Highly potent and selective inhibition of varicella-zoster virus by bicyclic furopyrimidine nucleosides bearing an aryl side chain

Article abstract of DOI:10.1021/jm000210m

In addition to our recent report on the potent anti-varicella-zoster virus (VZV) activity of some unusual bicyclic furopyrimidine nucleosides bearing long alkyl side chains, we herein report the further significant enhancement of the antiviral potency by

Full text of DOI:10.1021/jm000210m

J . Med. Chem. 2000, 43, 4993-4997
4993
High ly P oten t a n d Selective In h ibition of Va r icella -Zoster Vir u s by Bicyclic
F u r op yr im id in e Nu cleosid es Bea r in g a n Ar yl Sid e Ch a in
Christopher McGuigan,* Hubert Barucki, Sally Blewett, Antonella Carangio, J onathan T. Erichsen,‡
Graciela Andrei,† Robert Snoeck,† Erik De Clercq,† and J an Balzarini†
Welsh School of Pharmacy and Department of Optometry and Vision Sciences, Cardiff University, King Edward VII Avenue,
Cardiff CF10 3XF, U.K., and Rega Institute for Medical Research, Minderbroedersstraat 10, Leuven B3000, Belgium
Received September 12, 2000
In addition to our recent report on the potent anti-varicella-zoster virus (VZV) activity of some
unusual bicyclic furopyrimidine nucleosides bearing long alkyl side chains, we herein report
the further significant enhancement of the antiviral potency by inclusion of a phenyl group in
the side chain of these compounds. The target structures were prepared by the Pd-catalyzed
coupling of a series of para-substituted arylacetylenes with 5-iodo-2′-deoxyuridine, to give
intermediate 5-alkynyl nucleosides which were cyclized in the presence of Cu to give the desired
bicyclic systems. The compounds display extraordinary potency and selectivity for VZV; the
most active are ca. 10 000 times more potent than the reference compound acyclovir and ca.
100 times more potent than the alkyl analogues earlier reported by us. The current compounds
show little cytotoxicity, leading to selectivity index values G 1 000 000. From a range of DNA
and RNA viruses tested, only VZV was inhibited by these compounds indicating their extreme
selectivity for this target virus. The novelty of the molecules, coupled with their extreme potency
and selectivity, their desirable physicochemical properties, and their relative ease of synthesis,
makes them of considerable interest for potential drug development for VZV infections.
Ch a r t 1
In tr od u ction
We have recently reported on highly potent and
selective inhibitors of varicella-zoster virus (VZV) with
unusual bicyclic furopyrimidine structures.1 These novel
molecules displayed potencies against various strains
of VZV in cell culture which exceeded those of current
anti-VZV agents such as acyclovir by ca. 300-fold.1 An
unusual, but apparently critical, requirement for anti-
viral activity in this novel series of bicyclic nucleosides
is the presence of a long alkyl chain at the 6-position of
the furo ring, as typified by structures 1a -c (Chart 1).1
Although shorter alkyl chains do lead to antiviral
activity comparable to that of acyclovir (R ) C5-C6 for
example), longer chains (R ) C7-C10) were required for
optimal potency. In an effort to further explore the
structure-activity relationships (SARs) in this unusual
series of promising antivirals, we have prepared a small
series of ω-substituted compounds2 and have noted that
the ω-chlorononyl compound in particular displays
potency and selectivity at least comparable with that
of the parent decyl system.1,2 Substitution at the
terminus of the alkyl (nonyl) chain with other halogen
atoms (F, Br, I) is also acceptable, while more polar
groups such as hydroxyl lead to a significant diminution
of potency.3
effective. However, the corollary of this is that the
compounds display low water solubility. With this in
mind we have recently prepared a series of analogues
with (ether) oxygen substitution in the alkyl side chain.
Either one or two ether oxygens were incorporated, and
the position of these heteroatoms along the chain was
varied.4 The introduction of these oxygen atoms was
indeed highly successful in enhancing water solubility,
but unfortunately only at the expense of a significant
(ca. 1000-fold) reduction in antiviral potency.4
Taken together, all of these studies1-4 indicate the
absolute requirement for a nonpolar moiety at the C6
position of the furo ring system for potent anti-VZV
activity in these bicyclic nucleosides. To further probe
this requirement we now sought to replace a number
of the alkyl methylenes by alternative nonalkyl units.
In particular, we sought to constrain the highly flexible
alkyl systems into more defined conformations by the
use of ring moieties. Bearing in mind the above-
mentioned SARs we chose to incorporate a series of
para-substituted (predominantly alkyl) phenyl groups
at C6, and we herein report on the synthesis and
characterization of these novel analogues; in particular
we note their extreme potency and selectivity as inhibi-
tors of VZV.
The present compounds display high lipophilicity
values, as would be anticipated from their long alkyl
side chains. This may have the positive consequence
that their membrane permeation may be very efficient
and topical formulation and dosing may be notably
* To whom correspondence should be addressed. Tel: 44 2920 874
537. Fax: 44 29 2087 4537. E-mail: mcguigan@cardiff.ac.uk.
† Rega Institute for Medical Research.
‡ Department of Optometry and Vision Sciences.
10.1021/jm000210m CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/22/2000

4994 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
McGuigan et al.
