) to Gln-125 and via three hydrogen
bonds (involving the O6 and N7 of guanine) to Arg-176
(Figure 4). In the case of L-cyclohexenyl-G (6), only two
hydrogen bonds are involved in the interaction with Arg-
176. The sugar moieties of D- and L-cyclohexenyl-G have
one hydrogen bond interaction between the primary
hydroxyl group and Arg-163 and one hydrogen bond
interaction between the secondary hydroxyl group and
Glu-225. The hydrogen bond with Glu-225 is 1.3 Å
longer in the L-cyclohexenyl complex than in the
D-cyclohexenyl complex. A more detailed study of the
interaction energy between ligands and neighboring
residues reveals a slightly different binding energy for
both enantiomers. The interaction energies are given
in Table 3. The difference in binding is mainly due to a
different contribution of the sugar moiety. The interac-
tion between the secondary hydroxyl group of the
nucleosides and Glu-225 (and, to a lesser extent, be-
tween the primary hydroxyl group and Arg-163) ex-
plains the lower stability of the L-cyclohexenyl-G/
thymidine kinase complex versus the D-cyclohexenyl-
G/thymidine kinase complex. Table 3 also shows the
important contribution of Tyr-172 to the interaction
energy.
a
a. Adenine, DEAD, PPh3, dioxane, rt; b. K2CO3, MeOH, rt,
77%; c. 2-amino-6-chloropurine, DEAD, PPh3, dioxane, rt; d. TFA/
H2O (3:1), rt, 2 days, 58% from 11; e. NH3/MeOH, 80 °C, 2 days,
73%.
the conformational analysis in solution using NMR
spectrometry.23
The crystal structure of HSV-1 thymidine kinase
complexed with penciclovir, as described by the group
of Sanderson,30 was used as a starting model in the
docking experiment of the cyclohexenyl-G nucleosides
(after removal of water molecules together with penci-
clovir itself and adding hydrogen atoms). D- And
L-cyclohexenyl-G were docked in the active site of the
enzyme with the guanine base moiety anchored to Gln-
125 by double hydrogen bonds and the hydroxymethyl
group anchored to Arg-163 via hydrogen bonding. The
compounds having a conformation with a pseudo-axial
base moiety (Figure 3, N′-conformation) could not be
accommodated in the active side due to the difficulty of
obtaining close interactions between the hydroxymethyl
group of the nucleoside and Arg-163. On the contrary,
the cyclohexenyl nucleosides nicely fit in the active site
when the conformation is flipped to that with a pseudo-
equatorially oriented base moiety (Figure 3, S′-confor-
mation, and Figure 4). Both enantiomers adopt the syn
conformation (ø of 18.8° for 4 and -88.5° for 6). These
results are consistent with our previous observations
that hexitol nucleosides undergo chair inversions when
cocrystallized with HSV-1 thymidine kinase.3f
Con clu sion
The D- and L-cyclohexenyl-G nucleosides were syn-
thesized in a stereospecific manner starting from
R-(-)-carvone. D-Cyclohexenyl-G as well as L-cyclohex-
enyl-G exhibited potent and selective anti-herpesvirus
(HSV-1, HSV-2, VZV, CMV) activity. Their activity
spectrum is comparable to that of the known antiviral
drugs acyclovir and ganciclovir. D- And L-cyclohexenyl-G
represent the most potent antiviral nucleosides contain-
ing a six-membered carbohydrate mimic that have ever
been reported. They are as well the first example of two
enantiomeric nucleosides, both showing similar activity
against the whole series of classical herpesviruses. This
finding is rather unexpected for nucleosides having
three chiral centers.
The cyclohexenyl nucleosides have a flexible confor-
mation. In solution, they appear as an equilibrium
2
3
between the H3 and H2 conformations, and the energy
difference between the conformations is low. It is
expected that cyclohexenyl nucleosides can adapt their
conformation easily to different enzymatic require-
The guanine bases of 4, 6, and penciclovir are bound
in the same position and orientation. These guanine
bases are stacked against Tyr-172. The guanine base

740 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Wang et al.
Ta ble 1. Antiviral Activity of D- and L-Cyclohexenyl-G in Comparison with Approved Antiviral Drugs: 50% Inhibitory Concentration
(IC50, µg/mL)
D-cyclohexenyl-G (4)
L-cyclohexenyl-G (6)
virus
activity selectivity index activity selectivity index brivudin acyclovir ganciclovir cidofovir
HSV-1 (KOS)a
0.002b
0.002b
0.004b
0.05b
0.07b
0.07b
0.38b
0.01b
0.49d
0.64d
2.1d
>2 × 105
>2 × 105
>1 × 105
>8 × 103
>5 × 103
>5 × 103
>1 × 103
>4 × 104
0.003b
0.003b
0.004b
0.07b
0.1b
>5 × 103
>5 × 103
>4 × 103
>2.2 × 102
>1.6 × 102
>2.2 × 102
>12
0.001b
0.001b
0.001b
>80b
>80b
>80b
>80b
>80b
0.03d
0.003d
>20d
>50d
ND
0.01b
0.003b
0.005b
0.02b
0.02b
0.02b
9.6b
0.001b
0.001b
0.001b
0.002b
0.001b
0.001b
0.48b
0.01b
ND
NDe
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.08d
0.2d
HSV-1 (F)a
HSV-1 (McIntyre)a
HSV-2 (G)a
HSV-2 (196)a
HSV-2 (Lyons)a
HSV-1 (TK- KOS ACVr)a
HSV-1 (TK-/TK+ VMW1837)a
VZV (YS)c
0.07b
1.28b
0.01b
1.2d
>1.6 × 103
0.07b
1.1d
>40
>16
VZV (OKA)c
>30
>10
>7
>30
>25
1.9d
>10
0.8d
ND
ND
VZV (TK- 07/1)c
VZV (TK- YS/R)c
CMV (AD 169)c
CMV (Davis)c
5.8d
>3
13d
2.8d
6.8d
>3
28d
ND
0.6d
1.5d
>13
ND
ND
0.6d
0.8d
1.7d
>12
ND
0.8d
a
b
Activity determined in E6SM cell cultures. Minimum inhibitory concentration (µg/mL) required to reduce virus-induced cytopatho-
genicity by 50%. Virus input was 100 50% cell culture infective doses (CCID50). c Activity determined in HEL cells. Minimum inhibitory
d
concentration (µg/mL) required to reduce virus plaque formation by 50%. Virus input was 20 plaque-forming units (PFU). e ND: not
determined. The values are means of two independent determinations.
Ta ble 2. Cytotoxicity of D- and L-Cyclohexenyl-G in Four Different Cell Lines (concn in µg/mL)
cell line
D-cyclohexenyl-G
L-cyclohexenyl-G
brivudin
acyclovir
ganciclovir
cidofovir
HeLaa
Veroa
E6SMa
HELb
HELa
400
400
>400
>20
11
400
400
>16
>20
>20
g400
g400
g400
>50
NDc
ND
g400
>50
>200
ND
ND
>100
>50
>50
ND
ND
ND
>50
>50
>200
a
b
Minimum cytotoxic concentration causing a microscopically detectable alteration of cell morphology. Cytotoxic concentration required
to reduce cell growth by 50%. c ND: not determined.
F igu r e 3. Comparison of the northern h southern confor-
mational equilibrium between a furanose nucleoside and a
cyclohexene nucleoside.
cyclohexene nucleosides are bound in the active site in
a high-energy conformation (pseudo-equatorial orienta-
tion of the base moiety in the syn conformation). These
data confirm previous observations that HSV-1 thymi-
dine kinase may induce a conformational change in
nucleoside upon binding.3f In the X-ray structure of the
complex, the anhydrohexitol nucleoside is, likewise,
bound in a high-energy conformation.3f The present
study illustrates for the first time how enantiomeric
guanine-derived nucleosides can be bound in the active
site of HSV-1 thymidine kinase. Since the observations
of Fischer at the turn of the 20th century, it has been
believed that enzymes do show discrimination in the
way of binding of optical antipodes. However, this study
indicates that in special situations the binding mode of
F igu r e 2. Conformational globe of puckered cyclohexene
showing the different boat and half-chair forms.
ments. This flexibility may be an important structural
determinant in their potent antiviral activity. The
activity of D- and L-cyclohexenyl-G is lower against TK-
strains of HSV-1 and VZV than against TK+ strains.