Ta ble 1. Anti-VZV Activity, Cytotoxicity, and ClogP Values for Bicyclic Pyrimidines 4a -k and Reference Compounds 5, 6, and
1a -ca
EC50 (µM)
compd
R
ClogP
VZV OKA
VZV YS
VZV TK- 07
VZV TK- YS-R
MCC (µM)
CC50 (µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
Me
Et
Pr
0.35
0.85
1.38
1.91
2.44
2.97
3.50
4.03
4.56
1.07
1.22
0.28 ( 0.0
0.06 ( 0.0
0.09 ( 0.0
0.010 ( 0.001
0.0022 ( 0.001
0.0003 ( 0.0002
0.0005 ( 0.0003
0.0054 ( 0.002
0.040 ( 0.025
0.1
0.5
2.9
0.003
0.008
0.02
0.015
0.16 ( 0.0
0.06 ( 0.0
0.07 ( 0.0
0.008 ( 0.001
0.0005 ( 0.0003
0.0001 ( 0.0001
0.0001 ( 0.0001
0.003 ( 0.002
0.027 ( 0.023
0.1
0.5
1
0.003
0.024
0.02
0.008
>200
103
162
>200
>50
>20
>20
>5
>200
>200
200
G50
G20
G20
20
5
G20
200
>200
>200
123
>50
>50
G20
>20
>5
188
Bu
>200
>200
18
pentyl
hexyl
heptyl
octyl
Cl
>5
>5
>5
18
>20
>50
>50
74
>20
>50
>50
125
>200
4j
4k
nd
nd
Br
50
5 (acyclovir)
6 (BVDU)
1a
1b
1c
>200
>150
>50
G200
>50
>200
>400
G50
>200
>50
>150
>50
>200
>50
>150
>50
>200
>50
octyl
nonyl
decyl
2.46
2.99
3.52
a
EC50 values represent the concentration of compound in µM required to reduce VZV plaque formation after 5 days in HEL cell cultures
by 50% compared to untreated controls. Data are given for two thymidine kinase-competent strains of virus (OKA and YS) and for two
thymidine kinase-deficient strains (07 and YS-R). CC50 represents the 50% cytostatic concentration, defined as the compound concentration
in µM required to reduce the cell number by 50% after 5 days in the absence of virus. MCC (minimal cytotoxic concentration) is the
compound concentration required to cause a microscopically visible alteration of normal cell morphology. ClogP is the calculated logarithm
of the octanol-water partition coefficient P.9
Sch em e 1
ranged from 11-16% (un-optimized), being thus com-
parable to the overall yield of 4d (18%) and with a
significant saving of time in the purification of inter-
mediates. Target structures 4a -k were characterized
by 1H and 13C NMR, high-resolution mass spectrometry,
and microanalysis, all data being consistent with their
intended structures and confirming high chemical
purity.
An tivir a l Activity. The target bicyclic systems 4a -k
were evaluated for their ability to inhibit the replication
of herpes simplex virus (HSV-1, HSV-2), vaccinia virus
(VV), cytomegalovirus (CMV), and VZV according to
previously described methods.8 Data are shown in Table
1 for the activity of 4a -k versus two strains of thymi-
dine kinase-competent (TK+) VZV and also two strains
of thymidine kinase-deficient (TK-) VZV, with data also
included for the reference anti-herpes agents acyclovir
(5) and BVDU (6). We also include corresponding data
for the parent alkyl analogues 1a -c of the present
systems.1 Cytotoxicity data are also given for each
compound in two assays (Table 1). It is clear from these
data that the p-alkylaryl systems (4a -i) we herein
report are exquisitely potent and selective inhibitors of
VZV in these assays. As previously noted for the parent
alkyl analogues,1 there is a clear dependence on the
length of the p-alkyl substituent in the homologous
series 4a -i. However, whereas optimal alkyl chain
lengths were ca. C8-C11 for the parent alkyl series 1a -
c, we note that the inclusion of the phenyl ring in the
present compounds leads to a shift in optimal p-alkyl
chain length to ca. C4-C7. The p-pentylphenyl analogue
4f displays EC50 values against the two thymidine
kinase-competent strains of VZV at 0.1-0.3 nM con-
centrations, thus being ca. 10 000 times more potent
than the primary existing medicine for VZV (acyclovir)
and 10-30 times more potent than the exceptionally
potent BVDU (6). Cytotoxicity was in general noted for
the current agents only at concentrations G 20 to 200
µM; combined with the very high potency values these
data lead to selectivity index values (SIs) exceeding
1 000 000 for the most active compounds in the series.
Resu lts a n d Discu ssion
Ch em istr y. The synthetic route adopted by us for the
current target compounds (Scheme 1) followed closely
that we had adopted for the parent alkyl systems,1
involving the Pd-catalyzed coupling of arylacetylenes
with 5-iodo-2′-deoxyuridine (2) to give intermediate 5-(2-
arylethynyl)-2′-deoxyuridines 3a -k .5-7 We have previ-
ously noted1 that these intermediates either may be
isolated and purified prior to the second synthetic step
(cyclization) or may be cyclized in situ. In the first case
studied (3d ) we did isolate and purify the intermediate
alkyne (41%) and subjected this to cyclization with
copper catalysis (43%). However, in all subsequent cases
(3a -c,e-k ) the alkyne was not isolated and 2 was
converted to 4a -c,e-k in one step. The yields observed

Highly Potent and Selective Inhibition of VZV
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4995
at room temperature, under a nitrogen atmosphere, After this
time, the reaction mixture was concentrated in vacuo, and the
resulting residue was dissolved in dichloromethane and metha-
nol (1:1) (6 mL), whereupon an excess of Amberlite IRA-400
(HCO3- form) was added and stirred for 30 min. The resin was
filtered and washed with methanol, and the combined filtrate
was evaporated to dryness. The crude product was purified
by flash column chromatography (eluent: ethyl acetate). The
appropriate fractions were combined, and the solvent was
removed in vacuo to give 5-(4-n-propylphenylacetylene)-2′-
deoxyuridine as the pure product (350 mg, 41%): 1H NMR
(DMSO-d6; 300 MHz) δ 11.56 (1H, broad s, NH), 8.13 (1H, s,
Similarly, data for 4j,k indicate that a p-halogen atom
is tolerated in the phenyl ring, with similar potencies
to that noted for the parent phenyl (4a ) or toluyl (4b)
systems. The calculated log P values9 for 4j,k are
roughly in line with this observation (Table 1).