This indicates that the viral TK plays an important role
in their biological activity, at least against those viruses
for which these data are available. Therefore we inves-
tigated the binding of D- and L-cyclohexenyl-G in the
active site of HSV-1 thymidine kinase using molecular
modeling. This study revealed that both enantiomeric

Cyclohexene Ring System as Furanose Mimic
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 741
F igu r e 4. Plot of the minimized structure of HSV-1 thymidine kinase in complex with 4 (left) and 6 (right). Hydrogen bonds are
plotted if the distance donor-hydrogen to acceptor is less than 2.7 Å and are marked by dotted lines. In the complex with 4, the
double bond in the sugar points to the front, while in the complex at the right, this double bond points to the back.
Ta ble 3. Interaction Energy (kcal/mol) between D- and
difference in binding energy of both enantiomers is
primarily due to a difference in hydrogen-bonding
L-Cyclohexenyl-G and the Active Site Residuesa
D-cyclohexenyl-G
vdw electr total
L-cyclohexenyl-G
vdw electr total
interaction between the secondary hydroxyl groups of
the nucleoside and Glu-225. Whether this difference has
any biological relevance remains the subject of further
studies. Because an antiviral guanine nucleoside is
bound in the HSV-1 thymidine kinase in a slightly
different way than an antiviral pyrimidine nucleo-
side,5,30 it is to be expected that this study cannot be
extrapolated, per se, to all anti-herpesvirus nucleosides.
In summary, D-cyclohexenyl-G (4) and L-cyclohex-
enyl-G (6) are broad-spectrum anti-herpesvirus agents
with very similar antiviral activity. The flexibility of the
cyclohexene ring may be an important structural de-
terminant leading to the observed biological activity.
all 5 Å residues -22.6 -76.5 -99.1 -29.1 -49.9 -79.0
base
sugar
-16.1 -35.3 -51.4 -15.4 -36.6 -52.0
-6.5 -41.2 -47.8 -13.7 -13.3 -27.0
Glu-225
Arg-176
Gln-125
Arg-163
Tyr-172
Arg-222
Ile-100
Ile-97
Met-128
Trp-88
Met-231
His-58
6.2 -34.3 -28.1
0.3 -19.3 -19.0
1.0 -15.8 -14.5
-1.7
-8.5 -10.2
0.4 -20.3 -19.9
1.1 -15.9 -14.8
1.1 -10.5
-9.4
-7.8
-3.7
-2.9
-2.7
-2.3
-1.7
-1.7
-1.2
-1.4
-0.9
-0.7
-0.5
-0.5
-0.5
-0.2
-0.3
-0.2
0.1
0.9
-7.1
-1.6
-2.2
-2.3
-3.9
-2.0
-1.3
-2.1
-0.3
-0.6
-0.8
-0.5
-1.0
-0.2
-0.5
-0.2
-0.2
-0.7
-0.5
-1.6
-6.9
-0.6
-1.9
-0.4
0.2
0.5
0.0
-0.3
0.5
-0.6
0.4
0.0
0.0
0.6
-0.2
0.3
-0.1
0.2
0.8
-5.9
-7.8
-3.5
-2.6
-2.1
-3.3
-2.0
-1.6
-1.7
-0.9
-0.2
-0.8
-0.6
-0.4
-0.4
-0.2
-0.3
-0.1
0.1
-8.1
-0.3
-1.6
-0.3
0.2
-2.1
-2.6
-2.9
-2.9
-1.7
-1.4
-1.5
-0.3
-1.1
-0.8
-0.4
-0.9
-0.3
-0.5
-0.2
-0.2
-0.7
-0.4
-2.2
0.6
0.0
-0.3
0.3
Exp er im en ta l Section
Lys-62
-1.1
0.3
Gln-221
Ala-168
Ala-167
Glu-83
Ala-124
Pro-173
Met-182
Tyr-239
Met-121
Tyr-132
Tyr-101
Gen er a l Meth od s. Melting points were determined in
capillary tubes with a Bu¨chi-Tottoli apparatus and are uncor-
rected. Ultraviolet spectra were recorded with a Philips PU
0.1
-0.1
0.4
1
8740 UV/vis spectrophotometer. H and 13C NMR were deter-
-0.2
0.3
mined with a 200-MHz Varian Gemini apparatus or with a
Varian Unity 500 spectrometer with tetramethylsilane as
internal standard for the 1H NMR spectra and DMSO-d6 (39.6
ppm) or CDCl3 (76.9 ppm) for the 13C NMR spectra (s ) singlet,
d ) doublet, dd ) double doublet, t ) triplet, br s ) broad
singlet, br d ) broad doublet, m ) multiplet). Liquid secondary
ion mass spectra (LSIMS) with Cs+ as primary ion beam were
recorded on a Kratos Concept IH (Kratos, Manchester, U.K.)
mass spectrometer equipped with a MASPEC II32 data system
(Mass Spectrometry Services Ltd., Manchester, U.K.). Samples
were dissolved in glycerol (GLY)/thioglycerol (THGLY)/m-
nitrobenzyl alcohol (NBA) and the secondary ions were ac-
celerated at 7 kV. Scans were performed at 10 s/decade from
m/z 1000 down to m/z 50. Precoated Machery-Nagel Alugram
SIL G/UV254 plates were used for TLC; the spots were
examined with UV light and sulfuric acid/anisaldehyde spray.
Elemental analyses were done at the University of Konstanz,
Germany.
-0.1
0.0
0.8
0.9
2.7
0.4
0.5
0.8
1.4
0.3
-0.2
a
Residues having atoms at a distance less than 5 Å from the
ligand were included in the calculation; vdw ) van der Waals
energy contribution, electr ) electrostatic energy contribution.
enantiomers to a specific enzyme might be similar. The
amino acids in thymidine kinase involved in binding of
D- and L-cyclohexenyl-G are the same. Given the fact
that the guanine base moiety is bound to the enzyme
via a web of hydrogen bonds superimposed on a stacking
interaction, it is not unexpected that chemical modifica-
tions of the purine base of guanine nucleosides have
mostly led to a decrease of anti-herpes activity. The
9-[(1S,4R,5S)-5-(ter t-Bu tyld im eth ylsilyloxy)-4-(ter t-bu -
tyld im eth ylsilyloxym eth yl)-2-cycloh exen yl]-2-a m in o-6-

742 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Wang et al.
ch lor op u r in e (10). To a mixture of 9 (130 mg, 0.35 mmol),
2-amino-6-chloropurine (119 mg, 0.70 mmol), and PPh3 (184
mg, 0.70 mmol) in dry dioxane (7 mL) under N2 at room
temperature was added a solution of DEAD (110 µL, 0.70
mmol) in dry dioxane (3 mL) over a period of 1.5 h. The
reaction mixture was stirred at room temperature for 2 days
and concentrated. The residue was chromatographed on silica
gel (CH2Cl2-MeOH 50:1 then 20:1) to yield crude 10 (170 mg)
as a yellow foam: 1H NMR (CDCl3) δ -0.10 (s, 3H), -0.04 (s,
3H), 0.07 (s, 6H), 0.82 (s, 9H), 0.90 (s, 9H), 2.04 (t, 2H, J ) 5.6
Hz), 2.27 (m, 1H), 3.66 (dd, 1H, J ) 9.9, 5.1 Hz), 3.77 (dd, 1H,
J ) 9.9, 4.4 Hz), 3.98 (m, 1H), 5.21 (m, 1H), 5.43 (s, 2H, NH2),
5.79 (dm, 1H, J ) 9.9 Hz), 6.00 (dm, 1H, J ) 9.9 Hz), 7.79 (s,
1H); 13C NMR (CDCl3) δ -5.6 (q), -5.5 (q), -5.1 (q), -4.8 (q),
17.8 (s), 18.2 (s), 25.6 (q), 25.8 (q), 36.0 (t), 46.9 (d), 49.2 (d),
62.9 (t), 64.6 (d), 124.4 (d), 125.4 (s), 134.4 (d), 141.3 (d), 151.1
(s), 153.3 (s), 159.1 (s).