The precise mechanism of action of these compounds
remains unclear. However, the clear absence of antiviral
activity against thymidine kinase-deficient VZV strains
(Table 1) strongly implies an absolute need for VZV
thymidine kinase-mediated phosphorylation to the cor-
responding 5′-monophosphates as a prerequisite for
antiviral action. The present data cannot discriminate
between action as the monophosphate or further phos-
phorylation to the di- and triphosphates of these com-
pounds, but selective phosphorylation by VZV thymidine
kinase and/or specific inhibition of VZV DNA poly-
merase by the corresponding nucleoside triphosphates
may appear to be the simplest explanation of the very
high anti-VZV selectivity of these agents. Indeed, evalu-
ation of 4a -j against a series of other viruses (HSV-1,
HSV-2, VV, CMV) indicated a complete absence of
antiviral activity, suggesting a mode of action that is
entirely specific to one or more VZV-specific enzyme-
mediated steps. This mode of action may thus be similar
to that suggested for the parent alkyl analogues.
In conclusion we herein report p-alkylaryl analogues
of bicyclic furopyrimidine nucleosides as exquisitely
potent (subnanomolar) and selective inhibitors of VZV.
A number of features of these novel compounds indicate
that they may have considerable clinical potential for
evaluation as improved agents for the treatment of
herpes zoster and other VZV infections.
3
H-6), 7.42 (2H, Ha) - 7.25 (2H, Hb) (AB system, J ) 7.89 Hz,
3
3
4J ) 2.3 Hz), 6.18 (1H, dd, J ) 6.5 Hz, H-1′), 5.25 (1H, d, J
3
) 4.1 Hz, 3′-OH), 5.16 (1H, t, J ) 4.7 Hz, 5′-OH), 4.19 (1H,
m, H-3′), 3.73 (1H, m, H-4′), 3.49 (2H, m, H-5′), 2.37 (2H, t, 3J
) 6.9 Hz, R-CH2), 2.21 (2H, m, 2-H′a and 2-H′b), 1.51 (2H, sxt,
CH2, 3J ) 6.9 Hz), 0.85 (3H, t, 3J ) 6.9 Hz, CH3); 13C NMR
(DMSO-d6; 75 MHz) δ 13.8 (CH3), 19.5, 21.6, (C2H4), 40.7 (C-
2′), 60.8 (C-5′), 70.5 (C-3′), 73.7 (R-alkynyl), 85.4, 88.4 (C-1′,
C-4′), 93.8 (â-alkynyl), 99.9 (C-5), 124.3 (C-Hb), 129.5 (ipso-
C), 130.7 (C-Ha), 140.2 (para-C), 143.4 (C-6), 150.3 (C-2), 162.6
(C-4).
3-(2′-Deoxy-â-D-r ib ofu r a n osyl)-6-(4-n -p r op ylp h en yl)-
2,3-d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4d ). To a solution
of 5-(4-n-propylphenylacetylene)-2′-deoxyuridine (3d ) (200 mg,
0.54 mmol) in methanol and triethylamine (7:3) (20 mL) was
added copper iodide (20 mg, 0.102 mmol). The mixture was
refluxed for 4 h. The solvent was removed in vacuo, and the
crude product was purified by flash column chromatography
(initial eluent: ethyl acetate, followed by: ethyl acetate/
methanol (9:1)). The combined fractions were combined and
the solvent was removed in vacuo to give the crude product,
which was recrystallized from methanol to give pure 3-(2′-
deoxy-â-D-ribofuranosyl)-6-(4-n-propylphenylacetylene)-2,3-di-
hydrofuro[2,3-d]pyrimidin-2-one (4d ) (86 mg, 43%): 1H NMR
(DMSO-d6; 300 MHz) δ 8.72 (1H, s, H-4), 7.43 (2H, Ha) - 7.28
3
4
(2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz), 7.15 (1H, s,
Exp er im en ta l Section
3
3
H-5), 6.18 (1H, dd, J ) 6.15 Hz, H-1′), 5.31 (1H, d, J ) 4.0
Hz, 3′-OH), 5.12 (1H, t, 3J ) 5.01 Hz, 5′-OH), 4.31 (1H, m,
The bicyclic ring has been numbered in accordance with the
recommended IUPAC nomenclature guidelines. Both IUPAC
and accepted nomenclature for nucleoside chemistry has been
followed for the naming of the compounds described. For thin-
layer chromatography, precoated aluminum backed plates (60
F-54, 0.2 mm thickness; supplied by E. Merck AG, Darmstadt,
Germany) were used and were developed by the ascending
method. After evaporation of solvent, compounds were detected
by quenching of the fluorescence, at 254 or 366 nm, upon
irradiation with a UV lamp. For column chromatography, glass
columns were slurry packed in the appropriate eluent under
gravity, with silica gel (C-gel 60A, 40-60 µm; Phase Sep, U.K.).
Samples were applied as a concentrated solution, in the same
eluent, or preabsorbed onto silica gel. Fractions containing the
product were identified by TLC, “pooled” and concentrated in
vacuo. Flash column chromatography was performed using an
3
H-3′), 3.89 (1H, m, H-4′), 3.51 (2H, m, H-5′), 2.65 (2H, t, J )
6.9 Hz, R-CH2), 2.31 and 2.12 (2H, m, 2-H′a and 2-H′b), 1.58
(2H, sxt, CH2, 3J ) 6.9 Hz), 0.85 (3H, t, 3J ) 6.9 Hz, CH3); 13
C
NMR (DMSO-d6; 75 MHz) δ 13.2 (CH3), 20.1, 22.3, (C2H4), 41.5
(C-2′), 62.3 (C-5′), 71.6 (C-3′), 83.2, 88.4 (C-1′, C-4′), 100.4 (C-
5), 104.6 (C-4a), 125.3 (C-Hb), 128.4 (ipso-C), 131.8 (C-Ha),
141.2 (para-C), 138.5 (C-4), 154.6 (C-6), 159.1 (C-2), 172.3 (C-
7a); MS (ES+) m/e 393 (MNa+, 100%), 277 (baseNa+, 20%).
Accurate mass: C20H22N2O5Na requires 393.1426; found
393.1422. Found: C, 61.69; H, 6.23%; N, 7.13. C20H22N2O5‚
H2O requires: C, 61.85; H, 6.23; N, 7.21.
Gen er a l P r oced u r e for P r ep a r a tion of 3-(2′-Deoxy-â-
D-r ib ofu r a n osyl)-6-(4-n -a lk yllp h en yl)-2,3-d ih yd r ofu r o-
[2,3-d ]p yr im id in -2-on e An a logu es. To a stirred solution of
5-iodo-2′-deoxyuridine (2) (800 mg, 2.26 mmol) in anhydrous
dimethylformamide (8 mL) were added diisopropylethylamine
(584 mg, 0.8 mL, 4.52 mmol), the 4-n-alkylphenylacetylene
(6.76 mmol), tetrakis(triphenylphoshine)palladium(0) (261 mg,
0.266 mmol) and copper(I) iodide (86 mg, 0.452 mmol). The
mixture was stirred for 18 h, at room temperature, under a
nitrogen atmosphere, after which time TLC (ethyl acetate/
methanol 9:1), showed complete conversion of the starting
material. Copper(I) iodide (80 mg, 0.40 mmol), triethylamine
(15 mL) and methanol (20 mL) were then added to the mixture,
which was subsequently refluxed for 4 h. The reaction mixture
was then concentrated in vacuo, and the resulting residue was
dissolved in dichloromethane and methanol (1:1) (6 mL),
whereupon an excess of Amberlite IRA-400 (HCO3- form) was
added and stirred for 30 min. The resin was filtered and
washed with methanol, and the combined filtrate was evapo-
rated to dryness. The crude product was purified by flash
column chromatography (Initial eluent: ethyl acetate, followed
by: ethyl acetate/methanol (9:1)). The appropriate fractions
1
electrical pump. H and 13C NMR spectra were recorded on a
Bruker Avance DPX300 spectrometer (300 and 75 MHz,
respectively) and autocalibrated to the deuterated solvent
reference peak. All 13C NMR spectra were proton decoupled.
Mass spectra were performed by the service at the University
of Birmingham, U.K.. Elemental analyses were performed by
the service at the University of Wales, Swansea, U.K. All
solvents used were anhydrous and used as supplied from
Aldrich. All nucleosides and solid reagents were dried while
heating under high vacuum over phosphorus pentoxide. All
glassware was oven dried at 130 °C for several hours and
allowed to cool in a stream of dry nitrogen.
5-(4-n -P r op ylp h en yla cet ylen e)-2′-d eoxyu r id in e (3d ).
To a stirred solution of 5-iodo-2′-deoxyuridine (2) (800 mg, 2.26
mmol) in anhydrous dimethylformamide (8 mL) were added
diisopropylethylamine (584 mg, 0.8 mL, 4.52 mmol), 4-n-
propylphenylacetylene (0.97 g, 6.76 mmol), tetrakis(triph-
enylphoshine)palladium(0) (261 mg, 0.266 mmol) and copper(I)
iodide (86 mg, 0.452 mmol). The mixture was stirred for 18 h,

4996 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
McGuigan et al.
were combined, and the solvent was removed in vacuo, to give
the pure product.
(C-2′), 63.7 (C-5′), 73.6 (C-3′), 83.5, 88.7 (C-1′, C-4′), 100.8 (C-
5), 108.4 (C-4a), 125.3 (C-Hb), 128.4 (ipso-C), 131.8 (C-Ha),
141.2 (para-C), 138.5 (C-4), 154.6 (C-6), 159.1 (C-2), 170.9 (C-
7a); MS (ES+) m/e 407 (MNa+, 100%), 291 (baseNa+, 20%).
Accurate mass: C21H24N2O5Na requires 407.1583; found
407.1575. Found: C, 65.41; H, 6.48; N, 7.40. C21H24N2O5
requires: C, 65.61; H, 6.29; N, 7.29.
3-(2′-Deoxy-â-D-r ibofu r an osyl)-6-ph en yl-2,3-dih ydr ofu r o-
[2,3-d ]p yr im id in -2-on e (4a ). The procedure was carried out
using phenylacetylene (0.689 g, 6.76 mmol), which gave 3-(2′-
deoxy-â-D-ribofuranosyl)-6-phenylacetylene-2,3-dihydrofuro-
[2,3-d]pyrimidin-2-one (4a ) (123 mg, 16%), after purification
by column chromatography: 1H NMR (DMSO-d6; 300 MHz) δ
8.84 (1H, s, H-4), 7.81 (2H, Ha) - 7.62 (2H, Hb) (AB system,
3-(2′-Deoxy-â-D-r ib ofu r a n osyl)-6-(4-n -p en t ylp h en yl)-
2,3-d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4f). The procedure
was carried out using 4-n-pentylphenylacetylene (1.15 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-
pentylphenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-
one (4f) (137 mg, 15%), after purification by column chroma-
tography: 1H NMR (DMSO-d6; 300 MHz) δ 8.81 (1H, s, H-4),
4
3
3J ) 7.89 Hz, J ) 2.3 Hz), 7.25 (1H, s, H-5), 6.28 (1H, dd, J
3
) 6.15 Hz, H-1′), 5.41 (1H, d, J ) 4.0 Hz, 3′-OH), 5.23 (1H, t,
3J ) 5.01 Hz, 5′-OH), 4.33 (1H, m, H-3′), 3.91 (1H, m, H-4′),
3.72 (2H, m, H-5′), 2.40 and 2.15 (2H, m, 2-H′a and 2-H′b); 13
C
NMR (DMSO-d6; 75 MHz) δ 41.6 (C-2′), 60.9 (C-5′), 69.8 (C-
3′), 87.9, 88.5 (C-1′, C-4′), 99.12 (C-5), 107.1 (C-4a), 124.8 (C-
Hb), 128.4 (ipso-C), 129.7 (C-Ha), 140.2 (para-C), 138.5 (C-4),
154.1 (C-6), 160.3 (C-2), 171.4 (C-7a); MS (ES+) m/e 351 (MNa+,
100%). Accurate mass: C17H16N2O5Na requires 351.0957; found
351.0961. Found: C, 60.44; H, 5.18; N, 8.25. C17H16N2O5‚
0.5H2O requires: C, 60.53; H, 5.08; N, 8.30.