dichloromethane (20 mL) at 0 °C under N2 were added slowly
triethylamine (2.71 mL, 19.6 mmol, 5 equiv) and MsCl (456
µL, 5.89 mmol, 1.5 equiv) sequentially. After stirring at 0 °C
for 1 h, the reaction was quenched with ice. The reaction
mixture was poured into CH2Cl2 (250 mL), washed with a
saturated NH4Cl solution, water, and brine, dried over Na2-
SO4, and concentrated. The residue was chromatographed on
silica gel (n-hexanes-EtOAc 1:1) to afford 14 (2.17 g, 98%) as
a white foam: 1H NMR (CDCl3) δ 0.13 (s, 3H), 0.15 (s, 3H),
0.96 (s, 9H), 1.62 (td, 1H, J ) 12.0, 2.0 Hz), 1.85 (td, 1H, J )
12.0, 2.0 Hz), 2.26-2.58 (m, 3H), 2.98 (s, 3H), 4.34 (m, 1H),
4.50 (dd, 1H, J ) 11.4, 2.0 Hz), 4.60 (dd, 1H, J ) 11.4, 2.0
Hz), 5.28 (td, 1H, J ) 11.1, 4.8 Hz), 5.69 (td, 1H, J ) 11.0, 4.8
Hz), 7.42 (m, 4H), 7.57 (m, 2H), 8.03 (m, 4H); 13C NMR (CDCl3)
δ -5.3 (q), -5.2 (q), 17.8 (s), 25.5 (q), 37.8 (t), 38.1 (q), 39.9 (t),
46.7 (d), 58.9 (t), 65.9 (d), 67.8 (d), 75.9 (d), 128.5 (d), 129.6
(d), 129.9 (s), 133.1 (d), 165.4 (s), 166.3 (s); LSIMS (THGLY/
GLY) 563 (M + H)+; HRMS calcd for C28H39O8SSi (M + H)+
563.2135, found 563.2188.
9-[(1S,4R,5S)-5-Hyd r oxy-4-h yd r oxym eth yl-2-cycloh ex-
en yl]gu a n in e (4). Crude 10 (170 mg) was treated with TFA-
H2O (3:1, 10 mL) at room temperature for 2 days. The reaction
mixture was concentrated and coevaporated with toluene (2×).
The residue was chromatographed on silica gel (CH2Cl2-
MeOH 10:1 then 1:1) to afford 4 (45 mg, 46% overall yield
(1R,3S,4S,5R)-5-Ben zoyl-4-ben zoylm eth yl-3-(m eth a n e-
su lfon yloxy)cycloh exa n ol (15). To a solution of 14 (2.15 g,
3.82 mmol) in THF (50 mL) at room temperature was added
slowly a 1 M solution of TBAF (7.64 mL, 7.64 mmol, 2 equiv)
in THF. The reaction was stirred at room temperature for 2.5
h and quenched with ice. After standard workup and purifica-
tion on silica gel (n-hexanes-EtOAc 1:1), 15 (1.55 g, 86%) was
obtained as a white foam: 1H NMR (CDCl3) δ 1.71 (td, 1H, J
) 12.1, 2.2 Hz), 1.90 (td, 1H, J ) 12.2, 2.3 Hz), 2.29-2.65 (m,
4H), 3.00 (s, 3H), 4.41 (m, 1H), 4.52 (dd, 1H, J ) 11.7, 2.8
Hz), 4.61 (dd, 1H, J ) 11.7, 2.8 Hz), 5.27 (td, 1H, J ) 10.6, 4.8
Hz), 5.65 (td, 1H, J ) 10.6, 4.7 Hz), 7.42 (m, 4H), 7.55 (m,
4H), 8.02 (m, 4H); 13C NMR (CDCl3) δ 37.3 (t), 38.2 (q), 39.0
(t), 46.8 (d), 59.3 (t), 65.1 (d), 67.9 (d), 75.8 (d), 128.5 (d), 129.7
(d), 133.2 (d), 133.3 (d), 165.6 (s), 166.4 (s); LSIMS (THGLY/
TFA) 449 (M + H)+; HRMS calcd for C22H25O8S (M + H)+
449.1270, found 449.1244.
(4S,5R)-5-Ben zoyl-4-b en zoylm et h ylcycloh ex-2-en -1-
on e (17). A mixture of 15 (500 mg, 1.12 mmol) and PDC (2.1
g, 5.60 mmol, 5 equiv) in dry CH2Cl2 (30 mL) was stirred
vigorously at room temperature for 24 h. The reaction mixture
was filtered through Celite and washed with CH2Cl2. The
filtrate was concentrated and the residue was chromato-
graphed on silica gel (n-hexanes-EtOAc 2:1 then 1:2) to yield
starting material 15 (100 mg, 20%) and enone 17 (267 mg,
68%) as a light yellow oil: 1H NMR (CDCl3) δ 2.73 (dd, 1H, J
) 16.5, 8.8 Hz), 3.10 (dd, 1H, J ) 16.5, 4.4 Hz), 3.27 (m, 1H),
4.50 (dd, 1H, J ) 11.3, 4.7 Hz), 4.66 (dd, 1H, J ) 11.3, 5.5
Hz), 5.66 (ddd, 1H, J ) 8.8, 7.3, 4.4 Hz), 6.26 (dd, 1H, J )
10.2, 2.2 Hz), 6.96 (dd, 1H, J ) 10.2, 3.3 Hz), 7.40-7.63 (m,
6H), 8.01 (m, 4H); 13C NMR (CDCl3) δ 41.3 (d), 42.1 (t), 63.2
(t), 70.0 (d), 128.6 (d), 129.5 (2s), 129.7 (d), 129.8 (d), 131.4
(d), 133.5 (d), 146.5 (d), 165.5 (s), 166.4 (s), 195.8 (s).
1
starting from 9): mp >230 °C; UV λmax (MeOH) 253 nm; H
NMR (CD3OD) δ 1.94-2.27 (m, 3H), 3.77 (d, 2H, J ) 4.7 Hz),
3.85 (m, 1H), 5.17 (m, 1H), 5.88 (dm, 1H, J ) 10.2 Hz), 6.09
(dm, 1H, J ) 10.2 Hz), 7.73 (s, 1H); 13C NMR (CD3OD) δ 37.1
(t), 47.7 (d), 50.6 (d), 63.1 (t), 64.8 (d), 125.8 (d), 135.4 (d), 138.5
(d); LSIMS (THGLY/NBA) 278 (M + H)+; HRMS calcd for
C12H16N5O3 (M + H)+ 278.1253, found 278.1270. Anal. Calcd
for C12H15N5O3‚0.77H2O: C 49.49, H 5.73, N 24.05. Found: C
49.45, H 5.55, N 24.22.
(1S,2S,3R,5R)-3-Ben zoyl-2-b en zoylm et h yl-1,5-d i(ter t-
bu tyld im eth ylsilyloxy)cycloh exa n e (12). To a solution of
8b (2.2 g, 5.64 mmol) in dry dichloromethane (20 mL) at 0 °C
under N2 were added DMAP (3.44 g, 28.2 mmol, 5 equiv) and
Bz2O (3.83 g, 16.92 mmol, 3 equiv) sequentially and in portions.