3-(2′-Deoxy-â-D-r ib ofu r a n osyl)-6-(4-n -m et h ylp h en yl)-
2,3-d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4b). The procedure
was carried out using 4-n-methylphenylacetylene (0.791 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-me-
thylphenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4b)
(131 mg, 17%), after purification by column chromatography:
1H NMR (DMSO-d6; 300 MHz) δ 8.81 (1H, s, H-4), 7.79 (2H,
3
4
7.51 (2H, Ha) - 7.35 (2H, Hb) (AB system, J ) 7.89 Hz, J )
2.3 Hz), 7.18 (1H, s, H-5), 6.23 (1H, dd, 3J ) 6.15 Hz, H-1′),
5.37 (1H, d, 3J ) 4.0 Hz, 3′-OH), 5.31 (1H, t, 3J ) 5.01 Hz,
5′-OH), 4.34 (1H, m, H-3′), 3.79 (1H, m, H-4′), 3.41 (2H, m,
3
H-5′), 2.67 (2H, t, J ) 6.9 Hz, R-CH2), 2.34 and 2.14 (2H, m,
2-H′a and 2-H′b), 1.67 (2H, m, CH2), 1.51-1.32 (4H, m, CH2),
3
0.84 (3H, t, J ) 6.9 Hz, CH3); 13C NMR (DMSO-d6; 75 MHz)
δ 13.2 (CH3), 20.1, 22.3, 27.9, 28.4, (C4H8), 41.3 (C-2′), 62.6
(C-5′), 71.8 (C-3′), 83.4, 86.4 (C-1′, C-4′), 100.4 (C-5), 107.4 (C-
4a), 125.4 (C-Hb), 127.4 (ipso-C), 131.8 (C-Ha), 138.5 (C-4),
141.3 (para-C), 154.6 (C-6), 161.1 (C-2), 170.9 (C-7a); MS (ES+)
m/e 421 (MNa+, 100%), 305 (baseNa+, 40%). Accurate mass:
C
22H26N2O5Na requires 421.1739; found 421.1733. Found: C,
3
4
64.69; H, 6.67; N, 6.82. C22H26N2O5‚0.5H2O requires: C, 64.85;
H, 6.68; N, 6.87.
Ha) - 7.48 (2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz),
3
7.31 (1H, s, H-5), 6.22 (1H, dd, J ) 6.15 Hz, H-1′), 5.37 (1H,
d, J ) 4.0 Hz, 3′-OH), 5.19 (1H, t, J ) 5.01 Hz, 5′-OH), 4.29
(1H, m, H-3′), 3.87 (1H, m, H-4′), 3.65 (2H, m, H-5′), 2.41 and
3
3
3-(2′-Deoxy-â-D-r ibofu r a n osyl)-6-(4-n -h exylp h en yl)-2,3-
d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4g). The procedure
was carried out using 4-n-hexylphenylacetylene (1.26 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-hexy-
lphenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4g)
(124 mg, 13%), after purification by column chromatography:
1H NMR (DMSO-d6; 300 MHz) δ 8.85 (1H, s, H-4), 7.53 (2H,
3
2.19 (2H, m, 2-H′a and 2-H′b), 2.15 (3H, t, J ) 6.9 Hz, CH3);
13C NMR (DMSO-d6; 75 MHz) δ 15.2 (CH3), 40.5 (C-2′), 61.3
(C-5′), 70.3 (C-3′), 87.2, 89.1 (C-1′, C-4′), 100.2 (C-5), 106.3 (C-
4a), 125.3 (C-Hb), 127.4 (ipso-C), 128.8 (C-Ha), 138.2 (para-
C), 137.9 (C-4), 155.1 (C-6), 159.3 (C-2), 170.8 (C-7a); MS (ES+)
m/e 365 (MNa+, 100%), 249 (baseNa+, 10%). Accurate mass:
3
4
Ha) - 7.29 (2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz),
3
C
18H18N2O5Na requires 365.1113; found 365.1107. Found: C,
7.23(1H, s, H-5), 6.24 (1H, dd, J ) 6.15 Hz, H-1′), 5.58 (1H,
d, J ) 4.0 Hz, 3′-OH), 5.29 (1H, t, J ) 5.01 Hz, 5′-OH), 4.54
(1H, m, H-3′), 3.79 (1H, m, H-4′), 3.51 (2H, m, H-5′), 2.72 (2H,
t, J ) 6.9 Hz, R-CH2), 2.31 and 2.10 (2H, m, 2-H′a and 2-H′b),
1.62 (2H, m, CH2), 1.42-1.22 (6H, m, CH2), 0.87 (3H, t, J )
3
3
62.87; H, 5.39; N, 7.88. C18H18N2O5 requires: C, 63.15; H, 5.30;
N, 8.18.
3
3-(2′-Deoxy-â-D-r ibofu r a n osyl)-6-(4-n -eth ylp h en yl)-2,3-
d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4c). The procedure
was carried out using 4-n-ethylphenylacetylene (0.885 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-eth-
ylphenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4c)
(115 mg, 14%), after purification by column chromatography:
1H NMR (DMSO-d6; 300 MHz) δ 8.91 (1H, s, H-4), 7.76 (2H,
3
6.9 Hz, CH3); 13C NMR (DMSO-d6; 75 MHz) δ 13.2 (CH3), 20.1,
22.3, 27.9, 29.5, 30.2 (C5H10), 41.6 (C-2′), 62.3 (C-5′), 769.8 (C-
3′), 83.5, 88.7 (C-1′, C-4′), 99.1 (C-5), 107.2 (C-4a), 124.3 (C-
Hb), 126.4 (ipso-C), 129.3 (C-Ha), 138.5 (C-4), 141.2 (para-C),
154.6 (C-6), 160.9 (C-2), 171.3 (C-7a); MS (ES+) m/e 435 (MNa+,
100%), 319 (baseNa+, 40%). Accurate mass: C23H28N2O5Na
requires 435.1896; found 435.1897. Found: C, 64.28; H, 7.10;
N, 6.47. C23H28N2O5‚H2O requires: C, 64.17; H, 7.02; N, 6.51.
3
4
Ha) - 7.49 (2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz),
3
7.25 (1H, s, H-5), 6.26 (1H, dd, J ) 6.15 Hz, H-1′), 5.39 (1H,
d, J ) 4.0 Hz, 3′-OH), 5.24 (1H, t, J ) 5.01 Hz, 5′-OH), 4.34
(1H, m, H-3′), 3.98 (1H, m, H-4′), 3.71 (2H, m, H-5′), 2.71 (2H,
3
3
3-(2′-d eoxy-â-D-r ib ofu r a n osyl)-6-(4-n -h ep t ylp h en yl)-
2,3-d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4h ). The procedure
was carried out using 4-n-heptylphenylacetylene (1.35 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-hep-
tylphenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4h )
(129 mg, 13%), after purification by column chromatography:
1H NMR (DMSO-d6; 300 MHz) δ 8.91 (1H, s, H-4), 7.62 (2H,
3
t, J ) 6.9 Hz, R-CH2) 2.48 and 2.12 (2H, m, 2-H′a and 2-H′b),
1.2 (3H, t, J ) 6.9 Hz, CH3); 13C NMR (DMSO-d6; 75 MHz) δ
3
15.7 (CH3), 27.9 (CH2), 40.6 (C-2′), 61.0 (C-5′), 69.8 (C-3′), 87.9,
88.5 (C-1′, C-4′), 99.1 (C-5), 107.2 (C-4a), 124.9 (C-Hb), 126.5
(ipso-C), 128.8 (C-Ha), 145.7 (para-C), 138.2 (C-4), 154.1 (C-
6), 162.1 (C-2), 171.4 (C-7a); MS (ES+) m/e 379 (MNa+, 100%),
263 (baseNa+, 10%). Accurate mass: C19H20N2O5Na requires
379.1270; found 379.1272. Found: C, 62.77; H, 5.93; N, 7.61.
3
4
Ha) - 7.35 (2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz),
3
7.26 (1H, s, H-5), 6.28 (1H, dd, J ) 6.17 Hz, H-1′), 5.62 (1H,
d, J ) 4.1 Hz, 3′-OH), 5.32 (1H, t, J ) 5.12 Hz, 5′-OH), 4.52
(1H, m, H-3′), 3.81 (1H, m, H-4′), 3.62 (2H, m, H-5′), 2.71 (2H,
t, J ) 6.9 Hz, R-CH2), 2.35 and 2.14 (2H, m, 2-H′a and 2-H′b),
1.59 (2H, m, CH2), 1.48-1.21 (8H, m, CH2), 0.82 (3H, t, J )
3
3
C
19H20N2O5‚0.5H2O requires: C, 62.46; H, 5.79; N, 7.67.
3-(2′-Deoxy-â-D-r ibofu r a n osyl)-6-(4-n -bu tylp h en yl)-2,3-
3
d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4e). The procedure
was carried out using 4-n-butylphenylacetylene (1.072 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-bu-
tylphenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4e)
(140 mg, 16%), after purification by column chromatography:
1H NMR (DMSO-d6; 300 MHz) δ 8.76 (1H, s, H-4), 7.46 (2H,
3
6.9 Hz, CH3); 13C NMR (DMSO-d6; 75 MHz) δ 13.2 (CH3), 20.1,
22.3, 27.9, 28.5, 29.5, 30.2 (C6H12), 41.6 (C-2′), 61.5 (C-5′), 69.8
(C-3′), 87.9, 88.5 (C-1′, C-4′), 99.1 (C-5), 107.2 (C-4a), 124.3
(C-Hb), 126.2 (ipso-C), 129.3 (C-Ha), 138.2(C-4), 144.2 (para-
C), 154.6 (C-6), 160.7 (C-2), 170.6 (C-7a); MS (ES+) m/e 449
(MNa+, 100%), 333 (baseNa+, 30%). Accurate mass: C24H30N2O5-
Na requires 449.2052; found 449.2057. Found: C, 67.27; H,
7.26; N, 6.38. C24H30N2O5 requires: C, 67.59; H, 7.09; N, 6.57.