After stirring at 0 °C for 2 h, the reaction was quenched with
ice. The reaction mixture was poured into CH2Cl2 (250 mL),
washed with water and brine, dried over Na2SO4, and con-
centrated. The crude product was chromatographed on silica
gel (n-hexanes-EtOAc 10:1) to yield 12 (3.3 g, 98%) as a light
yellow oil: 1H NMR (CDCl3) δ 0.03 (s, 3H), 0.06 (s, 3H), 0.13
(s, 3H), 0.17 (s, 3H), 0.89 (s, 9H), 1.01 (s, 9H), 1.58 (m, 2H),
2.09 (m, 2H), 2.37 (m, 1H), 4.29 (br s, 1H), 4.40 (td, 1H, J )
10.3, 4.0 Hz), 4.49 (dd, 1H, J ) 11.4, 2.0 Hz), 4.61 (dd, 1H, J
) 11.4, 2.2 Hz), 5.66 (td, 1H, J ) 11.0, 4.6 Hz), 7.37-7.44 (m,
4H), 7.49-7.59 (m, 2H), 8.02 (m, 4H); 13C NMR (CDCl3) δ -5.3
(q), -5.2 (q), -5.1 (q), -4.5 (q), 17.8 (s), 25.6 (q), 25.7 (q), 38.2
(t), 42.4 (t), 49.5 (d), 60.0 (t), 65.2 (d), 66.4 (d), 68.5 (d), 128.3
(d), 129.6 (d), 130.3 (s), 130.4 (s), 132.8 (d), 165.6 (s), 166.5 (s).
(1S,2S,3R,5S)-3-Ben zoyl-2-b en zoylm et h yl-5-(ter t-b u -
tyld im eth ylsilyloxy)cycloh exa n ol (13). A solution of 1 M
TBAF in THF (5.38 mL, 5.38 mmol) was added slowly to a
solution of 12 (3.23 g, 5.38 mmol) in THF (50 mL) at 0 °C.
The reaction mixture was stirred at 0 °C for 2 h and further
at room temperature for 3 h. Ice was added and the reaction
mixture was poured into EtOAc (300 mL) which was washed
with NH4Cl solution, water, and brine, dried over Na2SO4, and
concentrated. The crude product was purified on silica gel (n-
hexanes-EtOAc 5:1 then 1:1) to yield 13 (1.93 g, 74%) as a
white foam: 1H NMR (CDCl3) δ 0.02 (s, 3H), 0.09 (s, 3H), 0.81
(s, 9H), 1.50-1.63 (m, 2H), 1.96 (m, 1H), 2.13 (m, 1H), 2.32
(m, 1H), 3.24 (d, 1H, J ) 4.8 Hz, -OH), 4.04 (m, 1H), 4.25 (m,
1H), 4.33 (dd, 1H, J ) 11.4, 2.2 Hz), 5.04 (dd, 1H, J ) 11.4,
2.2 Hz), 5.60 (td, 1H, J ) 11.0, 4.5 Hz), 7.36-7.60 (m, 6H),
8.06 (m, 4H); 13C NMR (CDCl3) δ -5.2 (q), 17.6 (s), 25.4 (q),
38.2 (t), 40.8 (t), 50.4 (d), 59.8 (t), 64.1 (d), 66.1 (d), 68.4 (d),
128.4 (d), 129.6 (d), 129.8 (d), 130.4 (s), 133.0 (d), 133.2 (d),
165.6 (s), 167.6 (s); LSIMS (THGLY/TFA) 485 (M + H)+;
HRMS calcd for C27H37O6Si (M + H)+ 485.2359, found 485.2376.
(1S,4S,5R)-5-Ben zoyl-4-ben zoylm eth ylcycloh ex-2-en -1-
ol (11). To a solution of 17 (267 mg, 0.76 mmol) in MeOH (10
mL) at room temperature under N2 was added CeCl3‚7H2O
(426 mg, 1.14 mmol, 1.5 equiv). The mixture was stirred for
0.5 h and a clear solution was obtained. NaBH4 (35 mg, 0.91
mmol, 1.2 equiv) was added in portions and H2 evolved. The
reaction mixture was stirred for 1 h and quenched with ice.
The mixture was stirred for 15 min and concentrated. The
residue was taken up into EtOAc, washed with H2O and brine,
dried over Na2SO4, and concentrated. The residue was chro-
matographed on silica gel (n-hexanes-EtOAc 10:1) to give 11
(200 mg, 75%) as a light yellow oil: 1H NMR (CDCl3) δ 1.77
(d, 1H, J ) 7.2 Hz), 1.93 (ddd, 1H, J ) 12.1, 10.2, 8.0 Hz),
2.54 (ddd, 1H, J ) 12.1, 5.8, 3.3 Hz), 3.00 (m, 1H), 4.32 (dd,
1H, J ) 11.4, 5.5 Hz), 4.44 (dd, 1H, J ) 11.4, 5.5 Hz), 4.50 (m,
1H), 5.35 (ddd, 1H, J ) 10.2, 7.3, 2.9 Hz), 5.78 (dt, 1H, J )
10.2, 1.8 Hz), 5.97 (dt, 1H, J ) 10.2, 2.5 Hz), 7.34-7.60 (m,
6H), 8.00 (m, 4H); 13C NMR (CDCl3) δ 36.6 (t), 40.9 (d), 46.6
(t), 65.8 (d), 69.9 (d), 126.6 (d), 128.4 (d), 128.5 (d), 129.7 (d),
129.8 (s), 130.9 (s), 132.7 (d), 133.1 (d), 133.2 (d), 166.0 (s),
166.5 (s); LSIMS (THGLY/TFA) 353 (M + H)+; HRMS calcd
for C21H21O5 (M + H)+ 353.1389, found 353.1440.
(1S,2S,3R,5S)-3-Ben zoyl-2-b en zoylm et h yl-5-(ter t-b u -
t yld im et h ylsilyloxy)-1-(m et h a n esu lfon yloxy)cycloh ex-
a n e (14). To a solution of 13 (1.90 g, 3.92 mmol) in dry

Cyclohexene Ring System as Furanose Mimic
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 743
9-[(1R,4S,5R)-5-Ben zoyl-4-b en zoylm et h yl-2-cycloh ex-
en yl]a d en in e (18a ). To a mixture of 11 (65 mg, 0.18 mmol),
adenine (48 mg, 0.36 mmol, 2 equiv), and PPh3 (94 mg, 0.36
mmol, 2 equiv) in dry dioxane (4 mL) under N2 at room
temperature was added a solution of DEAD (56 µL, 0.36 mmol,
2 equiv) in dry dioxane (3 mL) over a period of 1 h. The reaction
mixture was stirred at room temperature overnight and
concentrated. The residue was chromatographed on silica gel
(CH2Cl2-MeOH 50:1, 20:1, 10:1) to yield 18a (33 mg, 40%) as
a white solid: UV λmax (MeOH) 231 and 263 nm; 1H NMR
(CDCl3) δ 2.48 (ddd, 1H, J ) 13.6, 8.3, 5.8 Hz), 2.57 (ddd, 1H,
J ) 13.6, 6.0, 3.2 Hz), 4.50 (dd, 1H, J ) 10.4, 5.0 Hz), 4.63
(dd, 1H, J ) 10.4, 6.1 Hz), 5.53 (m, 2H), 5.92 (s, 2H), 6.09 (dm,
1H, J ) 10.0 Hz), 6.17 (dm, 1H, J ) 10.0 Hz), 7.41 (m, 4H),
7.57 (m, 2H), 7.86 (s, 1H), 8.04 (m, 4H), 8.35 (s, 1H); 13C NMR
(CDCl3) δ 32.4 (t), 40.6 (d), 48.7 (d), 64.3 (t), 68.2 (d), 120.1 (s),
126.8 (d), 128.5 (d), 128.6 (d), 129.6 (d), 129.7 (d), 131.0 (d),
133.4 (d), 138.8 (d), 149.8 (s), 153.1 (d), 155.8 (s), 165.8 (s),
166.5 (s); LSIMS (THGLY/NBA) 470 (M + H)+; HRMS calcd
for C26H24N5O4 (M + H)+ 470.1828, found 470.1845.
found 278.1247. Anal. Calcd for C12H15N5O3‚1.5H2O: C 47.35,
H 5.96, N 23.03. Found: C 47.46, H 5.64, N 22.87.
Molecu la r Mod elin g. F or ce field a n d p a r a m eter s: The
AMBER 4.1 all-hydrogen parameter database of 199431 was
used for thymidine kinase without modification. For the
substrates, the same parameter set was used but missing bond
and angle parameters were taken from comparable bonds or
angles from the AMBER force field. Particularly the param-
eters for the double bond in the sugar moiety (C5′, C6′, H5′,
and H6′, respectively) were extracted from the AMBER atom
types CM, and H4. Partial atomic charges for the substrates
were calculated using the standard procedure called RESP,32
of fitting the electrostatic potential at the 6-31G* level of theory
using GAMESS.33 All energy refinements and calculations
using the Sander and Anal modules of AMBER were done in
vacuum, with a distance-dependent dielectric constant and a
cutoff of 9 Å for the nonbonded interactions.
Sm a ll m olecu les: Molecules 4 and 6 were built starting
from a riboguanine monomer generated by the Nucgen pro-
gram in the AMBER software. A simple text editor was used
to modify the Protein Data Bank (pdb) structure files. The
energy minimization was performed in Sander to relax the
structures. A conformational search was performed on the
ligand 4 by the QCPE software DGEOM95 (QCPE-590).34,35
Two major low-energy conformational clusters were found, the
first group showing pseudo-axial and the second cluster having
all pseudo-equatorial conformations of the base moiety. The
energy as a function of the torsion angle C6′-C1′-N9-C4 was
calculated using the Sander module of AMBER. This calcula-
tion revealed that the most stable conformation for 4 is pseudo-
axial, with the torsion angle C6′-C1′-N9-C4 equal to 200°
(anti) and about 3 kcal/mol (in vacuum) more stable than the
semi-equatorial form. Since structure 6 is the mirror structure
of 5, the same low-energy conformations are expected, and
hence the different conformations of 8 were created by
coordinate transformations from structure 4.
9-[(1R,4S,5R)-5-Hyd r oxy-4-h yd r oxym eth yl-2-cycloh ex-
en yl]a d en in e (5). Compound 18a (33 mg, 0.07 mmol) was
treated with anhydrous K2CO3 (100 mg) in MeOH (3 mL) at
room temperature for 3 h. A small portion of silica gel was
added to the reaction mixture and the solvent was evaporated.
The residue was chromatographed on silica gel (CH2Cl2-
MeOH 10:1, 1:1) to give 5 (14 mg, 77%): 1H NMR (CD3OD) δ
2.02-2.32 (m, 3H), 3.79-3.90 (m, 3H), 5.35 (m, 1H), 5.93 (dm,
1H, J ) 9.9 Hz), 6.15 (dm, 1H, J ) 9.9 Hz), 8.09 (s, 1H), 8.21
(s, 1H); 13C NMR (CD3OD) δ 37.2 (t), 47.7 (d), 51.1 (d), 63.0
(t), 64.7 (d), 120.6 (s), 125.4 (d), 136.0 (d), 141.6 (d), 150.3 (s),
153.8 (d), 157.5 (s); LSIMS (THGLY/TFA) 262 (M + H)+;
HRMS calcd for
262.1323.
C
12H16N5O2 (M + H)+ 262.1304, found
9-[(1R,4S,5R)-5-Ben zoyl-4-b en zoylm et h yl-2-cycloh ex-
en yl]gu a n in e (20). Compound 11 (160 mg, 0.45 mmol) was
treated with 2-amino-6-chloropurine (153 mg, 0.90 mmol, 2
equiv) in the presence of PPh3 (235 mg, 0.90 mmol, 2 equiv)
and DEAD (140 µL, 0.90 mmol, 2 equiv) in dry dioxane (12
mL) at room temperature overnight. After concentration and
purification on silica gel (CH2Cl2-EtOAc 1:1), crude 19 (500
mg) was obtained, which was treated with CF3COOH/H2O (3:
1, 12 mL) at room temperature for 2 days. The reaction
mixture was concentrated and coevaporated with toluene. The
residue was purified on silica gel (CH2Cl2-MeOH 20:1) to yield
20 (126 mg, 58% over two steps) as a white solid: UV λmax
(MeOH) 251 and 256 nm; 1H NMR (500 MHz, DMSO-d6) δ
2.30 (ddd, 1H, J ) 13.6, 8.3, 5.9 Hz), 2.42 (ddd, 1H, J ) 13.6,
6.4, 3.2 Hz), 3.00 (m, 1H), 4.52 (m, 2H), 5.17 (m, 1H), 5.37 (m,
1H), 6.03 (dm, 1H, J ) 10.2 Hz), 6.11 (dm, 1H, J ) 10.2 Hz),
6.45 (s, 2H), 7.51 (m, 4H), 7.66 (m, 2H), 7.69 (s, 1H), 7.95 (m,
4H), 10.61 (s, 1H); 13C NMR (DMSO-d6) δ 31.4 (t), 40.0 (d,
overlapped with DMSO-d6 peak), 47.9 (d), 64.4 (t), 68.5 (d),
116.9 (s), 127.0 (d), 128.9 (d), 129.3 (d), 129.4 (d), 130.2 (d),
133.6 (d), 135.7 (d), 150.9 (s), 153.8 (s), 156.9 (s), 165.3 (s),
165.8 (s); LSIMS (THGLY/GLY) 486 (M + H)+; HRMS calcd
for C26H24N5O5 (M + H)+ 486.1777, found 486.1816.
En zym e: The 2.37 Å resolution crystal structure of HSV-1
thymidine kinase in complex with penciclovir, pdb entry
1KI3,30,36 was used as a starting point for all modeling
experiments because compounds 4 and 6 also contain the
guanine base moiety.
The first entity (A) in the asymmetric crystal unit was used.
All crystallographic waters were removed. Penciclovir was also
removed, but its coordinates were saved in a separate file to
be used as a fitting template in the docking experiments.
Residues with missing heavy atoms were removed; the final
enzyme contained residues 47-71, 77-149, 152-263, and
281-374. Hydrogens were added by the Edit module of the
AMBER 4.1 software, and the energy of the system was
relaxed until the energy gradient dropped below 0.01 kJ /mol‚
Å. During this minimization the non-hydrogen atoms were
kept fixed at their X-ray positions by a harmonic potential with
a force constant of 5000 kcal/mol. This final structure was used
as a starting model in the docking experiments.
Dock in g Exp er im en ts. During the docking experiments
the following interactions between 4, 6, and the viral TK were
respected: (1) the base moiety is anchored by double hydrogen
bonds to Gln-125; (2) the 5′-OH group is hydrogen bonding to
Arg-163 to keep it in the right position for phosphorylation.
In a first simulation the pseudo-equatorial and pseudo-axial
conformations of molecule 4 were fit on the penciclovir guanine
base coordinates using the Carnal module of AMBER. Those
structures were docked in the active site of thymidine kinase.
The compound with an axial base conformation could not be
accommodated in the active site because (1) position of the
primary hydroxyl (equivalent of O5′ in a natural nucleoside)
is not close to Arg-163 and (2) major steric clashes. This steric
hindrance in the axial conformation could not be removed by
turning the dihedral angle C6′-C1′-N9-C4. This procedure
was repeated for molecule 6, leading to the same conclusion.