3
4
Ha) - 7.31 (2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz),
3
7.20 (1H, s, H-5), 6.21 (1H, dd, J ) 6.15 Hz, H-1′), 5.37 (1H,
d, J ) 4.0 Hz, 3′-OH), 5.31 (1H, t, J ) 5.01 Hz, 5′-OH), 4.31
(1H, m, H-3′), 3.75 (1H, m, H-4′), 3.48 (2H, m, H-5′), 2.65 (2H,
3
3
3
t, J ) 6.9 Hz, R-CH2), 2.31 and 2.12 (2H, m, 2-H′a and 2-H′b),
3-(2′-Deoxy-â-D-r ibofu r a n osyl)-6-(4-n -octylp h en yl)-2,3-
d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4i). The procedure was
carried out using 4-n-octylphenylacetylene (1.45 g, 6.76 mmol),
1.62 (4H, m, CH2), 0.87 (3H, t, 3J ) 6.9 Hz, CH3); 13C NMR
(DMSO-d6; 75 MHz) δ 13.2 (CH3), 20.1, 22.3, 27.9 (C3H6), 42.5

Highly Potent and Selective Inhibition of VZV
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4997
which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-octylphenyl-
acetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4i) (111 mg,
11%), after purification by column chromatography: 1H NMR
(DMSO-d6; 300 MHz) δ 8.92 (1H, s, H-4), 7.61 (2H, Ha) - 7.33
An tivir a l Assa ys. Confluent HEL cells grown in 96-well
microtiter plates were inoculated with VZV at an input of 20
PFU (plaque-forming units)/well or with CMV at an input of
100 PFU/well with HSV at 100 CCID50 (50% cell culture
infective doses)/well. After a 1-2-h incubation period, residual
virus was removed and the infected cells were further incu-
bated with MEM (supplemented with 2% inactivated FCS, 1%
L-glutamine and 0.3% sodium bicarbonate) containing varying
concentrations of the compounds. Antiviral activity was ex-
pressed as EC50 (50% effective concentration), or compound
concentration required to reduce viral plaque formation (VZV
after 5 days) or virus-induced cytopathicity (CMV after 7 days;
HSV and VV after 3 days) by 50% compared to the untreated
control.
3
4
(2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz), 7.25 (1H, s,
3
3
H-5), 6.21 (1H, dd, J ) 6.19 Hz, H-1′), 5.59 (1H, d, J ) 4.1
Hz, 3′-OH), 5.272 (1H, t, 3J ) 5.12 Hz, 5′-OH), 4.39 (1H, m,
3
H-3′), 3.75 (1H, m, H-4′), 3.62 (2H, m, H-5′), 2.71 (2H, t, J )
6.9 Hz, R-CH2), 2.34 and 2.13 (2H, m, 2-H′a and 2-H′b), 1.61
3
(2H, m, CH2), 1.51-1.19 (10H, m, CH2), 0.82 (3H, t, J ) 6.9
Hz, CH3); 13C NMR (DMSO-d6; 75 MHz) δ 13.2 (CH3), 20.1,
21.39, 22.3, 27.9, 28.5, 29.5, 30.2 (C7H14), 41.7 (C-2′), 61.1 (C-
5′), 69.8 (C-3′), 87.9, 88.7 (C-1′, C-4′), 99.0 (C-5), 107.2 (C-4a),
124.8 (C-Hb), 126.2 (ipso-C), 129.3 (C-Ha), 138.2(C-4), 144.2
(para-C), 154.2 (C-6), 160.7 (C-2), 171.6 (C-7a); MS (ES+) m/e
463 (MNa+, 100%), 347 (baseNa+, 30%). Accurate mass:
Cytotoxicity Assa ys. Confluent monolayers of HEL cells
as well as growing HEL cells in 96-well microtiter plates were
treated with different concentrations of the experimental
drugs. Cell cultures were incubated for 3 (growing cells) or 5
(confluent cells) days. At the indicated times, the cells were
trypsinized and the cell number was determined using a
Coulter counter. The 50% cytostatic concentration (CC50) was
defined as the compound concentration required to reduce the
cell number by 50%.
C
25H32N2O5Na requires 463.2209; found 463.2215. Found: C,
67.83; H, 7.41; N, 6.28. C25H32N2O5 requires: C, 68.16; H, 7.32;
N, 6.36.
3-(2′-Deoxy-â-D-r ib ofu r a n osyl)-6-(4-n -ch lor op h en yl)-
2,3-d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4j). The procedure
was carried out using 4-n-chlorophenylacetylene (0.92 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-chlo-
rophenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4j)
(474 mg, 58%), after purification by column chromatography:
1H NMR (DMSO-d6; 300 MHz) δ 8.91 (1H, s, H-4), 7.88 (2H,
Ack n ow led gm en t. The authors are grateful to Mrs.
Anita Camps and Ms. Lies Vandenheurck for excellent
technical assistance. This research was supported by
grants from the Belgian Fonds voor Geneeskundig
Wetenschappelijk Onderzoek, Belgian Geconcerteerde
Onderzoeksacties, and Therapeutic Developments Ltd.
(TDL). We also thank Helen Murphy for excellent
secretarial assistance.
3
4
Ha) - 7.57 (2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz),
7.37 (1H, s, H-5), 6.19 (1H, dd, 3J ) 6.17 Hz, H1′), 5.35 (1H, d,
3J ) 4.1 Hz, 3′-OH), 5.24 (1H, t, 3J ) 5.12 Hz, 5′-OH), 4.26
(1H, m, H-3′), 3.95 (1H, m, H-4), 3.70 (2H, m, H-5′), 2.41 and
2.13 (2H, m, 2-H′a and 2-H′b); 13CNMR (DMSO-d6; 75 MHz) δ
41.6 (C-2′), 60.9 (c-5′), 69.8 (C-3′), 88.0, 88.5, (C-1′, C-4′), 100.8
(C-5), 107.0 (C-4a), 126.6 (C-Hb), 127.6 (ipso-C), 129.6 (C-
Ha), 134.2 (C-4), 152.8 (para-C), 154.1 (C-6), 161.2 (C-2), 171.8
(C-7a); MS (ES+) m/e 385 (MNa+, 100%), 269 (baseNa+, 10%).