The final docking experiments were done as follows. The
guanine base in the equatorial conformation of molecule 4 was
fitted onto the base of the penciclovir molecule using Carnal.
The resulting molecule was docked in the active site of the
9-[(1R,4S,5R)-5-Hyd r oxy-4-h yd r oxym eth yl-2-cycloh ex-
en yl]gu a n in e (6). A mixture of 20 (85 mg) in an ammonium
MeOH solution (75 mL) was sealed and heated at 80 °C for 2
days. After cooling to room temperature, the mixture was
concentrated and the residue was purified by reversed-phase
HPLC (4% CH3CN in water) to afford 6 (36 mg, 75%) as a
1
white powder: mp 255 °C dec; UV λmax (MeOH) 254 nm; H
NMR (500 HMz, DMSO-d6) δ 1.85 (m, 1H), 1.98 (m, 1H), 2.11
(m, 1H), 3.54 (dd, 1H, J ) 10.3, 5.5 Hz), 3.60 (dd, 1H, J )
10.3, 4.8 Hz), 3.70 (m, 1H), 4.68 (br s, 1H, -OH), 4.75 (br s,
1H, -OH), 4.99 (m, 1H), 5.77 (dm, 1H, J ) 9.8 Hz), 5.97 (dm,
1H, J ) 9.8 Hz), 6.57 (s, 2H, -NH2), 7.50 (s, 1H), 10.8 (br s,
1H, -NH); 13C NMR (125 MHz, DMSO-d6) δ 35.9 (t), 46.4 (d),
48.2 (d), 61.5 (t), 62.7 (d), 116.9 (s), 124.8 (d), 133.7 (d), 135.6
(d), 150.8 (s), 154.1 (s), 157.6 (s); LSIMS (THGLY/NBA) 278
(M + H)+; HRMS calcd for C12H16N5O3 (M + H)+ 278.1253,

744 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Wang et al.
prepared thymidine kinase structure. The dihedral angle C6′-
C1′-N9-C4 was rotated manually to make the primary
hydroxyl close to Arg-163. The complex was energy-minimized
in the Sander module. The substrate and residues with atoms
closer than 5 Å to the original penciclovir substrate were
allowed to move free; all others were fixed to their initial
positions (X-ray coordinates for heavy atoms, built coordinates
for hydrogens) by a force constant of 5000 kcal/mol. Three
additional distance restraints were added: two for the double
hydrogen bond from the guanine base to Gln-125 (atoms O6,
H1A-HE21, OE1, respectively) and one hydrogen bond from
the hydroxymethyl group to Arg-163 atom HH21 (force con-
stant ) 100 kcal/mol). After the whole system was relaxed,
the minimization was continued but now the distance re-
straints were removed. This procedure was repeated for
structure 6. The final structures are shown in Figure 4.
Con for m a tion . Both ligands fit well into the active site.
The molecules 4 and 6 have a pseudo-equatorial conformation
when bound to the TK enzyme. Both molecules adapt the syn
conformation, the torsion angle ø[C6′-C1′-N9-C4] is 18.8°
in 4 and -88.5° (or 271.5°) for molecule 6. Superimposing the
1KI3 structure, having penciclovir in the active site, onto the
structure with the predicted binding orientations of structures
4 and 6 reveals that the guanine base is located in the same
position and orientation in the three complexes.
Hyd r ogen bon d s: The guanine bases of the cyclohexenyl
nucleosides are at the expected location making a double
hydrogen bond from H1/N1 and O6 to Gln-125. The Gln-125
NH2 group is slightly rotated so that a double hydrogen bond
to O6 becomes possible. O6 is also involved in a hydrogen bond
to Arg-176 HE (hydrogen of NE). A hydrogen bond exists from
H2A (hydrogen of base nitrogen N2) to Gln-125 OE1. The base
nitrogen N7 is making two hydrogen bonds to Arg-176 HH21
(hydrogen of NH2) and HE (hydrogen of NE) in the case of 4.
In 6 only one hydrogen bond exists to HH21. The ‘sugar’ in 4
and 6 has similar hydrogen bonds: one hydrogen bond from
the primary hydroxyl group to Arg-163 HH21 and another
from 3′-OH to Glu-225 OE2. The Tyr-101 OH group is out of
reach of the sugar 3′-OH. Instead, it is hydrogen bonding to
the Glu-225 OE1 atom.
En er gies: From the hydrogen bond pattern it is not very
clear if there is a difference in binding strength between
molecules 4 and 6. Therefore the interaction energy between
ligands and neighboring residues in the active site was studied
in detail using the Anal module of AMBER. The results are
given in Table 3. The interaction of the active site with the
base moiety is the same for molecules 4 and 6. The sugar in 4
is more stabilized by the binding to thymidine kinase than 6.
The main contribution to this stabilization energy is coming
from the closer contact of the 3′-OH group of 4 to Glu-225 than
in the complex with 6 and hence results in a better interaction.
While the stacking of the guanine base with Tyr-172 is, like
in the penciclovir and acyclovir complexes, 1KI3 and 2KI5 pdb
entries, not as good as in the deoxythymidine complexes (1KIM
entry), there is clearly a stabilizing contribution (-8 kcal/mol)
from this interaction for the binding of D- and L-cyclohexenyl-
G.
An tivir a l Activity Assa ys. The antiviral assays were
based on an inhibition of virus-induced cytopathicity in either
E6SM, HeLa, Vero, or HEL cell cultures, following previously
established procedures.24,25 Briefly, confluent cell cultures in
microtiter trays were inoculated with 100 CCID50 of virus, 1
CCID50 being the virus dose required to infect 50% of the cell
cultures. After a 1-h virus adsorption period, residual virus
was removed and the cell cultures were incubated in the
presence of varying concentrations of the test compounds. Viral
cytopathicity was recorded as soon as it reached completion
in the control virus-infected cell cultures.
ven (GOA 97/11). We are indebted to Prof. M. Sanderson
for the coordinates of HSV-1 thymidine kinase. We
thank Chantal Biernaux for outstanding editorial help.
Refer en ces
(1) Elion, G. B.; Furman, P. A.; Fyfe, J . A.; De Miranda, P.;
Beauchamp, L.; Schaeffer, H. J . Selectivity of action of an
antiherpetic agent, 9-(2-hydoxy-ethoxymethyl)guanine. Proc.
Natl. Acad. Sci. U.S.A. 1977, 74, 5716-5720.
(2) Herdewijn, P. Structural requirements for antiviral activity in
nucleosides. Drug Discovery Today 1997, 2, 235-242.
(3) (a) Verheggen, I.; Van Aerschot, A.; Toppet, S.; Snoeck, R.;
J anssens, G.; Balzarini, J .; De Clercq, E.; Herdewijn, P. Syn-
thesis and anti-herpes activity of 1,5-anhydrohexitol nucleosides.
J . Med. Chem. 1993, 36, 2033-2040. (b) Verheggen, I.; Van
Aerschot, A.; Van Meervelt, L.; Rozenski, J .; Wiebe, L.; Snoeck,
R.; Andrei, G.; Balzarini, J .; Claes, P.; De Clercq, E.; Herdewijn,
P. Synthesis, biological evaluation, and structure analysis of a
series of new 1,5-anhydrohexitol nucleosides. J . Med. Chem.
1995, 38, 826-835. (c) Verheggen, I.; Van Aerschot, A.; Rozenski,
J .; J anssen, G.; De Clercq, E.; Herdewijn, P. Synthesis of 1,5-
anhydrohexitol nucleosides as mimics of AZT, D4T and DDC.
Nucleosides Nucleotides 1996, 15, 325-335. (d) Andersen, M.