Accurate mass:
C17H15N2O5ClNa requires 385.0567; found
385.0575. Found: C, 56.02; H, 4.39; N, 7.67. C17H15ClN2O5
requires: C, 56.29; H, 4.17, N, 7.72.
Refer en ces
(1) McGuigan, C.; Yarnold, C. J .; J ones, G.; Velazquez, S.; Barucki,
H.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini,
J . Potent and selective inhibition of Varicella-Zoster virus (VZV)
by nucleoside analogues with an unusual bicyclic base. J . Med.
Chem. 1999, 42, 4479-4484.
3-(2′-Deoxy-â-D-r ib ofu r a n osyl)-6-(4-n -b r om op h en yl)-
2,3-d ih yd r ofu r o[2,3-d ]p yr im id in -2-on e (4k ). The procedure
was carried out using 4-n-bromophenylacetylene (1.22 g, 6.76
mmol), which gave 3-(2′-deoxy-â-D-ribofuranosyl)-6-(4-n-bro-
mophenylacetylene)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4k)
(174 mg, 19%), after purification by column chromatography:
1H NMR (DMSO-d6; 300 MHz) δ 8.88 (1H, s, H-4), 7.78 (2H,
(2) McGuigan, C.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq,
E.; Balzarini, J . Bicyclic Nucleoside Inhibitors of Varicella-Zoster
Virus (VZV): The effect of a terminal halogen substitution in
the side chain. Bioorg. Med. Chem. Lett. 2000, 10, 1215-1217.
(3) McGuigan, C.; Yarnold, C. J .; J ones, G.; Vela´zquez, S.; Barucki,
H.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini,
J . Novel Bicyclic Fluorescent Nucleosides as Potent and Selective
Inhibitors of VZV. 13th International Conference on Antiviral
Research, Baltimore, MD, Apr 16-21, 2000; Presentation 121.
(4) Brancale, A.; McGuigan, C.; De Clercq, E.; Balzarini, J . Design,
Synthesis and Evaluation of New Antiviral Nucleosides. 13th
International Conference on Antiviral Research, Baltimore, MD,
Apr 16-21 2000; Presentation 128.
(5) Robins, M. J .; Barr, P. J . Nucleic-Acid Related-Compounds 0.39.
Efficient Conversion of 5-Iodo to 5-Alkynyl and derived 5-Sub-
stituted Uracil Bases and Nucleosides. J . Org. Chem. 1983, 48,
1854-1862.
(6) Robins, M. J .; Barr, P. J . Nucleic-Acid Related-Compounds 0.31.
Smooth and Efficient Palladium Copper Catalyzed Coupling of
Terminal Alkynes with 5-Iodouracil Nucleosides. Tetrahedron
Lett. 1981, 22, 421-424.
3
4
Ha) - 7.66 (2H, Hb) (AB system, J ) 7.89 Hz, J ) 2.3 Hz),
7.34 (1H, s, H-5), 6.14 (1H, dd, 3J ) 6.17 Hz, H1′), 5.31 (1H, d,
3J ) 4.1 Hz, 3′-OH), 5.19 (1H, t, 3J ) 5.12 Hz, 5′-OH), 4.65
(1H, m, H-3′), 3.92 (1H, m, H-4), 3.67 (2H, m, H-5′), 2.48 and
2.19 (2H, m, 2-H′a and 2-H′b); 13CNMR (DMSO-d6; 75 MHz) δ
41.6 (C-2′), 60.9 (c-5′), 69.8 (C-3′), 88.1, 88.5, (C-1′, C-4′), 100.9
(C-5), 107.0 (C-4a), 122.9 (C-Hb), 126.8 (ipso-C), 127.9 (C-
Ha), 139.0 (C-4), 152.8 (para-C), 154.1 (C-6), 160.9 (C-2), 171.3
(C-7a); MS (ES+) m/e 429 (MNa+, 100%), 431 (MNa+, 100%),
313 (baseNa+, 25%), 315 (baseNa+, 25%). Accurate mass:
C
17H15N2O579BrNa requires 429.0062; found 429.0061; C17H15
-
N2O581BrNa requires 431.0042; found 431.0052. Found: C,
49.89; H, 3.88; N, 6.63. C17H15BrN2O5‚0.5H2O requires: C,
49.04; H, 3.88, N, 6.73.
Ma ter ia ls a n d Exp er im en ta l P r oced u r es: Vir ology.
Cells. Human embryonic lung (HEL) fibroblasts and E6SM
cells were grown in minimum essential medium (MEM)
supplemented with 10% inactivated fetal calf serum (FCS),
1% L-glutamine and 0.3% sodium bicarbonate.
Vir u ses. The laboratory wild-type VZV strains OKA and
YS, the thymidine kinase-deficient VZV strains 07-1 and
YS-R, HSV-1 (KOS), HSV-2 (G), the thymidine kinase-deficient
HSV-1 strains B-2006 and VMW 1837, cytomegalovirus strains
Davis and AD-169, and vaccinia virus were used in the virus
inhibition assays.
(7) Crisp, G. T.; Flynn, B. L. Palladium-Catalyzed Coupling of
Terminal Alkynes with 5-(Trifluoromethanesulfonyloxy)-
Pyrimidine Nucleosides. J . Org. Chem. 1993, 58, 6614-6619.
(8) De Clercq, E.; Holy, A.; Rosenberg, I.; Sakuma, T.; Balzarini,
J .; Maudgal, P. C. A Novel Selective Broad-Spectrum Anti-DNA
virus Agent. Nature 1986, 324, 464-467, 293-297.
(9) Values calculated using ClogP 1.0, Biobyte Corp., Claremont,
CA.
J M000210M