W.; Daluge, S. M.; Kerremans, L.; Herdewijn, P. The synthesis
of modified D- and L-anhydrohexitol nucleosides. Tetrahedron
Lett. 1996, 37, 8147-8150. (e) Hossain, N.; Rozenski, J .; De
Clercq, E.; Herdewijn, P. Synthesis and antiviral activity of the
R-analogues of 1,5-anhydrohexitol nucleosides (1,5-anhydro-2,3-
dideoxy-D-ribohexitol nucleosides). J . Org. Chem. 1997, 62,
2442-2447. (f) 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: synthesis, antiviral activity, conformational
analysis and interaction with viral thymidine kinase. J . Med.
Chem. 1998, 41, 4343-4353. (g) Hossain, N.; Luyten, I.; Roth-
enbacher, K.; Busson, R.; Herdewijn, P. Synthesis and confor-
mational analysis of 1,5-anhydro-2,4-dideoxy-D-mannitol nucle-
osides. Nucleosides Nucleotides 1999, 18, 161-180.
(4) (a) Ezzitouni, A.; Marquez, V. E. Conformationally locked
carbocyclic nucleoside built on a bicyclo[3.1.0]hexane template
with a fixed southern conformation. Synthesis and antiviral
activity. J . Chem. Soc., Perkin Trans. I 1997, 1073-1078. (b)
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 a sugar pucker influence biological activity
in nucleosides and oligonucleotides. J . Med. Chem. 1996, 39,
3739-3747.
(5) Wouters, J .; Herdewijn, P. 5-Substituted pyrimidine 1,5-anhy-
drohexitols: conformational analysis and interaction with viral
thymidine kinase. Bioorg. Med. Chem. Lett. 1999, 9, 1563-1566.
(6) (a) Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Van
Aerschot, A.; Herdewijn, P. 1′,5′-anhydrohexitol oligonucle-
otides: synthesis, base pairing and recognition by regular
oligodeoxyribonucleotides and oligoribonucleotides. Chem. Eur.
J . 1997, 3, 110-120. (b) Hendrix, C.; Rosemeyer, H.; De Bouvere,
B.; Van Aerschot, A.; Seela, F.; Herdewijn, P. 1′,5′-anhydrohexi-
tol oligonucleotides: hybridization and strand displacement with
oligoribonucleotides, interaction with RNase H and HIV reverse
transcriptase. Chem. Eur. J . 1997, 3, 1513-1520. (c) De Winter,
H.; Lescrinier, E.; Van Aerschot, A.; Herdewijn, P. Molecular
dynamics simulation to investigate differences in minor groove
hydration of HNA/RNA hybrids as compared to HNA/DNA
complexes. J . Am. Chem. Soc. 1998, 120, 5381-5384.
(7) Maurinsh, Y.; Rosemeyer, H.; Esnouf, R.; Medvedovici, A.; Wang,
J .; Ceulemans, G.; Lescrinier, E.; Hendrix, C.; Busson, R.;
Sandra, P.; Seela, F.; Van Aerschot, A.; Herdewijn, P. Synthesis
and pairing properties of oligonucleotides containing 3-hydroxy-
4-hydroxymethyl-1-cyclohexanyl nucleosides. Chem. Eur. J .
1999, 5, 2139-2150.
(8) (a) Schaeffer, H. J .; Marathe, S.; Alks, V. Enzyme Inhibitors I.
Inhibition of adenosine deaminase by isosteric nucleosides. J .
Pharm. Sci. 1964, 53, 1368-1370. (b) Schaeffer, H. J .; Kaistha,
K. K.; Chakraborti, S. K. Enzyme Inhibitors II. Synthesis of
trans-3-(6-substituted-9-purinyl)cyclohexanols as adenosine deam-
inase inhibitors. J . Pharm. Sci. 1964, 53, 1371-1374. (c)
Schaeffer, H. J .; Godse, D. D.; Liu, G. Enzyme Inhibitors III.
Syntheses of cis-(6-substituted-9-purinyl)cycloalkylcarbinols as
adenosine deaminase inhibitors. J . Pharm. Sci. 1964, 53, 1510-
1515. (d) Schaeffer, H. J .; Vince, R. Enzyme inhibitors. XIX. The
synthesis of some 1-hydroxy-2-hydroxymethyl-4-(6-substituted-
9-purinyl)cyclohexanes as nucleo-side analogues. J . Med. Chem.
1968, 11, 15-20.
Cytotoxicity Assa ys. The cytotoxicity measurements were
based on microscopically visible alteration of normal cell
morphology (E6SM, HeLa, Vero) or inhibition of normal cell
growth (HEL), as previously described.37
(9) Halazy, S.; Kenny, M.; Dulworth, J .; Eggenspiller, A. Synthesis
and antiviral properties of new cycloalkanol derivatives of
guanine. Nucleosides Nucleotides 1992, 11, 1595-1606.
Ack n ow led gm en t. This work was supported by a
grant from the E.C. (BI04-CT98-0354) and from K.U.Leu-

Cyclohexene Ring System as Furanose Mimic
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 745
(10) Kitagawa, I.; Cha, B. C.; Nakae, T.; Okaichi, Y.; Takinami, Y.;
Yoshikawa, M. A new approach to the synthesis of optically
active cyclohexane analogues of nucleoside using a Michael-type
addition reaction to nitro-cyclohexenes as a key reaction. Chem.
Pharm. Bull. 1989, 37, 542-544.
(11) Aquilar, G. J .; Gelpi, M. E.; Cadenas, R. A. Nucleocyclitols.
Synthesis of substituted purinyl-muco-inositol derivatives. J .
Heterocycl. Chem. 1992, 29, 401-405.
(d) Gosselin, G.; Schinazi, R. F.; Sommadossi, J .-P.; Mathe´, C.;
Bergogne, M.-C.; Aubertin, A.-M.; Kirn, A.; Imbach, J .-L. Anti-
human immunodeficiency virus activities of the â-L-enantiomer
of 2′, 3′-dideoxycytidine and its 5-fluoro derivative in vitro.
Antimicrob. Agents Chemother. 1994, 38, 1292-1297.
(23) Wang, J .; Herdewijn, P. Enantioselective synthesis and confor-
mational study of cyclohexene carbocyclic nucleosides. J . Org.
Chem. 1999, 64, 7820-7827.
(12) Calvani, F.; Macchia, M.; Rossello, A.; Gismondo, M. R.; Drago,
L.; Fassina, M. C.; Cisternino, M.; Domiano, P. Synthesis and
antiviral activity of dihydrocyclohexyl pyrimidine and purine
carbocyclic nucleosides. Bioorg. Med. Chem. Lett. 1995, 5, 2567-
2572.
(13) (a) Mikhailov, S. N.; Blaton, N.; Rozenski, J .; Balzarini, J .; De
Clercq, E.; Herdewijn, P. Use of cyclohexene epoxides in the
preparation of carbocyclic nucleosides. Nucleosides Nucleotides
1996, 15, 867-878. (b) Maurinsh, Y.; Schraml, J .; De Winter,
H.; Blaton, N.; Peeters, O.; Lescrinier, E.; Rozenski, J .; Van
Aerschot, A.; De Clercq, E.; Busson, R.; Herdewijn, P. Synthesis
and conformational study of 3-hydroxy-4-(hydroxymethyl)-L-
cyclohexanyl purines and pyrimidines. J . Org. Chem. 1997, 62,
2861-2871. (c) Wang, J .; Busson, R.; Blaton, N.; Rozenski, J .;
Herdewijn, P. Enantioselective approach to the synthesis of
cyclohexane carbocyclic nucleosides. J . Org. Chem. 1998, 63,
3051-3058.
(14) Ramesh, K.; Wolfe, M. S.; Lee, Y.; Vander Velde, D. S.; Bor-
chardt, R. T. Synthesis of hydroxylated cyclohexenyl- and
cyclohexanyladenines as potential inhibitors of S-adenosylho-
mocysteine hydrolase. J . Org. Chem. 1992, 57, 5861-5873.
(15) Pe´rez-Pe´rez, M.-J .; Rozenski, J .; Busson, R.; Herdewijn, P.
Application of the Mitsunobu-type condensation reaction to the
synthesis of phosphonate derivatives of cyclohexenyl and cyclo-
hexanyl nucleosides. J . Org. Chem. 1995, 60, 1531-1537.
(16) Rosenquist, Å.; Kvarnstro¨m, I.; Classon, B.; Samuelsson, B.;
Synthesis of enantiomerically pure bis(hydroxymethyl)-branched
cyclohexenyl and cyclohexyl purines as potential inhibitors of
HIV. J . Org. Chem. 1996, 61, 6282-6288.
(17) Katagiri, N.; Ito, Y.; Shiraishi, T.; Maruyama, T.; Sato, Y.;
Kaneko, C. Deamination of 9-(hydroxymethylated cycloalkyl)-
9H-adenines (carbocyclic adenine nucleosides) by adenosine
deaminase: effect of high-pressure upon deamination rate and
enantioselectivity. Nucleosides Nucleotides 1996, 15, 631-647.
(18) (a)Konkel, M. J .; Vince, R. Synthesis and biological activity of
cyclohexenyl nucleosides. cis-5-(9H-purin-9-yl)-3-cyclohexenyl
carbinols and their 8-azapurinyl analogues. Nucleosides Nucle-
otides 1995, 14, 2061-2077. (b) Konkel, M. J .; Vince, R.
Cyclohexenyl nucleosides: synthesis of cis-4-(9H-purin-9-yl)-2-
cyclohexenylcarbinols. Tetrahedron 1996, 52, 799-808.
(19) Arango, J . H.; Geer, A.; Rodriguez, J .; Young, P. E.; Scheiner,
P. Cyclohexenyl nucleosides and related compounds. Nucleosides
Nucleotides 1993, 12, 773-784.
(20) Pe´rez-Pe´rez, M.-J .; Balzarini, J .; Rozenski, J .; De Clercq, E.;
Herdewijn, P. Synthesis and antiviral activity of phosphonate
derivatives of enantiomeric dihydro-2H-pyranyl nucleosides.
Bioorg. Med. Chem. Lett. 1995, 5, 1115-1118.
(21) Luyten, I.; Herdewijn, P. Synthesis and conformational behavior
of purine and pyrimidine â-D-threo-hex-3′-enopyranosyl nucleo-
sides. Tetrahedron 1996, 52, 9249-9262.
(22) (a) Mansuri, M. M.; Farina, V.; Starrett, J . E., J r.; Benigni, D.
A.; Brankovan, V.; Martin, J . C. Preparation of the geometric
isomers of DDC, DDA, D4C and D4Τ as potential anti-HIV
agents. Bioorg. Med. Chem. Lett. 1991, 1, 65-68. (b) Coates, J .
A. V.; Cammack, N.; J enkinson, H. J .; Mutton, I. M.; Pearson,
B. A.; Storer, R. J .; Cameron, J . M.; Penn, C. R. The separated
enantiomers of 2′-deoxy-3′-thiacytidine (BCH 189) both inhibit
human immunodeficiency virus replication in vitro. Antimicrob.
Agents Chemother. 1992, 36, 202-205. (c) Schinazi, R. F.; Chu,
C. K.; Peck, A. McMillan, A.; Mathis, R.; Cannon, D.; J eong, L.-
S.; Beach, J . W.; Choi, W.-B.; Yeola, S.; Liotta, D. C. Activities
of the four optical isomers of 2′,3′-dideoxy-3′-thiacytidine (BCH-
189) against human immunodeficiency virus type I in human
lymphocytes. Antimicrob. Agents Chemother. 1992, 36, 672-676.
(24) De Clercq, E.; Descamps, J .; Verhelst, G.; Walker, R. T.; J ones,
A. S.; Torrence, P. F.; Shugar, D. Comparative efficacy of
antiherpes drugs against different strains of herpes simplex
virus. J . Infect. Dis. 1980, 141, 563-574.
(25) De Clercq, E.; Ho´ly, A.; Rosenberg, I.; Sakuma, T.; Balzarini,
J .; Maudgal, P. C. A novel selective broad-spectrum anti-DNA
virus agent. Nature 1986, 323, 464-467.
(26) Cremer, D.; Szabo, K. J . Ab initio studies of six-membered
rings: present status and future development. In Conforma-
tional behavior of six-membered rings. Analysis, dynamics and
steroelectronic effects; J uaristi, E., Ed.; VCH Publishers Inc.:
New York, 1995; pp 59-135.
(27) (a) Saebø, S.; Boggs, J . E. Structure and conformation of
cyclohexene, 1,4-cyclohexadiene, and 1,3-cyclohexadiene. J . Mol.
Struct. 1981, 73, 137-144. (b) Saebø, S.; Cordell, F. R.; Boggs,
J . E. Structure and conformations of cyclopentane, cyclopentene
and cyclopentadiene. THEOCHEM 1983, 13, 221-232.
(28) Plavec, J .; Thibaudeau, C.; Chattopadhyaya, J . How does the
2′-hydroxy group drive the pseudorotational equilibrium in
nucleoside and nucleotide by the tuning of the 3′-gauche effect?
J . Am. Chem. Soc. 1994, 116, 6558-6560.
(29) (a) Doboszewski, B.; Blaton, N.; Herdewijn, P. Easy synthesis
and different conformational behavior of purine and pyrimidine
â-D-glycero-pent-2′-enopyranosyl nucleosides. J . Org. Chem.
1995, 60, 7909-7919. (b) Polak, M.; Doboszewski, B.; Herdewijn,
P.; Plavec, J . Conformational studies on 2′,3′-unsaturated pen-
topyranosyl nucleosides by 1H NMR spectroscopy. Impact of π
f σ* interactions on the axial preference of the purine versus
pyrimidine nucleobase. J . Am. Chem. Soc. 1997, 119, 9782-
9792.
(30) Champness, J . N.; Bennett, M. S.; Wien, F.; Visse, R.; Summers,
W. C.; Herdewijn, P.; de Clercq, E.; Ostrowski, T.; J arvest, R.
L.; Sanderson, M. R. Exploring the active site of herpes simplex
virus type-1 thymidine kinase by X-ray crystallography of
complexes with aciclovir and other ligands. Proteins Struct.
Funct. Gen. 1998, 32, 350-361.
(31) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K.
M., J r.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell,
J . W.; Kollman, P. A. A second generation force field for the
simulation of proteins, nucleic acids, and organic molecules. J .
Am. Chem. Soc. 1995, 117, 5179-5197.
(32) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. A well-
behaved electrostatic potential based method using charge
restraints for deriving atomic charges: the RESP model. J . Phys.
Chem. 1993, 97, 10269-10280.
(33) Schmidt, M. W.; Baldridge, K. K.; Boatz, J . A.; Elbert S. T.;
Gordon, M. S.; J ensen, J . H.; Koseki, S.; Matsunaga N.; Nguyen,
K. A.; Su, S.; Windus, T. L.; Dupuis M.; Montgomery, J . A.
General atomic and molecular electronic structure system. J .
Comput. Chem. 1993, 14, 1347-1363.
(34) QCPE, Department of Chemistry, Indiana University.
(35) Blaney, J . M.; Dixon, J . S. Distance Geometry in Molecular
Modeling. Rev. Comput. Chem. 1994, 5, 299-335.
(36) The 1KI3 pdb entry was obtained from the Protein Data Bank
at the Research Collaboratory for Structural Bioinformatics
(RCSB).
(37) De Clercq, E.; Balzarini, J .; Torrence, P. F.; Mertes, M. P.;
Schmidt, C. L.; Shugar, D.; Barr, P. J .; J ones, A. S.; Verhelst,
G.; Walker, R. T. Thymidylate synthetase as a target enzyme
for the inhibitory activity of 5-substituted 2′-deoxyuridines on
mouse leukemia L-1210 cell growth. Mol. Pharmacol. 1981, 19,
321-330.
J M991171L